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https://openalex.org/W2163196794
https://figshare.com/articles/journal_contribution/Supplementary_Figure_1_from_Cetuximab-Activated_Natural_Killer_and_Dendritic_Cells_Collaborate_to_Trigger_Tumor_Antigen_Specific_T-cell_Immunity_in_Head_and_Neck_Cancer_Patients/22454904/1/files/39906024.pdf
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Cetuximab-Activated Natural Killer and Dendritic Cells Collaborate to Trigger Tumor Antigen–Specific T-cell Immunity in Head and Neck Cancer Patients
Clinical cancer research
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31
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https://openalex.org/W2784295131
https://www.matec-conferences.org/10.1051/matecconf/201814502004/pdf
English
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Tensile behavior of humid aged advanced composites for helicopter external fuel tank development
MATEC web of conferences
2,018
cc-by
4,433
© The Authors, published by EDP Sciences. This is an open access article distributed under the terms of the Creative Commons Attribution License 4.0 (http://creativecommons.org/licenses/by/4.0/). Tensile behavior of humid aged advanced composites for helicopter external fuel tank development Mihaela Condruz1*, Alexandru Paraschiv2, Cristian Pușcașu2, and Ionuț Sebastian Vintilă1 1COMOTI - Romanian Research & Development Institute for Gas Turbines, Composite Materials Laboratory for Aeronautical Field and Space, 220 D Iuliu Maniu Av., Bucharest, Romania 2COMOTI - Romanian Research & Development Institute for Gas Turbines, Gas Turbine Special Components. Physics and Mechanic Testing Laboratory, 220 D Iuliu Maniu Av., Bucharest, Romania Abstract. Influence of humid aging on tensile properties of two polymeric composites was studied. The purpose of the study was to evaluate the suitability of the materials for a naval helicopter external fuel tank. Due to the application, the humid environment was kerosene and saline solution to evaluate the sea water effect on the composite tensile strength. The composite samples were immersed in kerosene for 168 hours, respective 1752 hours and in saline solution for 168 hours. Tensile tests were performed after the immersion. The composite sample tensile tests showed that kerosene and saline solution had no influence on the elastic modulus of the materials, but it was observed a slight improvement of the tensile strength of the two polymeric composites. * Corresponding author : raluca.condruz@comoti.ro MATEC Web of Conferences 145, 02004 (2018) NCTAM 2017 MATEC Web of Conferences 145, 02004 (2018) NCTAM 2017 https://doi.org/10.1051/matecconf/201814502004 1 Introduction Plastic fuel tanks should be fuel and environmental resistant (corrosion due to salt, moisture, ultraviolet radiation, thermal cycling in atmospheric conditions during normal service use and storage between -54°C and +71°C) [8-11]. Durability of FRP can be affected by the contact with water or other fluids [12]. Most polymeric materials (in the form of a composite matrix or a polymeric fibre) are capable of absorbing relatively small but potentially significant amounts of moisture from the surrounding environment. The physical mechanism for moisture gain, assuming there are no cracks or other wicking paths, is generally assumed to be the mass diffusion following Fick's Law (the moisture analogue to thermal diffusion) [13]. The humid aging is one of the main causes of long-term failure of organic composites, which can affect the material in several modes: matrix microcracking, plasticization, hydrolysis, delamination, blistering, void growth, swelling, localized damage at the fibre-matrix interface [12-18]. Epoxy resin is known to have affinity for water [12], moisture has significant effects on physical and chemical properties of the resin as well as on the final composite structures performance, especially in long- term utilization [19]. The moisture absorption effect on polymeric composites mechanical properties had been studied for decades [20,21], but until now it’s still a challenge to predict how a fibre reinforced composite will behave under a certain humid environment. In early technical studies regarding environmental influence on composites, it was observed that in case of carbon/epoxy composites, the filament properties show very little environmental sensitivity, while epoxy resin is affected by the absorbed moisture and temperature effects which can lead to a change in failure mode [20], also the mechanical properties of composites are reduced after humid exposure, but all moisture effects were completely reversed upon re-drying the specimens [21]. Most of the authors studied water humid aging influence on polymeric composites [22-28] and as a general conclusion, the water causes mechanical properties degradation of thermosetting composites in case of extended period of immersion which can be noticed by matrix osmotic, cracking, swelling, mass loss, fibre exposure, chemical alterations etc. Many information about the effect of other fluids than water on composite properties are not available in the literature. p p p Souza et.al. 1 Introduction Nowadays, fibre reinforced polymers (FRP) are a common material used in the aerospace field (for commercial/military aircrafts, helicopters, satellites etc.) due to their remarkable properties, such as strength, stiffness, low density and low coefficient of thermal expansion [1, 2]. An important factor that stays behind the greater use of composites is the cost reduction experienced by carbon fibres [1]. Also, in case of aerospace applications, the reduction of weight, costs and fuel along with the increase of resistance and durability of the components are key aspects. Usually, in helicopter applications, composite materials are used to manufacture rotor blades, which ensure a significant weight reduction over aluminium and provide a better control over the vibration characteristics of the blades [3]. Excepting rotor blades, polymeric composites were integrated in tank assemblies for launch vehicles and helicopters. Researches were conducted to manufacture and validate external composite fuel tanks for helicopters to replace the metallic tanks [4]. External fuel tanks have been introduced to extend the range of a helicopter or to provide refuelling capacity at a remote location (it can provide at least an additional hour of flight depending on its capacity [5]). The first attempt to replace metallic external tanks was in the middle of 1970 and they were replaced by a hybrid model, a Composite Overwrapped Pressure Vessel - MATEC Web of Conferences 145, 02004 (2018) NCTAM 2017 https://doi.org/10.1051/matecconf/201814502004 COPV [4] (structure with a thin metal liner that acts as a leakage barrier, overwrapped by composite winding layers [6]). Afterwards, the COPV tanks were replaced by all composite external tanks. For researchers, an opportunity is represented by designing and manufacturing of an external composite fuel tank for F-35 Lightning II fighters [4,7]. An important aspect for designing and manufacturing external and auxiliary fuel tanks is to study structure's requirements and specification, which are stated in MIL-T-7378A [8]. COPV [4] (structure with a thin metal liner that acts as a leakage barrier, overwrapped by composite winding layers [6]). Afterwards, the COPV tanks were replaced by all composite external tanks. For researchers, an opportunity is represented by designing and manufacturing of an external composite fuel tank for F-35 Lightning II fighters [4,7]. An important aspect for designing and manufacturing external and auxiliary fuel tanks is to study structure's requirements and specification, which are stated in MIL-T-7378A [8]. 2 Experimental procedure Two types of polymeric composite laminates were manufactured for this experiment. The laminates were manufactured by using metallic moulds and an autoclave (curing process under temperature, vacuum, pressure). The composite consisted in two prepreg precursors, a HexPly M49/42%/200T2X2/CHS-3K (referred as HexPly) and an EP 127-C20-45 T2 (referred as EP). The HexPly consisted in a twill 2x2 carbon fabric 3K HS preimpregnated with an epoxy resin system (42% resin content), and the EP was a twill 2x2 carbon fabric 3K HTA preimpregnated with a blend of epoxy resin and cyanate ester (45±3% resin content). After curing, samples were cut from laminates using a water jet cutting machine. The samples dimensions (250x25x3 mm with 1 mm thick tabs, type 3) were established based on SR EN ISO:527-4/2000 [33], and ASTM D3039/D 3039M/2000 [34]. To ensure the water removal from the cutting process and the curing of the tabs, all the samples were post-cured in a vacuum assisted oven (curing cycle: heating for 35 minutes until 120°C were reached, heating rate of 2°C/min., 90 minutes at 120°C and oven assisted cooling). All the samples were weighed before the immersion in saline solution and in kerosene at room temperature. To analyse the sea water effect on the composite tensile strength a saline solution was made by dissolving 35 g sea salt in a litre of distilled water to obtain a saline solution with 35 ‰ salinity (typical seawater salinity). The specimens were immersed in saline solution in an open glass recipient and in kerosene in a sealed container. The samples were kept at room temperature (20±2°C) in kerosene for 168 hours (7 days), respective 1752 hours (73 days) and in saline solution for 168 hours. In Fig. 1 can be observed the immersed samples in kerosene and saline solution. After immersion, the samples were wiped out with a cloth, weighed out to calculate the moisture content (or percent weight gain) of the material to establish if the material has kept its dimensional measurements (or has swollen). a b Fig. 1. Samples immersed in kerosene (a) and saline solution (b) a Fig. 1. Samples immersed in kerosene (a) and saline solution (b) Tensile tests were carried out by using a Universal mechanical testing machine Instron 8802 with a displacement rate of 2 mm/min, according to SR EN ISO: 527-4 [33]. 1 Introduction [29] evaluated the creep behaviour of an E-glass/polyester composite in water and lubricant oil and it was observed that after 6 and 14 months of exposure, Young’s modulus of the composite was reduced by 20% regardless the aging fluid when the test was performed at room temperature and in case of tests performed at 60°C, the effects of immersion in lubricant oil were more severe than those after immersion in water. Areef [30] performed an experimental study on different solution absorption behaviour of a sandwich glass fibres/epoxy composite. The results showed a maximum value of mass gain of the samples immersed in organic solutions (kerosene, benzene) compared with samples immersed in other solutions, the samples immersed in distilled water had a minimum mass gain. Curliss [31] studied the effect of jet fuel JP-4 on thermoplastic and thermosetting composites. The thermoplastic matrix composites absorbed more fuel compared with the thermoset matrix composites, but the absorption appeared to be reversible without notable degradation of the matrix polymer. There was no evidence that any component of the matrix polymers was leaching into the jet fuel or that any fraction of the polymers was dissolving into the jet fuel. Taghavi [32] studied the diffusion of moisture into glass/epoxy, carbon/epoxy and carbon/polyimide composites immersed in water and kerosene. The glass epoxy composites showed a loss of about 21% of their transverse tensile strength after 100 2 MATEC Web of Conferences 145, 02004 (2018) NCTAM 2017 https://doi.org/10.1051/matecconf/201814502004 hours of immersion in 80°C kerosene, but after this point no further changes were observed up to 3500 hours. The transverse modulus was not significantly changed even after 3500 hours of immersion. For carbon polyimide composites, even after 3500 hours of immersion in 80°C kerosene neither the transverse modulus nor the strength changed. The present research was focused on studying the kerosene and saline solution influence on the tensile properties of polymeric composite materials suitable for the manufacturing of an external fuel tank for a naval helicopter (IAR 330 PUMA). 2 Experimental procedure Example representation of typical failure modes in tensile tests of composites [34 2 Experimental procedure For evaluating the influence of the immersion in kerosene and salted water as a function of time it were used five specimens for each type of sample and for comparison were used 3 3 MATEC Web of Conferences 145, 02004 (2018) NCTAM 2017 https://doi.org/10.1051/matecconf/201814502004 reference samples from each lot of samples (not immersed). The fracture’s modes were coded using typical modes from ASTM D3039/D 3039M/2000 [34], in Table 1 are presented the tensile test failure codes. In Table 2 can be observed the fracture’s modes of composite specimens after tensile tests. reference samples from each lot of samples (not immersed). The fracture’s modes were coded using typical modes from ASTM D3039/D 3039M/2000 [34], in Table 1 are presented the tensile test failure codes. In Table 2 can be observed the fracture’s modes of composite specimens after tensile tests. Table 1. Tensile test failure codes/typical modes [34] Table 1. Tensile test failure codes/typical modes [34] First character Code Second character Code Third character Code Angled A Inside grip/tab I Bottom B edge Delamination D At grip/tab A Top T Grip/tab G <1W from grip/tab W Left L Lateral L Gage G Right R Multi – mode M Multiple areas M Middle M long. Splitting S Various V Various V eXplosive X Unknown U Unknown U Other O - - - Fig. 2. Example representation of typical failure modes in tensile tests of composites [34] Table 1. Tensile test failure codes/typical modes [34] First character Code Second character Code Third character Code Angled A Inside grip/tab I Bottom B edge Delamination D At grip/tab A Top T Grip/tab G <1W from grip/tab W Left L Lateral L Gage G Right R Multi – mode M Multiple areas M Middle M long. Splitting S Various V Various V eXplosive X Unknown U Unknown U Other O - - - Fig. 2. Example representation of typical failure modes in tensile tests of composites [34] Table 1. Tensile test failure codes/typical modes [34] First character Code Second character Code Third character Code Angled A Inside grip/tab I Bottom B edge Delamination D At grip/tab A Top T Grip/tab G <1W from grip/tab W Left L Lateral L Gage G Right R Multi – mode M Multiple areas M Middle M long. Splitting S Various V Various V eXplosive X Unknown U Unknown U Other O - - - Fig. 2. MATEC Web of Conferences 145, 02004 (2018) NCTAM 2017 MATEC Web of Conferences 145, 02004 (2018) NCTAM 2017 https://doi.org/10.1051/matecconf/201814502004 In comparison with their reference samples, the HexPly specimens showed an increase of the tensile strength with 3% after 1752 hours of kerosene immersion at room temperature. EP specimens showed an increase of the tensile strength with 5% after 1752 hours of kerosene immersion at room temperature. A critical factor that can affect the tensile strength of the polymeric composite materials immersed in kerosene is the temperature of kerosene, fact proved by Taghavi [32] in his thesis, in which the specimens immersed for 100 hours in kerosene at 80°C showed a 21% reduction of transverse tensile strength, but after this point no further changes were observed up to 3500 hours. Thereby, in case of room temperature immersion for this period of time (max. 1752 h), the kerosene had an opposite effect, it leads to a slight increase of tensile strength. It can be concluded that kerosene absorption could slightly improve the tensile strength on short term due to modifications of the internal structure of the polymer and on long term it could weaken the polymer due to breaking of hydrogen bonds because a decrease number of hydrogen bonds is a factor which influences the mechanical properties of the polymeric matrix. Regarding the influence of the immersion on saline solution the tensile properties of composites weren’t affected by the saline solution immersion on short term (168 h), results confirmed also by the literature. The studies regarding saline solution/seawater immersion of composite materials are limited, Roy Xu et. al. [28] observed that compression-after-impact strengths of the wet specimens were reduced by around 10% compared to the baseline dry specimens. Visco et. al. [22] studied seawater absorption ability and mechanical performance of two glass–fibre reinforced composites with different thermoset matrices (isophthalic polyester, vinyl ester) and concluded that vinyl ester resin composite had a lower water diffusion coefficient but higher chemical stability, stiffness and strength after immersion in seawater than the isophthalic one. Even if the tensile strength of the composite materials was increased it was observed that they maintain their Young’s modulus value, 68 GPa for HexPly samples, respective 62 GPa for EP samples. The elastic modulus could be maintained over a period of time in moist environment and afterwards it could be altered, the polymer becoming more brittle and easy to break. 3 Results and discussions Both materials (HexPly and EP) showed a moist weight gain of 0,10% after the first 168 hours of immersion in kerosene and 0,16% after the 1752 hours of immersion in kerosene. Regarding the immersion in saline solution, the moist weight gain of both composites was 0,13% after 168 hours of immersion. The composite materials gained more weight after the immersion in saline solution compared with kerosene immersion, those were different results compared with the findings of Areef [30] who observed a higher weight gained by the composite immersed in organic solution (kerosene) then in distilled water (this could be influenced by the fact that the composite material used by Areef [30] consisted in glass fibre reinforced polymer and the samples were immersed only in distilled water not in distilled water with salt). The moisture absorption of the HexPly and EP materials could increase after more hours of immersion, but the overall capacity of absorption of the composite wasn’t the goal of this study. The composite samples maintained their dimensional measurements after the immersion in kerosene and saline solution immersion, they didn’t modify the physical aspect of the samples (no peels or cracks were observed) and the polymeric materials 4 MATEC Web of Conferences 145, 02004 (2018) NCTAM 2017 https://doi.org/10.1051/matecconf/201814502004 weren’t dissolved after immersion in saline solution or kerosene. Similar physical observations regarding the effect of jet fuel on polymeric composite materials were made by Curtis et.al. [31]. g g j p y p by Curtis et.al. [31]. Table 2. Fracture’s modes of composite specimens after tensile tests Immersion conditions HexPly EP Reference - unimmersed 168 h saline 168 h kerosene 1752 h kerosene The tensile test results showed a slight increase of the tensile strength of laminates after each immersion phase in kerosene. by Curtis et.al. [31]. Table 2. Fracture’s modes of composite specimens after tensile tests Immersion conditions HexPly EP Reference - unimmersed 168 h saline 168 h kerosene 1752 h kerosene The tensile test results showed a slight increase of the tensile strength of laminates after each immersion phase in kerosene. The tensile test results showed a slight increase of the tensile strength of laminates after each immersion phase in kerosene. 5 5 MATEC Web of Conferences 145, 02004 (2018) NCTAM 2017 Regarding the analysis of the tensile failure modes, it was observed that in case of HexPly samples, the majority presented a lateral inside grip top (LIT) typical failure mode followed by lateral gage top (LGT) failure mode, while EP samples presented an angle gage middle (AGM) failure mode followed by angle gage bottom (AGB) failure mode. In Table 3 are specified the results of tensile test failure mode analysis of the composite specimens. It can be observed that in case of HexPly, the tested specimens present less scattered failure modes compared with the EP specimens. The predominant failure modes are typical for the matrix shear failure and brittle failure of polymeric matrix composites reinforced with fibres along with fibre pull-out. In Fig. 3 a, b it can be summarized the influence of kerosene immersion on the tensile strength of the two composite materials and in Fig. 3 c can be observed the influence of saline solution on their tensile strength after 168 hours of immersion. 6 6 https://doi.org/10.1051/matecconf/201814502004 MATEC Web of Conferences 145, 02004 (2018) NCTAM 2017 Table 3. Results of the tensile test failure mode analysis a b c Fig. 3. Influence of kerosene and saline solution immersion on composite tensile strength: a) in case of HexPly; b) in case of EP; c) results in case of saline immersion of the Material Typical failure mode Number of specimens HexPly LIT 10 specimens HexPly LGB 2 specimens HexPly LGT 3 specimens HexPly LAB 2 specimens HexPly LAT 1 specimen HexPly LIB 1 specimen HexPly MIT 1 specimen EP AGM 6 specimens EP AGB 3 specimens EP LIB 1 specimen EP LAT 2 specimens EP AMM 1 specimen EP LGT 1 specimen EP AGT 1 specimen EP LGB 2 specimens EP LIT 2 specimens EP LAB 1 specimen a b c Fig. 3. MATEC Web of Conferences 145, 02004 (2018) NCTAM 2017 Influence of kerosene and saline solution immersion on composite tensile strength: a) in case of HexPly; b) in case of EP; c) results in case of saline immersion of the composites Material Typical failure mode Number of specimens HexPly LIT 10 specimens HexPly LGB 2 specimens HexPly LGT 3 specimens HexPly LAB 2 specimens HexPly LAT 1 specimen HexPly LIB 1 specimen HexPly MIT 1 specimen EP AGM 6 specimens EP AGB 3 specimens EP LIB 1 specimen EP LAT 2 specimens EP AMM 1 specimen EP LGT 1 specimen EP AGT 1 specimen EP LGB 2 specimens EP LIT 2 specimens EP LAB 1 specimen b c Fig. 3. Influence of kerosene and saline solution immersion on composite tensile strength: a) in case of HexPly; b) in case of EP; c) results in case of saline immersion of the composites 7 7 https://doi.org/10.1051/matecconf/201814502004 MATEC Web of Conferences 145, 02004 (2018) NCTAM 2017 4 Conclusions The present study was focused on the tensile behaviour of two composite materials before and after aging in humid environment (saline solution and kerosene). The humid environments selected for the tests were kerosene due to the fact that jet fuels consist in kerosene and different types of additives and saline solution due to the fact that the tank is designed for a navel helicopter. Two types of composite laminates were tested and it was observed that kerosene slightly improves the tensile properties on short term exposure. g y p p p p HexPly M49/42%/ 200T2X2/CHS-3K showed an increase in tensile strength of 3% after 1752 hours of kerosene immersion and EP 127-C20-45 T2 showed an increase in tensile strength of 5% after 1752 hours of kerosene immersion. The saline solution exposure on short term (168 h) didn’t influence the tensile properties of the HexPly M49/42%/ 200T2X2/CHS-3K, but in case of EP 127-C20-45 T2 it was observed that the saline absorption increased the tensile properties with 5%. However, it was observed that the composite laminates maintain their Young modulus 68 GPa for HexPly samples, respective 62 GPa for EP samples. Peeling, cracking or matrix dissolution of polymeric composite immersed in saline solution or kerosene weren’t observed. Due to the fact that it can’t be predicted how a fibre reinforced composite will behave under a certain humid environment further studies will be made regarding the fuel compatibility with polymeric composites. Acknowledgment This study was carried out within Grant no. 293/2014, supported by the Romanian Minister of Research and Innovation. References [1] A.P. Mouritz, Introduction to aerospace materials, Woodhead Publishing, 303-337 (2012) [1] A.P. Mouritz, Introduction to aerospace materials, Woodhead Publishing, 303-337 (2012) [2] R.J.H. Wanhill, Aerospace Materials and Material Technologies : Aerospace Materials, 1, Springer (2017) [3] P.K. Mallick, Fiber-reinforced composites: Materials, manufacturing and design, CRC Press, 19 - 48 (2007) [4] M.R. Legault, http://www.compositesworld.com/articles/carrier-capable-all-composite- external-fuel-tank(2) (2011) [5] UH-60 A/L/M CRASHWORTHY EXTERNAL FUEL SYSTEM (CEFS) Brochure, http://www.robertsonfuelsystems.com/defense/crashworthy-external-fuel-system-cefs/ [6] M. Ren, X. Chang, H.Y. Xu, T. Li, Compos Struct, 160, 1339-1347 (2017) [7] E. Elkhershi, The Telegraph, http://www.telegraph.co.uk/news/2016/03/18/inside-the-f- 35-lightning-ii---the-invisible-fighter-jet/ (2015) [8] MIL-T-7378A. Military specification Tanks, Fuel, Aircraft, external, Auxiliary, Removable (1958) [9] MIL-DLT-5578D. Detail Specification Tanks, Fuel, Aircraft, Self-sealing (2008) [10] B. Wei, H. Cao, S. Song, Corros Sci, 53, 426-431 (2011) [11] S. Lampman, Characterization and Failure Analysis of Plastics, ASM International, 146-152 (2003) [12] J.A. Hinkley, J.W. Connell, Long-Term Durability of Polymeric Matrix Composites, Springer, 1-38 (2012) ( ) [2] R.J.H. Wanhill, Aerospace Materials and Material Technologies : Aerospace Materials, 1, Springer (2017) [3] P.K. Mallick, Fiber-reinforced composites: Materials, manufacturing and design, CRC Press, 19 - 48 (2007) [4] M.R. Legault, http://www.compositesworld.com/articles/carrier-capable-all-composite- external-fuel-tank(2) (2011) [5] UH-60 A/L/M CRASHWORTHY EXTERNAL FUEL SYSTEM (CEFS) Brochure, http://www.robertsonfuelsystems.com/defense/crashworthy-external-fuel-system-cefs/ [6] M. Ren, X. Chang, H.Y. Xu, T. Li, Compos Struct, 160, 1339-1347 (2017) [7] E. Elkhershi, The Telegraph, http://www.telegraph.co.uk/news/2016/03/18/inside-the-f- 35-lightning-ii---the-invisible-fighter-jet/ (2015) [5] UH-60 A/L/M CRASHWORTHY EXTERNAL FUEL SYSTEM (CEFS) Brochure, http://www.robertsonfuelsystems.com/defense/crashworthy-external-fuel-system-cefs/ [6] M R X Ch H Y X T Li C St t 160 1339 1347 (2017) E. Elkhershi, The Telegraph, http://www.telegraph.co.uk/news/2016/03/18/inside-the-f- -lightning-ii---the-invisible-fighter-jet/ (2015) g g g j ( ) [8] MIL-T-7378A. Military specification Tanks, Fuel, Aircraft, external, Auxiliary, Removable (1958) [9] MIL-DLT-5578D. Detail Specification Tanks, Fuel, Aircraft, Self-sealing (2008) [10] B. Wei, H. Cao, S. Song, Corros Sci, 53, 426-431 (2011) [10] B. Wei, H. Cao, S. Song, Corros Sci, 53, 426-431 (2011) [11] S. Lampman, Characterization and Failure Analysis of Plastics, ASM International, 146-152 (2003) [11] S. Lampman, Characterization and Failure Analysis of Plastics, ASM International, 146-152 (2003) [12] J.A. Hinkley, J.W. Connell, Long-Term Durability of Polymeric Matrix Composites, Springer, 1-38 (2012) 8 8 MATEC Web of Conferences 145, 02004 (2018) NCTAM 2017 https://doi.org/10.1051/matecconf/201814502004 [13] MIL-HDBK-17-1F. Composite Materials Handbook, Polimeric Matrix Composites Guidelines for Characterization of Structural Materials, 1, (2002) [14] B.C. Ray, J. Reinf. Plast. Compos., 25, (2016), pp. 1227-1240 [15] P. Vaddadi, T. Nakamura, R.P. Singh, Acta Mater, 51, (2013), pp. 177-193 [16] B.G. Kumar, R.P. Singh, T. References Nakamura, J. Compos. Mater., 36, (2002), pp. 2713-27 [16] B.G. Kumar, R.P. Singh, T. Nakamura, J. Compos. Mater., 36, (2002), pp. 2713-2733 [17] S. Roy, Long-Term Durability of Polymeric Matrix Composites, Springer, (2012), pp. 181 - 236 [18] I. Merdas, F. Thominette, A. TCharkhtchi, J. Verdu, Compos. Sci. Technol., 62, (2002), pp. 487-492 [19] E. Perez-Pacheco, J.I. Cauich – Cupul, A. Valadez-Gonzalez, P.J. Herrera-Franco, J. Mater. Sci, 48, (2013), pp. 1873-1882, [20] C.E. Browning, C.E. Husman, J.M. Whitney, Moisture effects in epoxy matrix composites, Interim Report AFML-TR-77-171976, (1976) [21] J.M. Augl, The effect of moisture on carbon fibre reinforced epoxy composite - Mechanical property changes, Technical Report, Naval Surface Weapons Center, White Oak Laboratory, Maryland, USA, (1977) [22] A.M. Visco, N. Campo, P. Cianciafara, Compos. Part A-Appl S, 42, (2011), pp. 123- 130 [23] P. Krishnan, M.S. Abdul Majid, M. Afendi, S. Yaacob, A.G. Gibson, Compos. Struct., 148, (2016), pp. 1-11 [24] A. Chilali, W. Zouari, M. Assarar, H. Kebir, R. Ayad, Compos. Struct., Article in press, (2017) [25] V. Fiore, L. Calabrese, G. DI Bella, T. Scalici, G. Galtieri, A. Valenza, E. Proverbio, Compos. Part B-Eng., 93, (2016), pp. 35-42 [26] A.P.C. Barbosa, A.P.P. Fulco, E.S.S. Guerra, F.K. Arakaki, M. Tosatto, M.C.B. Co J.D.D Melo, Compos Part B-Eng., 110, (2017), pp. 298-306 [26] A.P.C. Barbosa, A.P.P. Fulco, E.S.S. Guerra, F.K. Arakaki, M. Tosatto, M.C.B. Costa J.D.D Melo, Compos Part B-Eng., 110, (2017), pp. 298-306 [27] J. Naveen, T.P. Sathish Kumar, S. Satheesh Kumar, 3, (2014), pp. 1091-1095 [28] L. Roy Xu, A. Krishnan, H. Ning, U. Vaidya, Compos Part B-Eng, 43, (2012), pp. 2480-2486 [29] L.R. de Souza, A.T. marques, J.R.M. D’Almeida, Compos Struct, 168, (2017), pp. 285-291 [30] S.R. Areef, Eng. & Technology, 26, (2008), pp. 1235-1239 [30] S. . ee , g. & ec o ogy, 6, ( 008), pp. 35 39 [31] D.B. Curliss, The effect of Jet fuel exposure on advanced aerospace composites I: Thermal and Chemical Analysis, Report, Materials Directorate, Wright Laboratory, Air Force System Command, Wright-Patterson Air Force Base, Ohio, USA, (1991) [32] S.G. Taghavi, Moisture effects on high performance Polymer Composites, Master Thesis, Department of Chemical Engineering and Applied Chemistry, University of Toronto, 2000 [33] SR EN ISO:527-4/2000 Plastics – Determination of tensile properties-Part 4: Test conditions for isotropic and orthotropic fibre-reinforced plastic composites [34] ASTM D3039/D 3039M/2000 Standard Test Method for Tensile Properties of Polymer Matrix Composite Materials 9 9
https://openalex.org/W3172459663
https://wrap.warwick.ac.uk/157454/1/WRAP-grey-white-matter-volume-changes-preterm-birth-Wolke-2021.pdf
English
null
Grey and white matter volume changes after preterm birth: A meta-analytic approach
medRxiv (Cold Spring Harbor Laboratory)
2,021
cc-by
9,016
  10 Department of Psychiatry, School of Medicine, Technical University of Munich, Ismaninger Str. 22, 81675 Munich, Germany Citation: Schmitz-Koep, B.; Haller, B.; Coupé, P.; Menegaux, A.; Gaser, C.; Zimmer, C.; Wolke, D.; Bartmann, P.; Sorg, C.; Hedderich, D.M. Grey and White Matter Volume Changes after Preterm Birth: A Meta-Analytic Approach. J. Pers. Med. 2021, 11, 868. https://doi.org/10.3390/jpm11090868 * Correspondence: benita.schmitz-koep@tum.de Abstract: Cross-sectional studies have reported lower brain grey matter volumes (GMV) and white matter volumes (WMV) in preterm (PT) born individuals. While large MRI studies in the normative population have led to a better understanding of brain growth trajectories across the lifespan, such results remain elusive for PT born individuals since large, aggregated datasets of PT born individuals do not exist. To close this gap, we investigated GMV and WMV in PT born individuals as reported in the literature and contrasted it against individual volumetric data and trajectories from the general population. Systematic database search of PubMed and Web of Science in March 2021, and extraction of outcome measures were conducted by two independent reviewers. Individual data on full-term (FT) controls was extracted from freely available databases. Mean GMV, WMV, total intracranial volume (TIV), and mean age at scan were the main outcome measures. Of 532 identified records, nine studies were included with 538 PT born subjects between 1.1 and 28.5 years of age. Reference data was generated from 880 FT controls between 1 and 30 years of age. GMV was consistently lower in PT born individuals from infancy to early adulthood with no evidence for catch-up growth. While GMV changes followed a similar trajectory as FT controls, WMV was particularly low in adolescence after PT birth. Results demonstrate altered brain volumes after PT birth across the first half of lifespan. Future studies should address this issue in large aggregated datasets of PT born individuals. Received: 18 July 2021 Accepted: 27 August 2021 Published: 30 August 2021 Publisher’s Note: MDPI stays neutral with regard to jurisdictional claims in published maps and institutional affil- iations. Keywords: brain development; grey matter volume; white matter volume; structural magnetic resonance imaging; preterm birth Copyright: © 2021 by the authors. Licensee MDPI, Basel, Switzerland. This article is an open access article distributed under the terms and conditions of the Creative Commons Attribution (CC BY) license (https:// creativecommons.org/licenses/by/ 4.0/). Journal of Personalized Medicine Journal of Personalized Medicine Journal of Personalized Medicine Article Grey and White Matter Volume Changes after Preterm Birth: A Meta-Analytic Approach Benita Schmitz-Koep 1,2,* , Bernhard Haller 3 , Pierrick Coupé 4, Aurore Menegaux 1,2, Christian Gaser 5,6, Claus Zimmer 1,2, Dieter Wolke 7,8 , Peter Bartmann 9, Christian Sorg 1,2,10 and Dennis M. Hedderich 1,2 1 Department of Neuroradiology, School of Medicine, Technical University of Munich, Ismaninger Str. 22, 81675 Munich, Germany; aurore.menegaux@tum.de (A.M.); claus.zimmer@tum.de (C.Z.); christian.sorg@tum.de (C.S.); dennis.hedderich@tum.de (D.M.H.) 1 Department of Neuroradiology, School of Medicine, Technical University of Munich, Ismaninger Str. 22, 81675 Munich, Germany; aurore.menegaux@tum.de (A.M.); claus.zimmer@tum.de (C.Z.); christian.sorg@tum.de (C.S.); dennis.hedderich@tum.de (D.M.H.) 2 TUM-NIC Neuroimaging Center, School of Medicine, Technical University of Munich, Ismaninger Str. 22, 81675 Munich, Germany 3 Institute of Medical Informatics, Statistics and Epidemiology, School of Medicine, Technical University of Munich, Ismaninger Str. 22, 81675 Munich, Germany; bernhard.haller@tum.de y , g , , y; 4 Laboratoire Bordelais de Recherche en Informatique (LaBRI) UMR 5800, CNRS, Bordeaux INP, y g y 4 Laboratoire Bordelais de Recherche en Informatique (LaBRI) UMR 5800, CNRS, Bordeaux INP, University of Bordeaux, F-33400 Talence, France; pierrick.coupe@u-bordeaux.fr Laboratoire Bordelais de Recherche en Informatique (LaBRI) UMR 5800, CNRS, Bordeaux INP, University of Bordeaux, F-33400 Talence, France; pierrick.coupe@u-bordeaux.fr 5 Department of Psychiatry, University Hospital Jena, Am Klinikum 1, 07747 Jena, Germany; christian.gaser@uni-jena.de 6 Department of Neurology, University Hospital Jena, Am Klinikum 1, 07747 Jena, Germany 7 Department of Psychology, University of Warwick, University Road, Coventry CV4 7AL, UK; D.Wolke@warwick.ac.uk 7 Department of Psychology, University of Warwick, University Road, Coventry CV4 7AL, UK; D.Wolke@warwick.ac.uk 8 Warwick Medical School, University of Warwick, University Road, Coventry CV4 7AL, UK 9 9 Department of Neonatology, University Hospital Bonn, Venusberg-Campus 1, 53127 Bonn, Germany; Peter.Bartmann@ukbonn.de 2.2. Study Selection Criteria The following inclusion criteria were used to assess eligibility: (1) participants were born PT (<37 weeks of gestation); (2) MRI was used to determine mean total intracranial volume (TIV), total GMV, and total WMV; (3) mean age at MRI scan was reported and >1 year; and (4) the study was published in a peer-reviewed journal. If multiple studies reported on volumetric data of the same cohort at the same age, the study with the larger sample was included. Records were screened by two independent reviewers. p y p Studies with mean age lower than one year were excluded because of particular rapid brain growth in this period, in which small changes in postnatal age correspond to large volumetric changes [13]. 1. Introduction Preterm (PT) birth, defined as birth <37 weeks of gestation, has a high worldwide prevalence of almost 11% [1]. Prematurity is related to alterations in brain development in general, and in grey matter volume (GMV) and white matter volume (WMV) in particular, https://www.mdpi.com/journal/jpm J. Pers. Med. 2021, 11, 868. https://doi.org/10.3390/jpm11090868 J. Pers. Med. 2021, 11, 868 2 of 11 which has been documented at various age groups from infancy and childhood until early adulthood [2–7]. Large population studies and open datasets have led to a better understanding of brain development and aging in the general population across the lifespan [8–10]: GMV rapidly increases during infancy and early childhood until it peaks at school age. A fast decrease until around 40 years of age follows with a subsequent plateau phase. Lastly, there is an accelerated decrease of GMV around 80 years of age. Development of WMV follows an inverted U-shape. After fast growth in early ages, WMV peaks at around 30 to 40 years of age followed by a volume decrease. Characterization of age-dependent grey and white matter development in healthy subjects has enabled modelling pathological alterations in patients. For example, deviations from normal brain volume trajectories have been described in pathologic states such as Alzheimer’s disease and may be used as a diagnostic tool [11]. However, brain structure alterations after PT birth have mostly been studied in cross- sectional designs with narrow age ranges. Data from longitudinal studies with several magnetic resonance imaging (MRI) examinations are scarce and shared large datasets do not exist for PT populations. Hence, aggregated evidence about brain growth trajectories of the lifespan after PT birth remains elusive. To close this gap, we investigated GMV and WMV changes observed in cross-sectional studies of PT subjects over the first half of lifespan from infancy to early adulthood and contrasted it against brain growth trajectories of a large normative cohort of FT individuals. 2.1. Search Strategy The electronic databases PubMed and Web of Science were systematically searched for articles published before 30 March 2021. Key words: (birth OR born) AND (preterm OR prematur *) AND (magnetic resonance imaging OR MRI) AND (brain OR intracranial) AND (volume). 2.2. Study Selection Criteria 2. Materials and Methods This meta-analysis was conducted following the Preferred Reporting Items for Sys- tematic Reviews and Meta-Analyses (PRISMA) guidelines, see Table S1 [12]. It was not registered. A review protocol was not prepared. 3.1. Study Selection and Characteristics The search strategy identified 532 records (Figure 1, Table 1). After screening of abstract and title, 167 articles were identified as potentially relevant. Based on the inclusion criteria, nine studies were eligible for the meta-analysis [5,7,17–23]. Most of the studies excluded did not report on all variables of interest, i.e., TIV, GMV and WMV. For example, a recent study tracking regional brain growth up to age 13 in children born term and very preterm (VP) appeared to meet the inclusion criteria. However, while it did report mean age at scan, mean TIV and mean WMV, it did not report mean global GMV [24]. Therefore, it was excluded. Another study investigating regional brain volume abnormalities and long-term cognitive outcome in PT infants reported adjusted marginal mean of specific brain regions, however, it did not report mean TIV, WMV or GMV [25]. Therefore, it was excluded. Seven studies were excluded because they reported on data from cohorts that were already covered by other studies. In summary, 538 PT subjects collected in studies with mean age between 1.1 and 28.5 years, and data from 880 FT controls between 0.7 and 30 years of age were analyzed. Please see Figure 1 for a flowchart depicting study selection. Study characteristics of the nine studies included are shown in Table 1. 2.3. Data Collection Process and Data Extraction Data was extracted from the studies by two independent reviewers using a standard- ized form including author and year of publication, sample size, age at MRI scan (in years), mean and SD of TIV, GMV and WMV (in cm3), gestational age (GA, in weeks), birth weight (BW, in grams), percentages of male participants, year of birth, country of origin, and methodology of brain volume estimation. If PT samples were separated into groups (e.g., GA subgroups, positive or normal cranial ultrasound (cUS) findings and with or without intrauterine growth restriction (IUGR), volumes were reported and analyzed separately. If age was reported in months, it was divided by twelve to obtain age in years. If volume was reported in mm3, it was divided by 1000 to obtain volume in cm3. If volume was reported J. Pers. Med. 2021, 11, 868 3 of 11 in dm3, it was multiplied with 1000 to obtain volume in cm3. One study [7] reported mean GMV/TIV-ratio and mean WMV/TIV ratio. Mean GMV and WMV values were calculated from these ratios. If BW was reported in kilograms, it was multiplied with 1000 to obtain weight in grams. 2.4. Data on Full-Term Controls Data on FT controls was extracted from freely available databases: Cincinnati MR Imaging of NeuroDevelopment (C-MIND, https://research.cchmc.org/c-mind/ (accessed on February 2015)), National Database for Autism Research—NIH MRI Study of Normal Brain Development, Pediatric MRI Data Repository (NDAR-NIHPD, https://www.bic.mni. mcgill.ca/nihpd_info/info2/data_access.html (accessed on February 2015)), International Consortium for Brain Mapping (ICBM, http://www.loni.usc.edu/ICBM/ (accessed on February 2015)), and Information eXtraction from Images (IXI, http://brain-development. org/ixi-dataset/ (accessed on February 2015)). TIV, GMV and WMV were calculated from these four datasets as previously described by Coupé et al. [10] Overall, 880 FT con- trols between 0.7 and 30 years of age were included: 236 participants from C-MIND (mean age = 8.4 years, age range = 0.7–18.9 years, male = 45.3%), 375 from NDAR- NIHPD (mean age = 11.9 years, age range = 1.1–29.0 years, male = 53.6%), 169 from ICBM (mean age = 23.9 years, age range = 18–30 years, male = 52.7%), and 100 from IXI (mean age = 25.7 years, age range = 20.0–29.8 years, male = 44%). 2.5. Statistical Analysis Statistical analysis was performed with R [14]. A nonlinear regression model (Model 6, the cubic hybrid model, from Coupé et al. [10]: Vol = β4 (1 −e−Age/β5) + β0 + β1Age + β2Age2 + β3Age3 + ε) was used to model GMV, WMV, TIV, GMW/TIV, and WMV/TIV for FT controls in dependence of age using the R library minpack.lm [15]. Grey areas sur- rounding the curves show 95%-confidence intervals (CI), dotted lines show 95%-prediction intervals (PI), which were calculated using the R library propagate [16]. 3.2. Development of Grey Matter Volume after Preterm Birth While mean absolute GMV decre between adolescence and early adulthood in both PT subjects and FT controls, this crease appeared steeper in the PT studies. Figure 3a illustrates GMV/TIV, i.e., relative GMV, from the PT studies contra against individual data of FT controls. Relative GMV decreased from infancy thro early adulthood. Relative GMV calculated from data on infants with and without IU reported by Padilla et al. [20] was above the curve describing FT development. All o values were below the 95%-CI. Data from three PT studies of school-aged children adolescents with brain injury [19] and adults [17] were below the 95%-PI. In summary GMV was lower after PT birth from infancy through early adulth between adolescence and early adulthood in both PT subjects and FT controls, this decrease appeared steeper in the PT studies. REVIEW 4 of 12 ean absolute GMV extracted from the PT studies c f l b l ll f h below mean GMV in FT development. O infants appropriate for gestational age school age. While mean absolute GM hood in both PT subjects and FT con dies. y adulthood. Relative GMV calcul orted by Padilla et al. [20] was abov Figure 1. Flowchart of study selection. Figure 1. Flowchart of study selection. mmary, GMV was lower after PT birth owing a similar trajectory as FT control Figure 1. Flowchart of study selection. Figure 1. Flowchart of study selection. mmary, GMV was lower after PT birth owing a similar trajectory as FT control Figure 2. Mean absolute grey and white matter volumes after preterm birth compared to full-term controls. The four different datasets on FT controls are illustrated in different colors. C-MIND is presented in red, NDAR-NIHPD in in cyan, ICBM in green, and IXI in blue. Mean absolute volumes from the PT studies are included as diamonds. The size of each diamond is proportional to the sample size of the study. (a) Mean absolute GMV after PT birth compared to FT controls. The graph shows mean absolute GMV extracted from the PT studies contrasted against the GMV trajectory of FT controls with 95%-CI (grey areas) and 95%-PI (dotted lines). (b) Mean absolute WMV after PT birth compared to FT controls. The graph shows mean absolute WMV extracted from the PT studies contrasted against the WMV trajectory of FT controls with 95%-CI (grey areas) and 95%-PI (dotted lines). Figure 2. 3.2. Development of Grey Matter Volume after Preterm Birth C-MIND is presented in red, NDAR-NIHPD in in cyan, ICBM in green, and IXI in blue. Mean absolute volumes from the PT studies are included as diamonds. The size of each diamond is proportional to the sample size of the study. (a) Mean absolute GMV after PT birth compared to FT controls. The graph shows mean absolute GMV extracted from the PT studies contrasted against the GMV trajectory of FT controls with 95%-CI (grey areas) and 95%-PI (dotted lines). (b) Mean absolute WMV after PT birth compared to FT controls. The graph shows mean absolute WMV extracted from the PT studies contrasted against the WMV trajectory of FT controls with 95%-CI (grey areas) and 95%-PI (dotted lines). Figure 2. Mean absolute grey and white matter volumes after preterm birth compared to full-term controls. The four different datasets on FT controls are illustrated in different colors. C-MIND is presented in red, NDAR-NIHPD in in cyan, ICBM in green, and IXI in blue. Mean absolute volumes from the PT studies are included as diamonds. The size of each diamond is proportional to the sample size of the study. (a) Mean absolute GMV after PT birth compared to FT controls. The graph shows mean absolute GMV extracted from the PT studies contrasted against the GMV trajectory of FT controls with 95%-CI (grey areas) and 95%-PI (dotted lines). (b) Mean absolute WMV after PT birth compared to FT controls. The graph shows mean absolute WMV extracted from the PT studies contrasted against the WMV trajectory of FT controls with 95%-CI (grey areas) and 95%-PI (dotted lines). between adolescence and early adulthood in both PT subjects and FT controls, this de appeared steeper in the PT studies. Med. 2021, 11, x FOR PEER REVIEW ers. Med. 2021, 11, x FOR PEER REVIEW 6 3.2. Development of Grey Matter Volume after Preterm Birth Figure 2a illustrates mean absolute GMV extracted from the PT studies contra against individual GMV data of FT controls. Mean absolute GMV in all of the PT stu was below mean GMV in FT development. One study [20] reported mean absolute G of PT infants appropriate for gestational age (AGA) at about 1 year of age just within 95%-CI. All other studies reported mean absolute GMV below the 95%-CI. The hig mean absolute GMV was reported at school age. 3.2. Development of Grey Matter Volume after Preterm Birth Figure 2a illustrates mean absolute GMV extracted from the PT studies contrasted against individual GMV data of FT controls. Mean absolute GMV in all of the PT studies was below mean GMV in FT development. One study [20] reported mean absolute GMV of PT infants appropriate for gestational age (AGA) at about 1 year of age just within the 95%-CI. All other studies reported mean absolute GMV below the 95%-CI. The highest mean absolute GMV was reported at school age. While mean absolute GMV decreased J. Pers. Med. 2021, 11, 868 4 of 11 Pers. Med. 2021, 11, 868 4 of between adolescence and early adulthood in both PT subjects and FT controls, this decrea appeared steeper in the PT studies. Pers. Med. 2021, 11, x FOR PEER REVIEW 4 of Figure 1. Flowchart of study selection. Figure 1. Flowchart of study selection. J. Pers. Med. 2021, 11, x FOR PEER REVIEW 6 of 12 3.2. Development of Grey Matter Volume after Preterm Birth Figure 2a illustrates mean absolute GMV extracted from the PT studies contrasted against individual GMV data of FT controls. Mean absolute GMV in all of the PT studies was below mean GMV in FT development. One study [20] reported mean absolute GMV of PT infants appropriate for gestational age (AGA) at about 1 year of age just within the 95%-CI. All other studies reported mean absolute GMV below the 95%-CI. The highest mean absolute GMV was reported at school age. While mean absolute GMV decreased between adolescence and early adulthood in both PT subjects and FT controls, this de- crease appeared steeper in the PT studies. Figure 3a illustrates GMV/TIV, i.e., relative GMV, from the PT studies contrasted against individual data of FT controls. Relative GMV decreased from infancy through early adulthood. Relative GMV calculated from data on infants with and without IUGR reported by Padilla et al. [20] was above the curve describing FT development. All other values were below the 95%-CI. Data from three PT studies of school-aged children [5], adolescents with brain injury [19] and adults [17] were below the 95%-PI. In summary, GMV was lower after PT birth from infancy through early adulthood while following a similar trajectory as FT controls. Figure 2. Mean absolute grey and white matter volumes after preterm birth compared to full-term controls. The four different datasets on FT controls are illustrated in different colors. 3.2. Development of Grey Matter Volume after Preterm Birth Mean absolute grey and white matter volumes after preterm birth compared to full-term controls. The four different datasets on FT controls are illustrated in different colors. C-MIND is presented in red, NDAR-NIHPD in in cyan, ICBM in green, and IXI in blue. Mean absolute volumes from the PT studies are included as diamonds. The size of each diamond is proportional to the sample size of the study. (a) Mean absolute GMV after PT birth compared to FT controls. The graph shows mean absolute GMV extracted from the PT studies contrasted against the GMV trajectory of FT controls with 95%-CI (grey areas) and 95%-PI (dotted lines). (b) Mean absolute WMV after PT birth compared to FT controls. The graph shows mean absolute WMV extracted from the PT studies contrasted against the WMV trajectory of FT controls with 95%-CI (grey areas) and 95%-PI (dotted lines). J. Pers. Med. 2021, 11, 868 5 of 11 Table 1. Characteristics of preterm studies included. Abbreviations: AGA, appropriate for gestational age; BW, birth weight; cUS, cranial ultrasound; GA, gestational age; GMV, grey matter volume; IUGR, intrauterine growth restriction; SD, standard deviation; TIV, total intracranial volume; WMV, white matter volume. * corrected age. und; GA, gestational age; GMV, grey matter volume; IUGR, intrauterine growth restriction; SD, standard deviation; TIV, Abbreviations: AGA, appropriate for gestational age; BW, birth weight; cUS, cranial ultrasound; GA, gestational age; GM total intracranial volume; WMV, white matter volume. * corrected age. 3.2. Development of Grey Matter Volume after Preterm Birth Relative GMV calculated from data on infants with and without IUGR reported by Padilla et al. [20] was above the curve describing FT development. All other values were below the 95%-CI. Data from three PT studies of school-aged children [5], adolescents with brain injury [19] and adults [17] were below the 95%-PI. j y In summary, GMV was lower after PT birth from infancy through early adulthood while following a similar trajectory as FT controls. REVIEW 7 of 12 Figure 3. Relative grey and white matter volumes, and total intracranial volume after preterm birth compared to full-term controls. The four different datasets on FT controls are illustrated in different colors. C-MIND is presented in red, NDAR- NIHPD in in cyan, ICBM in green, and IXI in blue. Data points from the PT studies are included as diamonds. The size of each diamond is proportional to the sample size of the study. (a) Relative GMV after PT birth compared to FT controls. The graph shows GMV/TIV extracted from the PT studies contrasted against the GMV/TIV trajectory of FT controls with 95%-CI (grey areas) and 95%-PI (dotted lines). (b) Relative WMV after PT birth compared to FT controls. The graph shows WMV/TIV extracted from the PT studies contrasted against the WMV/TIV trajectory of FT controls with 95%-CI (grey areas) and 95%-PI (dotted lines). (c) TIV after PT birth compared to FT controls. The graph shows mean TIV extracted from the PT studies contrasted against the TIV trajectory of FT controls with 95%-CI (grey areas) and 95%-PI (dotted lines). 3.3. Development of White Matter Volume after Preterm Birth Figure 2b illustrates mean absolute WMV extracted from the PT studies contrasted Figure 3. Relative grey and white matter volumes, and total intracranial volume after preterm birth compared to full-term controls. The four different datasets on FT controls are illustrated in different colors. C-MIND is presented in red, NDAR- NIHPD in in cyan, ICBM in green, and IXI in blue. Data points from the PT studies are included as diamonds. The size of each diamond is proportional to the sample size of the study. (a) Relative GMV after PT birth compared to FT controls. The graph shows GMV/TIV extracted from the PT studies contrasted against the GMV/TIV trajectory of FT controls with 95%-CI (grey areas) and 95%-PI (dotted lines). (b) Relative WMV after PT birth compared to FT controls. 3.2. Development of Grey Matter Volume after Preterm Birth Author (Year) Group Sample (n) Age at Scan (Years) TIV Mean (cm3) TIV SD (cm3) GMV Mean (cm3) GMV SD (cm3) WMV Mean (cm3) WMV SD (cm3) GA (Weeks) BW (g) Male (%) Year of Birth Country of Origin Methodology of Brain Volume Estimation Pascoe (2019) [17] 150 28.5 1504 140 669 61 513 61 28.8 1077 41.3 1986 New Zealand CAT12 toolbox (SPM12) Lemola (2017) [18] 57 10.0 1397 148 777 70 464 67 29.7 1447 65.3 1998–2006 Switzerland New segment toolbox (SPM8) Meng (2016) [7] 85 26.5 1385 609.4 554 30.67 1356 55.3 1985–1986 Germany VBM8 toolbox (SPM8) Northam (2011) [19] positive cUS 27 16 1601 222 757 57 418 46 27.1 1081 44.4 1989–1994 United Kingdom VBM5 toolbox (SPM5) Northam (2011) [19] normal cUS 22 16.3 1480 193 765 65 438 49 28.1 1098 31.8 1989–1994 United Kingdom VBM5 toolbox (SPM5) Padilla (2011) [20] IUGR 18 1.1 * 969.6 101.8 683.7 64.5 243.5 36.9 32.1 1060 38.9 2006–2007 Spain VBM5 toolbox (SPM5) Padilla (2011) [20] AGA 15 1.1 * 1001.1 95.4 714.0 57.0 240.7 38.0 31 1580 73.3 2006–2007 Spain VBM5 toolbox (SPM5) Soria-Pastor (2009) [5] 20 9.3 1641.2 172.6 821.7 84.9 419.2 53.8 32.5 1754 55 1996–1998 Spain SPM5 Narberhaus (2007) [21] GA ≤27 9 14.1 1354.8 174.1 733.4 54.7 312.1 57.9 26.4 899 77.8 1983–1994 Spain SPM2 Narberhaus (2007) [21] GA 28–30 19 14.6 1488.5 164.9 771.3 133.2 377.2 57.9 29 1140 42.1 1983–1994 Spain SPM2 Narberhaus (2007) [21] GA 31–33 25 13.8 1445.3 146.4 778.1 72.1 372.0 52.1 31.7 1534 44 1983–1994 Spain SPM2 Narberhaus (2007) [21] GA 34–36 11 13.55 1473.3 148.4 780.3 69.9 389.8 45.1 34.6 2446 63.6 1983–1994 Spain SPM2 Gimenez (2006b) [23] 50 14.5 1488.8 148.8 787.8 80.8 377.2 47.4 29.9 1327 48 1982–1994 Spain SPM2 Gimenez (2006a) [22] 30 14.3 1460 140 780 70 360 50 29.1 1108 n.a. n.a. Spain SPM2 Abbreviations: AGA, appropriate for gestational age; BW, birth weight; cUS, cranial ultrasound; GA, gestational age; GMV, grey matter volume; IUGR, intrauterine growth restriction; SD, standard deviation; TIV, total intracranial volume; WMV white matter volume * corrected age Table 1. Characteristics of preterm studies included. J. Pers. Med. 2021, 11, 868 6 of 11 Figure 3a illustrates GMV/TIV, i.e., relative GMV, from the PT studies contrasted against individual data of FT controls. Relative GMV decreased from infancy through early adulthood. 3.2. Development of Grey Matter Volume after Preterm Birth (c) TIV after PT birth compared to FT controls. The graph shows mean TIV extracted from the PT studies contrasted against the TIV trajectory of FT controls with 95%-CI (grey areas) and 95%-PI (dotted lines). ve grey and white matter volumes, and total intracranial volume after preterm birth compared to full-term diff d FT l ill d i diff l C MIND i d i d NDAR e grey and white matter volumes, and total intracranial volume after preterm birth compared to full-term Figure 3. Relative grey and white matter volumes, and total intracranial volume after preterm birth compared to full-term controls. The four different datasets on FT controls are illustrated in different colors. C-MIND is presented in red, NDAR- NIHPD in in cyan, ICBM in green, and IXI in blue. Data points from the PT studies are included as diamonds. The size of each diamond is proportional to the sample size of the study. (a) Relative GMV after PT birth compared to FT controls. The graph shows GMV/TIV extracted from the PT studies contrasted against the GMV/TIV trajectory of FT controls with 95%-CI (grey areas) and 95%-PI (dotted lines). (b) Relative WMV after PT birth compared to FT controls. The graph shows WMV/TIV extracted from the PT studies contrasted against the WMV/TIV trajectory of FT controls with 95%-CI (grey areas) and 95%-PI (dotted lines). (c) TIV after PT birth compared to FT controls. The graph shows mean TIV extracted from the PT studies contrasted against the TIV trajectory of FT controls with 95%-CI (grey areas) and 95%-PI (dotted lines). 3 3 Development of White Matter Volume after Preterm Birth Figure 3. Relative grey and white matter volumes, and total intracranial volume after preterm birth compared to full-term controls. The four different datasets on FT controls are illustrated in different colors. C-MIND is presented in red, NDAR- NIHPD in in cyan, ICBM in green, and IXI in blue. Data points from the PT studies are included as diamonds. The size of each diamond is proportional to the sample size of the study. (a) Relative GMV after PT birth compared to FT controls. The graph shows GMV/TIV extracted from the PT studies contrasted against the GMV/TIV trajectory of FT controls with 95%-CI (grey areas) and 95%-PI (dotted lines). (b) Relative WMV after PT birth compared to FT controls. 3.2. Development of Grey Matter Volume after Preterm Birth The graph shows WMV/TIV extracted from the PT studies contrasted against the WMV/TIV trajectory of FT controls with 95%-CI (grey areas) and 95%-PI (dotted lines). (c) TIV after PT birth compared to FT controls. The graph shows mean TIV extracted from the PT studies contrasted against the TIV trajectory of FT controls with 95%-CI (grey areas) and 95%-PI (dotted lines). 3.3. Development of White Matter Volume after Preterm Birth Figure 3. Relative grey and white matter volumes, and total intracranial volume after preterm birth compared to full-term controls. The four different datasets on FT controls are illustrated in different colors. C-MIND is presented in red, NDAR- NIHPD in in cyan, ICBM in green, and IXI in blue. Data points from the PT studies are included as diamonds. The size of each diamond is proportional to the sample size of the study. (a) Relative GMV after PT birth compared to FT controls. The graph shows GMV/TIV extracted from the PT studies contrasted against the GMV/TIV trajectory of FT controls with 95%-CI (grey areas) and 95%-PI (dotted lines). (b) Relative WMV after PT birth compared to FT controls. The graph shows WMV/TIV extracted from the PT studies contrasted against the WMV/TIV trajectory of FT controls with 95%-CI (grey areas) and 95%-PI (dotted lines). (c) TIV after PT birth compared to FT controls. The graph shows mean TIV extracted from the PT studies contrasted against the TIV trajectory of FT controls with 95%-CI (grey areas) and 95%-PI (dotted lines). 3 3 Development of White Matter Volume after Preterm Birth Figure 3. Relative grey and white matter volumes, and total intracranial volume after preterm birth compared to full-term controls. The four different datasets on FT controls are illustrated in different colors. C-MIND is presented in red, NDAR- NIHPD in in cyan, ICBM in green, and IXI in blue. Data points from the PT studies are included as diamonds. The size of each diamond is proportional to the sample size of the study. (a) Relative GMV after PT birth compared to FT controls. The graph shows GMV/TIV extracted from the PT studies contrasted against the GMV/TIV trajectory of FT controls with 95%-CI (grey areas) and 95%-PI (dotted lines). (b) Relative WMV after PT birth compared to FT controls. The graph shows WMV/TIV extracted from the PT studies contrasted against the WMV/TIV trajectory of FT controls with 95%-CI (grey areas) and 95%-PI (dotted lines). 3.2. Development of Grey Matter Volume after Preterm Birth The graph shows WMV/TIV extracted from the PT studies contrasted against the WMV/TIV trajectory of FT controls with 95%-CI (grey areas) and 95%-PI (dotted lines). (c) TIV after PT birth compared to FT controls. The graph shows mean TIV extracted from the PT studies contrasted against the TIV trajectory of FT controls with 95%-CI (grey areas) and 95%-PI (dotted lines). 3.3. Development of White Matter Volume after Preterm Birth Figure 2b illustrates mean absolute WMV extracted fr 3.3. Development of White Matter Volume after Preterm Birth 4. Discussion This meta-analysis investigated changes of GMV and WMV after PT birth compared to FT controls across the first half of lifespan. We found lower GMV after PT birth from infancy until adulthood, indicating lastingly altered brain development. GMV changes of PT born individuals followed a similar trajectory as FT controls with no evidence for catch-up growth. WMV was particularly low in adolescence after PT birth. Furthermore, the present study highlights the need for large longitudinal datasets to compare PT and FT brain development across the lifespan. 4.1. Changes of Grey and White Matter Volume after Preterm Birth across the First Half of Lifespan We found lower GMV after PT birth compared to FT controls from infancy through early adulthood. Similar to their FT peers, GMV in PT subjects peaked at school age and decreased between adolescence and early adulthood. However, relative GMV in infancy was higher compared to FT peers, possibly reflecting delayed maturation. Yet this speculation is based on only one study. At school age, in adolescence and in early adulthood GMV was lower. There was no evidence for catch-up growth. Furthermore, WMV after PT birth was particularly low in adolescence. p y Comparing our results to the scarce longitudinal data available is difficult because of methodological differences. Ment et al. [26] observed reduction in cerebral GMV and an increase in WMV between 8 and 12 years of age. However, compared to FT controls, there was lower GMV reduction and less WMV gain over time [26]. In contrast, our results show that GMV in PT individuals mostly follow the FT reference curve, albeit on an overall lower level and that WMV is particularly low in early adolescence. Mostly in line with our results, Parker et al. [27] reported significantly smaller GMV and WMV in VP subjects compared to FT controls in adolescence (15 years) and in early adulthood (19 years). Furthermore, they reported similar growth patterns between these two timepoints [27]. Karolis et al. [28] observed GMV alterations in VP subjects between adolescence (15 years), early adulthood (20 years) and adulthood (30 years), indicating accelerated brain maturation. Similarly, our findings of lower relative GMV at school age, in adolescence and early adulthood might indicate early aging. Consistent with our findings, Karolis et al. [28] found no evidence for developmental catch-up. In line with our results, de Kieviet et al. 3.4. Total Intracranial Volume after Preterm Birth Figure 3c illustrates mean TIV extracted from the PT studies contrasted against indi- vidual TIV data of FT controls. While TIV reported by some studies in infancy, childhood and early adulthood was below the curve describing FT development, other PT studies reported higher values. 3.3. Development of White Matter Volume after Preterm Birth Figure 2b illustrates mean absolute WMV extracted f 3.3. Development of White Matter Volume after Preterm Birth Figure 2b illustrates mean absolute WMV extracted from the PT studies contrasted against individual WMV data of FT controls. Except for one study in early adulthood [7], mean absolute WMV in all of the PT studies was below the curve describing mean FT development. Mean absolute WMV of infants with and without IUGR reported by Padilla et al. [20] was within the 95%-CI. All other studies reported mean absolute WMV below Figure 2b illustrates mean absolute WMV extracted from the PT studies contrasted against individual WMV data of FT controls. Except for one study in early adulthood [7], mean absolute WMV in all of the PT studies was below the curve describing mean FT development. Mean absolute WMV of infants with and without IUGR reported by J. Pers. Med. 2021, 11, 868 7 of 11 Padilla et al. [20] was within the 95%-CI. All other studies reported mean absolute WMV below the 95%-CI. Mean absolute WMV reported in adolescence was particularly low, moving away from the curve describing mean FT development. Padilla et al. [20] was within the 95%-CI. All other studies reported mean absolute WMV below the 95%-CI. Mean absolute WMV reported in adolescence was particularly low, moving away from the curve describing mean FT development. Figure 3b illustrates WMV/TIV, i.e., relative WMV, from the PT studies contrasted against individual data of FT controls. Relative WMV calculated from data on infants with and without IUGR reported by Padilla et al. [20] and from data in early adulthood [7] was above the curve describing FT development. Data from one PT study at school age [18] was below the curve describing FT development but just within the 95%-CI. All other values were below the curve. The other PT study at school age [5] and all PT studies in adolescents were below the 95%-PI. In summary, we observed particularly low WMV in adolescence, while WMV in infancy and early adulthood were closer to the trajectory describing FT development. 3.4. Total Intracranial Volume after Preterm Birth 4. Discussion [29] found reductions in GMV and WMV compared to FT controls in their investigation of brain volume throughout childhood and adolescence in subjects born VP and with very low birthweight in a meta-analysis. J. Pers. Med. 2021, 11, 868 8 of 11 8 of 11 Proposed reasons for aberrant brain development after PT birth include several pro- cesses: Inflammatory, hypoxic-ischemic and/or stress-related events are potential causes for injury to preoligodendrocytes, axons, thalamus, subplate neurons or migrating gamma- aminobutyric acid-ergic neurons [6,30]. Remarkably, our results suggest a significant impact of prematurity on WMV in adolescence. On a cellular level, pre-oligodendrocytes, precursors of mature oligodendrocytes, are critical for white matter myelination. Pre- oligodendrocytes are specifically vulnerable to insults, such as ischemia and inflammation, resulting in cell injury or death and subsequent replenishment but dysmaturation [6,30–32]. Hence, our results showing preferential alteration of WMV might emphasize the signifi- cance of pre-oligodendrocyte vulnerability and dysmaturation in the context of prematurity. GMV and WMV changes have been associated with functional outcome such as cognitive development in children, adolescents, and young adults [5,7,25,33]. Therefore, characterization of GMV and WMV changes across the first half of lifespan could help identify PT individuals at increased risk for impaired cognitive development. In conclusion, PT birth has lasting effects on the development of GMV and WMV compared to FT controls with particularly low WMV in adolescence. We found no evidence for GMV catch-up growth. 4.2. What Is Needed in the Future? PT cohorts have been followed from birth and well investigated, however, to date longitudinal imaging data on PT subjects are scarce and only cover specific age ranges. Shared large datasets do not exist. Methodological differences in image processing as well as statistical analysis make it difficult to compare results. Thanks to large, open datasets on healthy subjects, FT developmental trajectories have been well characterized [10], facili- tating insights in diseases affecting brain structure, for example Alzheimer’s Disease [11]. Similarly, PT developmental trajectories could be used to identify PT adults with acceler- ated aging at risk for cognitive decline and/or neurodegenerative diseases. To achieve these goals, access to original imaging data of large PT populations across all age groups as well as shared individual patient data with information on parameters like birthweight, duration of neonatal intensive care unit exposure, and days of mechanical ventilation, and information about possible brain damage, such as intraventricular hemorrhage, is critical. 4.3. Limitations There are limitations of this meta-analysis. First, only few studies fit the inclusion criteria (see also Sections 2.2 and 2.3). One reason for exclusion was that raw volumes were not reported. For example, one study [2] investigated GMV and WMV in adolescents born VP, however, not raw volumes but volumes after controlling for whole brain volume were reported. Furthermore, not TIV but whole brain volume was reported [2]. Another study [26] investigated longitudinal brain volume changes in PT and FT subjects during late childhood and adolescence, however, volumes were not reported as raw values but as model-based least square means. Furthermore, not TIV but whole brain volume was reported [26]. A third study [34] investigated brain volumes and developmental outcome in childhood following fetal growth restriction leading to VP birth, however, mean age at scan was not reported. Another reason for exclusion was the use of other analysis methods, for example, investigating regional group differences instead of global volumes [25]. Second, only data on limited age groups was available. Hence, PT volume changes of GMV and WMV in comparison to FT trajectories remain less clear in some age groups, for example between about 2 and 7 years. However, it is inherently difficult to obtain a brain MRI in this age group since this is generally not possible without general anesthesia. Third, as mentioned above, different methods of processing images make it very difficult to compare data. All PT studies included in this meta-analysis used toolboxes within SPM (https://www.fil.ion.ucl.ac.uk/spm/) for segmentation, while FT images were processed with volBrain [35]. Volumes obtained with volBrain showed high correlation with volumes obtained by manual segmentations [35]. Comparing subcortical segmenta- J. Pers. Med. 2021, 11, 868 9 of 11 9 of 11 tion of volBrain with state-of-the-art methods like Freesurfer or the FSL-based tool FIRST, and with manual segmentation showed competitive results in terms of accuracy and repro- ducibility. However, the possibility of systematic error cannot be ruled out. Only access to raw imaging data could improve this major limitation and, as discussed, is needed. However, contrasting individual MRI scans and derived measures of GMV and WMV to reference data from open datasets is increasingly used in clinical routine. Lastly, GMV and WMV are global measures representing brain development. 4.3. Limitations How- ever, determination of developmental changes of other measures such as gyrification and individual volumes of subcortical structures are necessary to characterize PT brain development, but data is not available. 5. Conclusions Conflicts of Interest: The authors declare no conflict of interest. 5. Conclusions In conclusion, GMV is lower after PT birth from infancy through early adulthood, indicating lastingly altered brain development. GMV changes were similar to the trajectory of FT controls with no evidence for catch-up growth. WMV was particularly low in adolescence after PT birth. Large longitudinal datasets are crucial to compare PT with FT brain development across the lifespan. Supplementary Materials: The following are available online at https://www.mdpi.com/article/10 .3390/jpm11090868/s1, Table S1: PRISMA Checklist. Author Contributions: Conceptualization, D.M.H.; methodology, B.H., P.C., D.M.H.; software, B.H.; formal analysis, B.H., P.C.; investigation, D.M.H., B.S.-K.; resources, P.C., C.Z.; data curation, B.H., P.C., D.M.H., B.S.-K.; writing—original draft preparation, B.S.-K.; writing—review & editing, D.M.H., B.H., P.C., A.M., C.G., C.Z., D.W., P.B., C.S.; visualization, B.H., B.S.-K.; supervision, C.S., D.M.H.; project administration, D.M.H.; funding acquisition, D.W., P.B., C.S., D.M.H., B.S.-K. All authors have read and agreed to the published version of the manuscript. Author Contributions: Conceptualization, D.M.H.; methodology, B.H., P.C., D.M.H.; software, B.H.; formal analysis, B.H., P.C.; investigation, D.M.H., B.S.-K.; resources, P.C., C.Z.; data curation, B.H., P.C., D.M.H., B.S.-K.; writing—original draft preparation, B.S.-K.; writing—review & editing, D.M.H., B.H., P.C., A.M., C.G., C.Z., D.W., P.B., C.S.; visualization, B.H., B.S.-K.; supervision, C.S., D.M.H.; project administration, D.M.H.; funding acquisition, D.W., P.B., C.S., D.M.H., B.S.-K. All authors have read and agreed to the published version of the manuscript. Funding: This work was supported by the Deutsche Forschungsgemeinschaft (SO 1336/1-1 to C.S.), German Federal Ministry of Education and Science (BMBF 01ER0801 to P.B. and D.W., BMBF 01ER0803 to C.S.), the RECAP preterm project, an EU Horizon 2020 study (supported by grant 733280; D.W. and P.B.), and the Kommission für Klinische Forschung, Technische Universität München (KKF 8765162 to C.S., KKF 8700000474 to D.M.H., and KKF 8700000620 to B.S.-K.). 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: Please see references for the studies included in Section 3.1, and for data on full-term controls in Section 2.4. Data Availability Statement: Please see references for the studies included in Section 3.1, and for data on full-term controls in Section 2.4. Acknowledgments: We would like to thank all investigators involved in collecting following datasets: C-MIND (https://research.cchmc.org/c-mind/ (accessed on February 2015)), NDAR-NIHPD (https: //www.bic.mni.mcgill.ca/nihpd_info/info2/data_access.html (accessed on February 2015)), ICBM (http://www.loni.usc.edu/ICBM/ (accessed on February 2015)), and IXI (http://brain-development. org/ixi-dataset/ (accessed on February 2015)). Conflicts of Interest: The authors declare no conflict of interest. 1. Chawanpaiboon, S.; Vogel, J.P.; Moller, A.-B.; Lumbiganon, P.; Petzold, M.; Hogan, D.; Landoulsi, S.; Jampathong, N.; Kongwat- tanakul, K.; Laopaiboon, M.; et al. Global, regional, and national estimates of levels of preterm birth in 2014: A systematic review and modelling analysis. Lancet Glob. Health 2019, 7, e37–e46. [CrossRef] y g [ ] 4. Inder, T.E.; Warfield, S.K.; Wang, H.; Hüppi, P.S.; Volpe, J.J. Abnormal cerebral structure is present at term in premature infants. 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Dimensions of Online Conflict: Towards Modeling Agonism
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Abstract Not all conflicts are equal, and the confusion over the political and social value of conflict partly stems from its diverse nature. Conflict exists on a sliding scale ranging from antagonistic conflict be- tween enemies (which is often silencing, undemo- cratic, and hateful because it is focused on delegit- imizing opponents’ rights and status) to agonistic conflict between adversaries (which has productive potential for the emergence of democratic dialogue, dissent, and trust in the public and political sphere, since the struggle is over interpretation and not le- gitimacy to speak) (Mouffe, 2002; Wenman, 2013). As many across the political spectrum have noted, democracy needs agonism to widen voices and pre- vent public spaces from becoming totalitarian or meaningless consensus hubs. But how do we know which conflicts are agonistic and which hateful? How do we know what kinds of counterspeech ef- fectively facilitate dialogue and which destroy it? Which follow the spirit of platforms’ terms of use and which do not? Agonism plays a vital role in democratic di- alogue by fostering diverse perspectives and robust discussions. Within the realm of online conflict there is another type: hateful antago- nism, which undermines constructive dialogue. Detecting conflict online is central to platform moderation and monetization. It is also vital for democratic dialogue, but only when it takes the form of agonism. To model these two types of conflict, we collected Twitter conversations related to trending controversial topics. We introduce a comprehensive annotation schema for labelling different dimensions of conflict in the conversations, such as the source of con- flict, the target, and the rhetorical strategies deployed. Using this schema, we annotated ap- proximately 4,000 conversations with multiple labels. We then trained both logistic regression and transformer-based models on the dataset, incorporating context from the conversation, including the number of participants and the structure of the interactions. Results show that contextual labels are helpful in identifying con- flict and make the models robust to variations in topic. Our research contributes a conceptu- alization of different dimensions of conflict, a richly annotated dataset, and promising results that can contribute to content moderation. To begin answering these questions, we present an in-depth exploration of conflict online, using the platform once known as Twitter (now “X”) as a case study. Our work builds upon related Natural Language Processing (NLP) research fields such as abusive language, persuasion, and constructive comments. Abstract We use Twitter because, unlike the controlled environment of a priori conflictual dis- cussions like those in Reddit’s ‘Change My View’ (Monti et al., 2022; Srinivasan et al., 2019), it offers a more organic setting. Dimensions of Online Conflict: Towards Modeling Agonism 1Digital Democracies Institute, Simon Fraser University, Canada, matthew_canute@sfu.ca 2Department of Computer Science, University of Sheffield, UK, m.jin@sheffield.ac.uk Findings of the Association for Computational Linguistics: EMNLP 2023, pages 12194–12209 December 6-10, 2023 ©2023 Association for Computational Linguistics 2.1 Conflict and agonism Within this project, we understand conflict as the generative ground that spans hate to agonism. Ac- cording to political theorist Chantal Mouffe (2013), democratic speech or dialogue always bears traces of the conflicts from which it emerges. Democracy, therefore, necessarily entails conflict and negotia- tion; to expand who counts as a citizen, to negotiate differing claims to freedom or rights, and to vali- date collective decisions. This is agonistic conflict. Conflict can also be antagonistic: unproductive and undemocratic, when it silences individuals and groups, by shutting them out or harassing them with hateful speech. Democratic institutions are responsible for creating the space to allow con- flicts to take an agonistic form, in which opponents are not enemies but adversaries among whom con- flictual consensus may emerge (see also Mouffe, 2002; Rancière, 1999, 2010; Wenman, 2013). This generative aspect of conflict has been neglected in discussions around content moderation on social media platforms, which frame the problem as free- dom of speech versus censorship (Douek, 2022; Gillespie et al., 2020; Gillespie, 2018). Next, we trained logistic regression and trans- former models (BERT and GPT-3) on this dataset, to predict dimensions of conflict. The models are trained on human annotations of the conversations, enhanced with a) previously proposed labels for online conversation: constructiveness and toxicity; and b) contextual aspects such as cardinality (partic- ipant counts) and topology (interaction structure). We show that all models are sensitive to the specific words in the conversation, making them less gener- alizable across topics and domains. Incorporating conversational context, however, makes the models more robust, showing that cardinality and topol- ogy are important dimensions in the prediction of online conflict. The potential use cases of this work include the measurement of productive (agonistic) versus un- productive (antagonistic) conflicts, providing in- sights into where learning and constructive dis- course can be fostered. This work ties in with attempts at reflective content moderation, where the goal is not simply to delete harmful content or make it less visible (Zeng and Kaye, 2022), but also to identify and promote content that can be con- structive and productive towards democratic goals (Mouffe, 2013; Gillespie et al., 2020; Morrow et al., 2022). This kind of analysis has implications for detecting early signs of scapegoating and unproduc- tive disputes—cases where patterns of discourse do not necessarily break the terms of service, but can nonetheless bring harm over time. 1 Introduction Conflict is everywhere online. From political protests to spirited debates over the latest TikTok trend, these conflicts are simultaneously celebrated as promoting democracy and condemned for fos- tering polarization and undermining public insti- tutions. Conflicts—ideas, arguments, or attitudes that oppose each other—are also central to platform moderation and monetization: from the amplifica- tion of certain controversies to provoke user en- gagement, to content takedowns to comply with a platform’s terms of use or national laws (Gillespie, 2022, 2018; Morrow et al., 2022; Douek, 2022; Zeng and Kaye, 2022). To determine the nature of a conflict, context is central (Zosa et al., 2021; Hu et al., 2022; Ghosh et al., 2018). Currently, though, automatic content moderation mainly focuses on a single utterance rather than on the ongoing conversation. Whether a message fosters agonism depends on various di- mensions, including which groups are participating, how many participants, their level of interaction, and their relative power differences, all subject to 12194 Findings of the Association for Computational Linguistics: EMNLP 2023, pages 12194–12209 December 6-10, 2023 ©2023 Association for Computational Linguistics ual conversations on Twitter; c) a detailed schema to annotate conversations for various dimensions of conflict and the resulting annotations, with good inter-annotator agreement; and d) a set of experi- ments that show the usefulness of contextual infor- mation in predicting online conflict. change over time. We introduce a methodology for collecting and curating a dataset of English Twitter conversations embodying various aspects of dimensions of con- flict. We collected conversations about trending events, as these often serve as catalysts, prompting individuals to interrogate their stance on current is- sues and deliberate their self-conception in relation to their views. We then annotated the conversations following our own coding schema, created with antagonistic and agonistic conflict in mind. These annotations took context into consideration rather than focusing on an isolated tweet. 2.1 Conflict and agonism Our findings will be instrumental in shaping online discourse, aiming to harness conflict as a driver of democratic conversation (agonism) rather than as a destructive silencing element (antagonism). 2.4 Constructive comments Research into high-quality online content has shown that constructiveness is a useful dimension. Research into high-quality online content has shown that constructiveness is a useful dimension. Constructive comments build on and contribute to the conversation, providing points of view and jus- tification for a particular opinion. They are not necessarily conflictual in nature, since they may simply build on the ongoing conversation. In a study of online news comments, Kolhatkar et al. (2023) propose that constructive comments seek to create a civil dialogue, with remarks that are relevant to the article and not merely emotional provocations. Comments identified as constructive can be presented to future posters as prompts or examples of desirable behaviour or as nudges to de- polarize conversations (Stray, 2022). Our work on identifying conflict can contribute to the growing body of research on how to present content in such a way that it contributes to productive, civil, and also agonistic discussion. We emphasize, however, that we do not necessar- ily correlate the absence of toxicity or abuse with the presence of productive conflict. Abusive lan- guage research tends to characterize healthy and/or civil conversations as those that are absent of tox- icity (e.g., Smith et al., 2021; Hede et al., 2021). While that may be the case, healthy conversations are not necessarily agonistic. Agonism requires a certain level of disagreement as a source of political discussion and engagement. 3.1 Data collection We are interested in online conversations on con- tentious topics, so we used the Twitter Academic API v2 elevated access to gather replies containing certain keywords, starting with controversial topics. Then we consulted subject experts and representa- tives of equity-seeking groups as a way to increase the topic diversity within the dataset. This led to a set of keywords as search terms (in Appendix A). After selecting tweet replies in English contain- ing the relevant keywords from each topic, we then extracted their surrounding conversation trees us- ing two traversal methods: depth and breadth. The former involved recursively collecting a reply’s ref- erenced tweet until it reached the root message, or the 7-message limit (since length 7 is the last most frequent distribution before the start of the long tail of conversation thread). Breadth traversal involved capturing adjacent messages of a conversation by recursively creating new queries based on each re- ply’s tagged author and the conversation ID of the reply. We are interested in online conversations on con- tentious topics, so we used the Twitter Academic API v2 elevated access to gather replies containing certain keywords, starting with controversial topics. Then we consulted subject experts and representa- tives of equity-seeking groups as a way to increase the topic diversity within the dataset. This led to a set of keywords as search terms (in Appendix A). Modeling work includes attempts to find argu- ments in text, an area known as argumentation min- ing (Mochales and Moens, 2011; Lawrence and Reed, 2019; Harris and Di Marco, 2017). The goals include: a) identifying controversial topics in debates, news, Wikipedia articles, or online dis- cussions (Boltuži´c and Šnajder, 2014; Kittur et al., 2007; Choi et al., 2010; Swanson et al., 2015; Stab and Gurevych, 2017); b) forecasting conversational derailment (Zhang et al., 2018); c) identifying con- versational strategies that will change the interlocu- tor’s mind as in r/ChangeMyView (Monti et al., 2022; Srinivasan et al., 2019); d) detecting conflict outside the conversation, as in r/AmITheAsshole posts (Welch et al., 2022). After selecting tweet replies in English contain- ing the relevant keywords from each topic, we then extracted their surrounding conversation trees us- ing two traversal methods: depth and breadth. 2.3 Persuasion, argumentation, derailed conversations Persuasion styles, rhetorical strategies, and argu- mentation styles all play a role in how we perceive and interpret conflict. Research in this area has pro- duced manual annotations of rhetorical strategies such as framing, hedging, modality, repetition, and rhetorical questions (Peldszus, 2014; Green, 2014; Hirst et al., 2014). These approaches tend to fo- cus on understanding which rhetorical approaches will be most effective in changing someone’s mind (Habernal and Gurevych, 2016; Hidey and McKe- own, 2018). 2.2 Abusive language Abusive language online is a broad term that covers various forms of harmful or offensive communica- tion on the internet, such as hate speech, cyber- bullying, trolling, or flaming (Fortuna et al., 2020; Pachinger et al., 2023). Detecting and preventing abusive language online is an important challenge for natural language processing (NLP) and social computing, and an extensive literature on the topic exists. In addition to the challenges of detecting a social phenomenon that the perpetrators often try to disguise, current industry solutions suffer from a lack of interpretability, undermining their credibil- ity (MacAvaney et al., 2019). Equally dangerous is the over-policing of certain communities and topics online (Saleem et al., 2016).i Our main contributions are: a) a conceptualiza- tion of conflict online on a scale between antago- nism and agonism; b) a process to retrieve conflict- Further, supervised classifiers require high- quality annotated data that may harm the annotators 12195 sarily have a successful outcome in terms of per- suading interlocutors. and that may contain their biases (Sap et al., 2022; Vidgen and Derczynski, 2021). We know context is also crucial in obtaining high-quality annotations (Ljubeši´c et al., 2022), and that some disagreement among annotators is to be expected (Leonardelli et al., 2021). All this prior work informs our annota- tions and explorations of machine learning models. 3.2 Coding schema To label the dataset, we developed an original cod- ing protocol based on an interdisciplinary review of literature on conflict, including media studies, political science, conflict resolution studies, and critical race theory (e.g., Oetzel and Ting-Toomey, 2006; Lamberti and Richards, 2019; D’Errico et al., 2015; Itten, 2019; Yardi and boyd, 2010; Han et al., 2023). Figure 1: Coding schema for conflict session during which annotators were introduced to the project and its goals and were taught how to use the labelling platform (LabelStudio; see Appendix E for screenshots). During the first week, the an- notators and two members of the research team worked together and labelled a subset of about 400 tweets. The annotation was conducted in person, and each session had planned moments for dis- cussing disagreements and clarifying the gray areas of the annotation protocol. At the end of the train- ing session, we assigned a subset of the dataset to be codde. The research co-leaders held weekly in- dividual check-in meetings with annotators to trou- bleshoot issues and gather their feedback. These meetings also served to assess the emotional im- pact of annotation and to externalize the thoughts and emotions annotators encountered during their work. The entire research team also met every two weeks, to compare different annotation styles and discuss edge cases as a way to test the protocol’s reliability. Each message was annotated by two annotators. The initial protocol was first tested on a subset of conversations (see Section 3.3). We revisited the coding protocol twice throughout the course of the project based on coders’ feedback and discussions over disagreements. The final coding protocol fol- lowed a decision tree structure, where answering in the positive to one question led to a set of follow- up questions, as shown in Figure 1. Appendix E contains an extensive discussion of each of the con- cepts in the figure, with examples. 3.1 Data collection The former involved recursively collecting a reply’s ref- erenced tweet until it reached the root message, or the 7-message limit (since length 7 is the last most frequent distribution before the start of the long tail of conversation thread). Breadth traversal involved capturing adjacent messages of a conversation by recursively creating new queries based on each re- ply’s tagged author and the conversation ID of the reply. While this previous research on persuasiveness informs ours, its goal is to identify successful and unsuccessful argumentation styles. We are, first, looking for conflict, to then try and pinpoint exam- ples of agonistic discussions, which do not neces- The annotated dataset contains an equal mix of depth and breadth traversals. While the former en- ables more efficient data collection, the latter is useful for capturing the chaotic nature of conversa- 12196 Figure 1: Coding schema for conflict tions on most platforms, such as the one depicted in Figure 2. Only conversations of length 3-7 messages in English were stored. This iterative process contin- ued over a period of three years (January 2020 - December 2022), yielding a total of 220,626 con- versations.1 Based on a random sample of 1,000 conversa- tions, roughly 30% of these conversations likely involved first-time interactions2, suggesting that these topics were contentious enough to spark de- bates among strangers in the comments of large accounts, creating virtual public forums. 1Raw data, annotations, and code used to extract the conversations are available in our repository, which also includes all the code for the experiments in Section 4: https://github.com/Digital-Democracies- Institute/Dimensions-of-Online-Conflict 1Raw data, annotations, and code used to extract the conversations are available in our repository, which also includes all the code for the experiments in Section 4: https://github.com/Digital-Democracies- Institute/Dimensions-of-Online-Conflict 2First-time interactions were approximated by examining each account’s prior 200 messages and checking if any of the accounts had interacted with each other previously. 2First-time interactions were approximated by examining each account’s prior 200 messages and checking if any of the accounts had interacted with each other previously. 3.3 Annotation and agreement We recruited a team of four annotators. In selecting candidates, we aimed to maximize demographic di- versity and cultural background. The team included two graduate and two undergraduate students from three departments at our university: Communica- tion, Political Science, and International Studies. The self-identified gender split was three women and one man, and the age ranges were: three 20-25 and one 26-30. At the beginning of the project, three members of the research team led a training Throughout the duration of the annotation cam- paign (May-December 2022), annotators labelled 4,022 conversations involving 9,472 individual au- thors with 22 labels. It was a two-tiered labelling system, where the annotators would first read the entire conversation and indicate whether there was conflict in the last message of the conversation given the context of the previous messages. They 12197 would then annotate further aspects if the answer was ‘yes’, following Figure 1. age’). Appendix C provides further information on the rate of conflict per topic. In summary, most topics contained some form of conflict, due to the way they were collected (trending topics). This makes the dataset possibly unbalanced, but also a rich source of how conflict develops online. Since each binary label was annotated by two annotators, we can compute inter-annotator agree- ment using Cohen’s kappa, κ (Cohen, 1960). The initial question, ‘did the last message in the conver- sation contain conflict?’ had κ = 0.65, a moderate to substantial level of agreement (Landis and Koch, 1977). The kappa values for the follow-up ques- tions in Table 1 show that some of the other labels foster less consensus. Annotators agreed, in gen- eral, whether the conflict is internal or external to the tweet (with internal more difficult to adjudicate). A conflict is internal when all involved parties are also engaged in the conversation, i.e., it is conflict among the participants. An external conflict is dis- agreement about somebody else not in the conversa- tion, e.g., a public figure (see Appendix E for more detail). Annotators also often agreed that conversa- tional context was needed. The level of agreement for rhetorical strategies (sarcasm, explicit directives and calls to action, association and metaphor analo- gies) was quite low, although consistent with the well-documented difficulty in annotating sarcasm (González-Ibáñez et al., 2011; Oprea and Magdy, 2020). 3.3 Annotation and agreement In this paper, we mainly use the binary label for conflict in the tweet, leaving other features for further study. We kept only conversations where the two annotators agreed on whether there was conflict. This yielded a total of 3,577 data points. One could argue that, by keeping only cases with clear agreement, we are in effect making the task ‘easier’. Given the small size of the dataset, we follow this approach in order to eliminate noise. 3Names from the LEDIR repository (Sanders et al., 2020). 4 Conflict predictive models If there is a path from C to A, that means that A received a no- tification from C. For the above example, Deniz’s message to Boróka sends a notification to both Amal and Boróka, but not to Carlu. Intuitively, it should help to know the directed graph’s structure of a conversation. It seems important to know, for example, that an utterance is part of a larger con- versation sending out a notification to five different people versus a back-and-forth conversation be- tween just two people. For the experiments, we en- coded this feature as a binary ‘has_bidirectionality’ feature (i.e., is there back-and-forth interaction), but the dataset we will release has a full representa- tion of this dynamic. For instance, the conversation in Figure 2 is represented as: [(B, A), (D, B), (B, D), (C, A), (E, D), (E, C)], which is also converted to a matrix form in the annotated dataset. who received a notification from whom. If there is a path from C to A, that means that A received a no- tification from C. For the above example, Deniz’s message to Boróka sends a notification to both Amal and Boróka, but not to Carlu. Intuitively, it should help to know the directed graph’s structure of a conversation. It seems important to know, for example, that an utterance is part of a larger con- versation sending out a notification to five different people versus a back-and-forth conversation be- tween just two people. For the experiments, we en- coded this feature as a binary ‘has_bidirectionality’ feature (i.e., is there back-and-forth interaction), but the dataset we will release has a full representa- tion of this dynamic. For instance, the conversation in Figure 2 is represented as: [(B, A), (D, B), (B, D), (C, A), (E, D), (E, C)], which is also converted to a matrix form in the annotated dataset. We also postulate (Hypothesis 1c) that the bag- of-words approach can be enhanced with additional labels. These labels are derived from other models that classify messages according to their construc- tiveness (Kolhatkar et al., 2023) and toxicity (Hanu and Unitary Team, 2020).4 We added logit scores from each of these existing models into the same feature matrix. 4 Conflict predictive models Table 2 shows a confusion matrix for the bag-of-words (BOW) model with construc- tiveness and toxicity on the 716 conversations in our test set (we used a standard 80-20 split for train- ing and testing). We can see that this model is quite good at identifying both non-conflict and conflict, but it overpredicts conflict, as it is the majority in the imbalanced dataset. Non-Conflict Conflict Total Non-Conflict 27 87 114 Conflict 0 602 602 Total 27 689 716 Table 2: Confusion matrix for BOW LR model with constructiveness and toxicity (Hypothesis 1c) 4https://github.com/unitaryai/detoxify 5https://huggingface.co/bert-base-uncased 4 Conflict predictive models For now, we focus only on finding conflict, leaving the issue of whether the conflict is agonistic or not for future work (although we make some sugges- tions in Section 5). Our starting hypothesis is that we can find signals of conflict in the data. The tar- get variable of interest in this paper is the presence of conflict. Predictive features include: words in the text (bag of words model); constructiveness and toxicity labels; and context from the conversation, namely cardinality (number of participants) and topology (structure of participant interactions). By conversational topology we refer to the multi- threaded nature of online conversations, which have been described as polylogues (Marcoccia, 2004). Let us examine it with an example, rep- resented in Figure 2. Amal sends out a message about a new mural in their city. Boróka angrily replies that this mural is a waste of tax-payer money. Deniz replies to Boróka with a meme making fun of Amal, and Boróka sends the laugh emoji back to Deniz in response. Carlu tells Amal that a sec- tion of the mural is controversial and divisive and shouldn’t have been publicly-funded. Eryl replies to Deniz clarifying that the section of the mural they are referring to was not actually funded by the city. Eryl then says the same thing to Carlu.3 Table 1: Cohen’s kappa κ values for different features Table 1: Cohen’s kappa κ values for different features Feature κ Conflict (overall) 0.65 Target - Individual 0.90 External 0.58 Context - Conversational 0.58 Internal 0.48 Rhetorical strategy - Sarcasm 0.36 Rhetorical strategy - Explicit 0.35 Rhetorical strategy - Association 0.19 Context - Media 0.18 Context - Cultural 0.02 Amal Boróka Carlu Deniz Eryl Figure 2: An example conversation represented as a graph Amal Eryl Appendix B provides a trend line for level of activity per topic over time. We saw that some top- ics, like ‘Social Distancing’ were discussed over long periods of time, whereas other topics peaked and declined quickly, perhaps having to do with specific events (‘Will Smith Slap’, ‘Rogers Out- Figure 2: An example conversation represented as a graph Each directed edge embeds information about 3Names from the LEDIR repository (Sanders et al., 2020). Each directed edge embeds information about 3Names from the LEDIR repository (Sanders et al., 2020). 12198 who received a notification from whom. 4.1 Bag of Words model Table 2: Confusion matrix for BOW LR model with constructiveness and toxicity (Hypothesis 1c) Table 2: Confusion matrix for BOW LR model with constructiveness and toxicity (Hypothesis 1c) The first basic hypothesis (Hypothesis 1a) is that the presence of some words (unigrams and bi- grams) is predictive of conflict. We used a logis- tic regression classifier with L2 regularization to predict whether the entire conversation represented conflict or non-conflict. It used a TF-IDF vectorizer to extract unigram and bigram features from the last message in the conversation, the message that we first extracted using keywords. We also used not just the last message, but the entire conversational context, with the same model (Hypothesis 1b). When we ran this model, we saw that some terms had coefficients with very high absolute values in predicting the presence of conflict. Although some of these terms are intuitively indicative of general conflict, such as the unigram ‘people’, some of the terms are most likely hyper-specific for particular conflicts that will have only happened once, such as the unigram ‘smith’, referring to the Will Smith slapping incident. To reduce the risk of overfitting this model to particular topics on a new dataset or domain, and to have the model learn topic-agnostic linguistic patterns of conflict, we removed topic- related unigrams and bigrams by selecting the top 10 c-TF-IDF words from each topic and then re- moving those that seemed highly topic-specific (see Appendix D for lists of words removed by topic). We use this topic-agnostic dataset (Dataset 2) for the fine-tuning experiments in the next sec- tion. Dataset 1 contains all the words, without filtering. 4.3 Comparison of model results Even more interesting is the effect of additional labels. When we incorporate constructiveness and toxicity, there is an improvement over the baseline of the last message and a slight decline from just including all the messages. However, the model is more robust to the removal of topic words. The models with cardinality and topology have similar topic-independent robustness. Cardinality leads to the lowest drop in performance (3.47 points) for Dataset 2, and topology also seems to show some topic independence. The GPT-3 model performs worse compared to all BERT combinations, and also suffers from a drop in Dataset 2. We should note that the GPT-3 model that we use was trained on data collected roughly up to the end of 2019 (Brown et al., 2020), so it lacks knowledge about most of the topics in our data, which was collected later. It serves, thus, as a good test for topic inde- pendence. Table 3 shows F1 score results for the two main approaches we took, following the hypotheses de- scribed above. We first show results for a BOW logistic regression model under the three different conditions (last message, all messages, all mes- sages with constructiveness and toxicity). We then test the performance of BERT with the same condi- tions, plus we incorporate cardinality and topology. We also tested a simple GPT-3 fine-tuned model using OpenAI’s Curie fine-tuning API. Our test data comes from the human annotations (Dataset 1), and the same data but with topic-related words removed (Dataset 2). The latter is more likely to be generalizable to new data about different top- ics, which is why we are interested in performance changes relative to Dataset 1. Results in the table are F1 score averages (average of three runs with random seeds 42, 43, 44). Model Dataset 1 Dataset 2 LR Last Msg (H1a) 38.30 33.82 LR All Msg (H1b) 26.87 23.73 LR All Msg + constr, tox (H1c) 38.30 24.81 BERT Last Msg (H2a) 94.58 85.26 BERT All Msg (H2b) 96.06 85.42 BERT All Msg + constr, tox (H2c) 95.43 87.26 BERT All Msg + cardinality (H2d) 92.83 89.36 BERT All Msg + topology (H2e) 94.40 88.92 GPT-3 Fine-tune All Msg 91.95 85.85 Table 3: F1 scores across datasets. 4.2 Transformer model Our second main hypothesis (Hypothesis 2a) is that we can detect conflict using a transformer model, the BERT (Devlin et al., 2019) implementa- tion from HuggingFace.5 Further, we propose that, beyond the specific words in the message, the con- text of the conversation contributes to its likelihood of becoming conflictual. We examine different types of contextual information: the previous mes- sages (Hypothesis 2b); the previous messages with constructiveness and toxicity labels, as we saw in Section 4.1 (Hypothesis 2c); the cardinality of the conversation (Hypothesis 2d); and the topology, or structure of the conversation (Hypothesis 2e). We use a technique inspired by Jin and Aletras (2021) to incorporate contextual information into the BERT model. This approach has proven effec- tive for complaint severity classification by inject- ing linguistic features. The word representations from the embedding layer are combined with the contextual information using an attention gate to control the influence of different features. The com- bined representations are passed through the BERT encoder, followed by an output layer. We set the 12199 max length to 256 and keep the parameters in the attention gate the same as Jin and Aletras (2021). with no topic words. This improvement with all the messages likely results from BERT’s ability to account for contextual sequences, as compared to the LR model. 4.3 Comparison of model results constr = construc- tiveness labels; tox = toxicity labels We conclude that information about the conver- sational context is useful in pinpointing conflict, ad- ditionally contributing the type of contextual infor- mation that the model needs to be robust to changes in topics and individual words. 6 Conclusions and future work qualitative analysis of all the conversations in that green zone, to investigate whether they have traces of agonism. The long-term goal of our program of research is to identify agonistic conflict and distinguish it from less productive and democratic forms of conflict. The work presented here contributes a definition of agonism and its operationalization in a coding schema, an important step in approaching content moderation as a task of fostering agonistic dialogue, a productive form of conflict that is essential to democratic dissent. Furthering our analysis, we sampled the top 100 bidirectional conversations from the highest PU and PA scores. A different set of annotators was then tasked with categorizing each conversation as either agonistic, antagonistic, or neither. This re- sulted in an inter-annotator agreement quantified by κ = 0.44, indicating a moderate level of agreement. The distribution of resulting labels in agreement is shown in Table 4. The work presented here contributes a definition of agonism and its operationalization in a coding schema, an important step in approaching content moderation as a task of fostering agonistic dialogue, a productive form of conflict that is essential to democratic dissent. We introduce a richly annotated dataset of online conversations containing conflict. Using this data, we test methods that can identify conflict from con- versations, crucially incorporating contextual in- formation. We experiment with dimensions of the context that we believe can be proxies for agonistic conflict, including the presence of constructiveness and toxicity, the number of participants, and the topology of the conversation, which includes the level and direction of interaction. We show that the contextual information is key to identifying con- flict, especially because it helps the model remain topic-agnostic. This contextual approach can be helpful not just in identifying conflict and agonism, but also in detecting abusive language, as it pro- vides a wider view of the conversation, rather than whether an utterance is abusive or not in isolation. Table 4: Distribution of conversational labels Table 4: Distribution of conversational labels Label Percentage Agonistic 34% Antagonistic 32% Neither 35% 76.5% of the conversations coded as Agonism were sampled from the top 100 PA set, and 80% of the conversations coded as Antagonism were sampled from the top 100 PU set. These findings suggest that while our proxy scores could do mod- erately well at discerning between agonistic and antagonistic conversations, there is still room for improvement. 6https://github.com/Digital-Democracies- Institute/Dimensions-of-Online-Conflict 5 Approximating agonism We can attempt to approximate agonism as well as other categories of these conversations given the three dimensions shown in Figure 3 by defining PA as the Possibly Agonism Score, PU as the Possibly Unproductive Score, and PS as the Possibly Small- Talk Score as follows: Table 3: F1 scores across datasets. constr = construc- tiveness labels; tox = toxicity labels Table 3: F1 scores across datasets. constr = construc- tiveness labels; tox = toxicity labels We can see from Table 3 that the simple logistic regression model (LR) results are quite low, but have a drop for Dataset 2 that is comparable to that of the BERT models. There are no gains whatso- ever for the LR model from including more context, in the form of knowing the prior messages in the conversation, or more information, such as the con- structiveness and toxicity labels. This might result from the absence of word sequence modelling in LR, which may hinder its ability to capture contex- tual dependencies between words. PA = 1 − p (T −0.0)2 + (S −1.0)2 + (C −1.0)2 (1) PU = 1 − p (T −1.0)2 + (S −0.0)2 + (C −1.0)2 (2) PS = 1 − p (T −0.0)2 + (S −0.0)2 + (C −0.0)2 (3) PS = 1 − p (T −0.0)2 + (S −0.0)2 + (C −0.0)2 (3) where T is the Toxicity Likelihood, S is the Constructiveness Likelihood, and C is the Conflict Likelihood. The results from BERT are more interesting. First of all, a simple model with just the last mes- sage shows F1 = 94.58, with a 0.32 drop if topic words are not present. Including all messages helps considerably, raising the score to its highest level, 96.06, but with an ever larger drop for Dataset 2, Using these proxy scores, we can compare the ra- tios of unproductive versus agonistic conversations across different trending topics over time, shown in Figure 3. Part of our future work involves a 12200 6 Conclusions and future work Implementing a secondary machine learning model on a larger dataset with labelled data from this secondary annotation exercise ap- pears to be a promising next step in our goal to- wards modeling agonism. We have made data and code available in a repos- itory.6 This includes: the dataset of 4,022 conversa- tions with annotations, the code to collect conver- sations, the LabelStudio annotation scheme, and the code for all the experiments described. The appendices in this paper include detailed informa- tion about the data collection process, the coding schema, and multiple examples of annotated con- versations from the dataset. The repository also contains links to raw data, a larger dataset of con- versations we collected but have not annotated (the entire dataset contains 220,626 conversations). We will, additionally, make available a demo web appli- cation linked in the repository to experiment with the model results. Figure 3: Conversations plotted by conflict, construc- tiveness, and toxicity likelihoods. We are postulating the green volume as the ‘zone of agonism’. The next steps in our research program involve deploying the other dimensions in the data (us vs. them conflict; individual/group conflict, etc.). We also plan to perform a qualitative analysis of con- versations with high conflict, high constructiveness, and low toxicity, which we have defined as a poten- tial zone of agonism. Further experiments will ex- Figure 3: Conversations plotted by conflict, construc- tiveness, and toxicity likelihoods. We are postulating the green volume as the ‘zone of agonism’. Figure 3: Conversations plotted by conflict, construc- tiveness, and toxicity likelihoods. We are postulating the green volume as the ‘zone of agonism’. 12201 that we are at the early stages of a long and complex research process. tend this model to other topics in our larger dataset, to test whether it can be used for semi-automatic an- notation. We also plan to gather additional topics that generated discussion by querying keywords from monthly snapshots of the Wikipedia Por- tal:Current_events and the Top_25_Report through the Wayback Machine. Beyond identifying individual comments as con- structive, productive, or leading to agonism, it is also important to acknowledge the role of user and interface design in how comments are produced and presented (Masullo et al., 2022). We also note more common limitations, includ- ing the source of the data (Twitter), the language of study (English), and the lack of demographic information about the participants in the discus- sions. Ethics Statement We adhere to the ACL Ethics Policy. In particular, we strive to contribute to societal well-being and the public good by studying how conflict evolves in online conversations. We take the directive to avoid harms quite seriously. To avoid individual harm and respect privacy, we anonymize the tweets be- fore releasing them publicly (although the Twitter ids will provide a link to the original). We also take into account duty of care for our researchers and annotators, and have provided them with opportuni- ties to debrief and protect their mental health when they read conflictual and hateful material. We are concerned about the damage to the envi- ronment caused by training and fine-tuning large language models. We mitigated, in part, by using only pre-trained models. The BERT model was fine-tuned on a data center powered by hydroelec- tric energy, thus producing fewer CO2 emissions. Precisely because there is so much conflict on- line, a dataset with about 4,000 conversations is not a representative sample. The method of collection, where we started with topics likely to generate con- flict (both hateful and agonistic), may also result in biased data. One alternative we could contemplate is to draw from datasets that have been labelled for toxicity, as those are more likely to contain conflict. Such data, however, does not necessarily contain agonistic conflict. Furthermore, as we mention in the conclusion section, we have not yet reached the stage of identifying agonism automatically. We hope that, by detecting conflict overall, we can ex- tract many instances automatically, leading us to a method for distinguishing antagonistic conflict from agonism. The main limitation of our study is Although our current system is far from perfect in detecting conflict, and we have not yet produced a method to detect agonistic conflict, one risk of such systems lies in their misuse by employers, governments, or other social agents to quell agonis- tic and productive conversations. For example, an employer may wish to suppress agonistic discourse that could lead to employee unionization. Limitations Research on conflict consistently draws attention to its complex nature. Peace studies scholar Giorgio Gallo (2013) states that conflict can be character- ized by multiple, diverse, at times hidden, unde- fined, and evolving objectives. Thus, translating such a complex phenomenon into a set of labels output by a machine learning system is reductionist at best. Gallo notes that most research on conflict tends to isolate it from its context, thus oversim- plifying the inquiry. We note that ours is one such simplification. The nature of online conflict, with long conversations unbounded by time and space limitations, unlike face-to-face communication and debate, renders more fine-grained and contextual- ized approaches impossible. We nevertheless at- tempt to incorporate context beyond the words in the individual messages or message threads, by ex- amining features of the conversation, the number of participants, and the topology of the conversation. 6 Conclusions and future work We do not include information about lan- guage varieties and rhetorical strategies that may be characteristic of some online groups and linguis- tic communities and not in the mainstream. We do not know whether the conversations are repre- sentative of some mythical mainstream culture or of demographic groups with their own norms of debate and argumentation. Defining and identifying agonism in conversa- tions is a difficult task. 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In Proceedings of the 16th Annual Meet- ing of the Special Interest Group on Discourse and (1) #billc11, #cancon, #CRTC, #onlinecensorship, #online- harmsreduction, #StopAsianHate, #stopC11, bean dad, bell hooks, biden loans, bill gates divorce, canadian truck- ers, capitol insurrection, china gamer ban, coastal gas link, convoy, defund the police, depp heard, el salvador bitcoin, elon twitter, evergrande, gamestop, groomers, hbo test, india pakistan missile, ivermectin, libsoftiktok, metaverse, ocean fire, online harms, pandora papers, re- aldonaldtrump, rittenhouse, robb elementary, roe, rogers, sci_hub, social distancing, suez canal, taliban, tigray, tmx pipeline, west elm caleb, will smith slap, vaccine B Appendix: Topic distribution over time • defund-the-police: police, defund • defund-the-police-realdonaldtrump: police, realdon- aldtrump, defund • depp-heard: depp, heard, amber, johnny • elon-twitter: twitter, elon, musk, elonmusk • groomer: govrondesantis, groomers, travlingsnowman • india-pakistan-missile: pakistan, india, indian • libsoftiktok: libsoftiktok, taylorlorenz • metaverse: metaverse, crypto, bsc • protests-social-distancing: protests, distancing • queen-elizabeth: us • roe-v-wade: roe, abortion, wade • russia-ukraine: ukraine, russia • salman-rushdie-attack: rushdie, salman • social-distancing: distancing • tigray-ethiopia: tigray, ethiopia, tplf • vaccine: vaccine, covid Figure 4 shows the number of conversations related to each topic over time. We can see that certain topics exhibit a more enduring presence in the dis- course (Social Distancing), while others appear to be more transient, capturing attention for only a day or two (Will Smith Slap, Rogers Outage). Each topic was sampled from its peak of discussion. We have observed that topics with high peaks (Will Smith Slap or India Pakistan Missile) tend to have a higher level of conflict than topics that last over longer periods of time. A Appendix: Search keywords The following are the list of keywords used for the search described in Section 3.1. The following are the list of keywords used for the search described in Section 3.1. Christian Stab and Iryna Gurevych. 2017. Parsing argu- mentation structures in persuasive essays. Computa- tional Linguistics, 43(3):619–659. Jonathan Stray. 2022. Designing recommender systems to depolarize. First Monday, 27(5):https://doi. org/10.5210/fm.v27i5.12604. Reid Swanson, Brian Ecker, and Marilyn Walker. 2015. Argument mining: Extracting arguments from online dialogue. In Proceedings of the 16th Annual Meet- ing of the Special Interest Group on Discourse and 12205 12205 E.1 Is there conflict? A yes/no answer, based on the definition of conflict provided (see Section 2.1). Annotators were in- structed to label only the last message (e.g., the last tweet in Figure 5), but use the context (the previous tweets) if necessary. A ‘yes’ answer triggers all the decisions below. H = 12 N(N + 1) X j R2 j nj −3(N + 1) (4) (4) E.2 Internal/external If the annotators decided that there was conflict, they had to label the conflict as internal to the con- versation (‘when it involves people/entities directly engaged in the conversation’) or external. For ex- ample, if people are discussing Black Lives Matter as an organization, then the conflict is external. But if they discuss people in the conversation involved in BLM, then it’s internal. A tweet can be both internal and external, so both labels are allowed. This label had a relatively high level of agreement (see Table 1), so we feel this was a valid distinction. Examples (2) and (3) show instances of each. This calculation obtained a p-value < .005. We can confidently say that topics are clearly differ- ent in their rate of conflict, with discussions about Biden’s loan forgiveness program being 100% con- flictual, and conversations about Facebook’s Meta- verse being low in conflict (8% of the conversations in that topic had conflict). E Appendix: Coding schema Content warning: Examples in this section con- tain offensive language. Table 5 presents summary statistics for the top top- ics (a total of 2,718 conversations, out of the 4,022 in the dataset). The first column shows the total number of conversations in that topic. The next few columns display the percentage of conversations in that topic with the label (e.g., 62% of the Social Distancing conversations contained conflict, and 11% had sarcasm). We see that most topics con- tained some form of conflict (making the dataset unbalanced), but that presence of other dimensions of conflict varies widely. Using the percentage of conflict per topic in Table 5, we can test for statisti- cally significant differences in conflict rates across topics. Assuming non-normality and non-equal variances, we can use the Kruskal-Wallis H test defined as follows: Annotators were presented with a LabelStudio interface that provided: the tweet to annotate, the previous 3-7 messages in the conversation, and the labels to choose from, as seen in Figure 5. Labels were always for the last tweet in the conversation, but in the context of the entire thread. Here, we elaborate on the descriptions for each annotation decision, from the schema in Figure 1. (2) [Internal] You are going to arrst me for standing 4’ away from someone? Go for it (3) [External] Honestly at this point second wave is gonna have to happen for it to get through their THICK SKULLS that they completely and utterly fucked this up E.3 Who/what is the target? (6) [Policy/institutions] That assumption is based on a busy car park. With miles of beach I am sure it’s easy to stay far enough away from others. Visiting Tesco’s is 100x more dangerous The target of the conflict can be more than one, so multiple labels are possible here. Some examples are presented in (4)-(6). • Individual The following is the list of keywords removed from each topic as described in Section 3.1. The following is the list of keywords removed from each topic as described in Section 3.1. • Jan-6th-insurrection: capitol, insurrection, trump (2) [Internal] You are going to arrst me for standing 4’ away from someone? Go for it • TMX-pipeline/coastal-gaslink: pipeline, tmx, trudeau • biden-loans: loans student biden • TMX-pipeline/coastal-gaslink: pipeline, tmx, trudeau • biden-loans: loans, student, biden (3) [External] Honestly at this point second wave is gonna have to happen for it to get through their THICK SKULLS that they completely and utterly fucked this up • canada-day: canada • canadian-truckers: truckers, canadian, convoy • canadian-truckers, protests: truckers, protests, canadian, canada 12206 2020-03-01 2020-09-01 2021-03-01 2021-09-01 2022-03-01 2022-09-01 2023-03-01 India Pakistan Missile Rogers Outage Vaccine Tigray Ethiopia Roe v Wade Metaverse Defund the Police Biden Loans Libsoftiktok Canadian Truckers Depp Heard Elon Twitter Will Smith Slap Social Distancing Figure 4: Volume of messages for some of the top topics over time Figure 4: Volume of messages for some of the top topics over time Topic N Conflict Sarcasm Metaphors Directives Target Social Distancing 615 0.62 0.11 0.36 0.29 0.40 Will Smith Slap 320 0.97 0.20 0.76 0.05 0.96 Elon Twitter 268 0.90 0.23 0.67 0.14 0.91 Depp Heard 233 0.97 0.11 0.70 0.08 0.95 Canadian Truckers 230 0.97 0.10 0.67 0.15 0.53 Libsoftiktok 191 0.97 0.18 0.55 0.09 0.80 Biden Loans 190 1.00 0.13 0.72 0.23 0.78 Defund the Police 157 0.94 0.06 0.41 0.42 0.33 Metaverse 131 0.08 0.18 0.50 0.05 0.59 Roe v Wade 91 0.95 0.10 0.38 0.19 0.43 Tigray Ethiopia 90 1.00 0.06 0.68 0.40 0.31 Vaccine 76 0.89 0.20 0.24 0.30 0.45 Rogers Outage 69 0.48 0.15 0.20 0.30 0.41 India Pakistan Missile 57 1.00 0.21 0.61 0.12 0.46 Total/average 2,718 0.84 0.12 0.49 0.16 0.56 Table 5: Top topics and percentage of conversations in those topics that were given select annotation labels Topic N Conflict Sarcasm Metaphors Directives Target Table 5: Top topics and percentage of conversations in those topics that were given select annotation labels E.4 Authority evoked • Group This question asked whether the position, stance, or claim is recognized, consistent with, legitimated, or supported by some form of authority. Annotators were instructed to consider how different forms of authority are invoked by the participants, again focusing on the last tweet in the conversation. • Policy/institutions • Systemic issue or cause • Systemic issue or cause • Events y • Events • Events • Other (specify) (4) [Individual] Cummings should be sacked! And prose- cuted in criminal law. • Personal experience. This could be an individ- ual encounter, an understanding, or an insight, usually derived from proximity and epistemic positions. (5) [Group] Its unreasonable and unrationale to say that we can’t let emotions take over. Imagine being a black person in America right now. We should be angry, we shouldn’t be pushing down or hiding our feelings. We want justice, everything else is not important right now. 12207 Figure 5: Annotation interface This label had a very low level of agreement with a kappa value of 0.02. We believe the explanation of this distinction, as shown above, was unclear to annotators, perhaps also because these oppositional relations are not often explicitly stated. Some ex- amples are provided below. Note the dual label in (10). (10) [Us above them], [Us/them disjuntive] idk why y’all continue to defend people who prove themselves over and over again the rich will always find ways to hide their money and avoid paying their dues, if we continue to let them Figure 5: Annotation interface Figure 5: Annotation interface Figure 5: Annotation interface (11) [Us/them disjunctive] Please sign our petition to fire RCMP Commissioner Brenda Lucki here, and maybe we can finally get some accountability for an organization that has gone completely off the rails. Keep fighting for what is right, (11) [Us/them disjunctive] Please sign our petition to fire RCMP Commissioner Brenda Lucki here, and maybe we can finally get some accountability for an organization that has gone completely off the rails. Keep fighting for what is right, • Common sense. Authority is evoked by refer- ring to common sense knowledge, some sense of what is shared in the specific or cultural context (regardless whether common sense is actually true). E.5 Groups being discussed This is a binary question, asking whether the con- versation involves clearly identified groups or fac- tions, presented as an attempt to render conflict as isolated incompatibility between groups. If the an- swer is ‘yes’, this triggers another set of questions, about the relationships among those groups and how they are being discussed: (14) [Sarcasm] So doxxing people who disagree with you is OK, but not doxxing people who agree with you. Got it. E.6 Rhetorical strategy • Factual/distant expertise or institution. Par- ticipants support their statements by referring to experts, facts, or (statistics provided by) institutions. A ‘yes’ answer here means that the annotator saw persuasion or appeals to sensibilities and mean- ings that were grounded in linguistic techniques, language moves, or other linguistic mechanisms. If the answer was ‘yes’, then they were asked to specify what type of strategy was deployed: • Authority is rejected. Participants reject or challenge authorities invoked by others. • Other (specify) • Explicit directives and calls to action (7) [Personal experience] Pretty sure for my son to go to boarding school he needed proof of vaccinations (7) [Personal experience] Pretty sure for my son to go to boarding school he needed proof of vaccinations • Associations, metaphors, or analogies • Sarcasm • Sarcasm (8) [Common sense] The mothers who raise these men play a role. They, too, are responsible. Many of them support the Taliban. It is naive to believe otherwise • Other (specify) (12) [Explicit directives], [Sarcasm] Yo ACLU and Amnesty International, calm down. Why sudden panic about “free speech” when Elon wanna buy Twitter? (9) [Factual/distant expertise or institution] All indications are that the practice of sex with social dis- tancing will have a profound effect on population control. Perhaps even larger that what ‘the pill’ had in its day when it first came out. (13) [Associations, metaphors, or analogies] So seems like evergrande stroke a deal which is good for now (kicking can down the road) and news just came out of China about reducing coal. Now just the Fed gotta worry about. Besides the fact that charts are looking ugly. E.7 Meta questions Finally, regardless whether the answer to the con- flict question was yes or no, annotators were asked three further questions, about context and about their own reaction. • Us above them • Us below them • Us/them conjunctive. The two or more groups being discussed or involved in the conversa- tion are presented as being allied, connected, or somehow related. The first question was whether more context was needed, beyond the thread. As we see in Figure 5, annotators were asked to label the last message and were allowed to read the previous messages on the screen. But they also had the possibility to click on the tweet and look at the context on • Us/them disjuntive. The two or more groups are presented as not allied, connected, or re- lated. 12208 12208 the Twitter platform, including any media. If they clicked, they were asked to answer ‘yes’ to this question. Further, they could specify what kind of context was needed: • Cultural. The annotator needed to know more about the issue at stake. This could be current news topics, subcultural trends online, or other aspects of the topic. Figure 6: Conversation, with labels shown in Figure 7 • Conversational. The conversation was miss- ing some elements, perhaps earlier than the messages included in the annotation platform, which made it difficult to interpret. • Missing content. Tweets in the thread had been deleted. • Media. Videos or images in the thread were not available, but seemingly necessary for in- terpretation. Figure 6: Conversation, with labels shown in Figure 7 The second meta question asked about the emo- tional reaction of the annotator, simply asking ‘How did you feel when reading the conversation?’ Some suggestions were provided: • Shock • Sadness • Confusion • Indifference Figure 7: Full annotation for conversation in Figure 6 • Entertained • Hopeful The third and final question was about level of confidence (‘How confident are you about your analysis?’). Annotators were provided a 1-5 scale, with 1 being not confident at all (more context was needed, tweets had been removed, tweet was inde- cipherable), and 5 being a high level of confidence or that the interpretation was straightforward. E.8 Full annotation Figure 6 displays a message (last message) and its context, to show the richness and complexity of the annotation scheme. Labels for the annotation of this example are given in Figure 7. Figure 7: Full annotation for conversation in Figure 6 12209
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Argumentation-Based Health Information Systems: A Design Methodology
IEEE intelligent systems
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Argumentation-Based Health Information Systems: A Design Methodology Helena Lindgren, Timotheus Kampik , Esteban Guerrero Rosero , Madeleine Blusi , and Juan Carlos Nieve Umea  University, 90187 Umea , Sweden In this article, we present a design methodology for argumentation-based health information systems. With a focus on the application of formal argumentation, the methodology aims at eliciting requirements in regard to argumentation reasoning behavior, knowledge and user models, and business logic on levels below and above the argumentation layer. We highlight specific considerations that need to be made dependent on the system type, i.e., for clinical decision-support systems, patient-facing systems, and administration systems. In addition, we outline challenges in regard to the design of argumentation-based intelligent systems for healthcare, considering the state of the art of argumentation research, health information systems, and software design methods. For each challenge, we outline a mitigation strategy. F F ormal argumentation has emerged as a promis- ing method for automated reasoning. While a large body of works exists on theoretical aspects of formal argumentation,1 the application of the method to real-world use cases is—despite some success stories—still at an early stage. Hence, it is important to advance research that closes the gap between the theoretical knowledge the community is accumulating and real-world applications. clinical pathways recommends the intake of a, based on treatment plan d. In contrast, a medical specialist recommends the intake b, based on treatment plan c. Because stimulate medication and antidepressants counteract each other, only one of the treatment options can be chosen. The practitioner who is responsible for treating the patient needs to decide which advise to follow. Figure 1 shows the argumenta- tion graph of the example. Formal argumentation approaches are frequently proposed in the context of health information sys- tems2 to derive conclusions from conflicting, incon- sistent, or uncertain information. The following (simplified) example highlights the usefulness of argumentation in healthcare (using the abstract argumentation approach).3 A patient shows symp- toms that could either indicate attention-deficit/ hyperactivity disorder (ADHD) (the treatment of which we denote by argument c) or depression (whose treatment we denote by d). ADHD can be treated with stimulant medication (argument b), depression with antidepressants (argument a). A decision support system based on standardized In formal argumentation, problems of this type can be expressed in a mathematical model, which can be solved using a formal method, e.g., a so-called argu- mentation semantics. Published by the IEEE Computer Society March/April 2021 Published by the IEEE Computer Society DESIGN METHODOLOGY DESIGN METHODOLOGY The design methodology can be divided into following three phases. The design methodology can be divided into following three phases. 1) Use case and architecture identification. 2) Iterative system design and implementation. In the past, formal argumentation has been highlighted as a potential solution to a set of common challenges that arise when designing clinical decision support systems.4 Still, and although the formal argu- mentation community is thriving, no success stories that report on the wide-spread adoption or large-scale clinical trial of argumentation-based systems in healthcare exist. Indeed, much of the research on formal argumentation and healthcare is limited to the definition and (some- times) the implementation of running examples, without the involvement of domain experts, the creation of open- sourced software artifacts, and the empirical evaluation of the developed prototype applications. 3) Empirical evaluation. The end of each phase (and end of the Phase 2 iteration cycle) represents an inflection point, at which a preliminary evaluation of the system design is con- ducted that informs the decision on how to proceed further. Also, each phase results in the creation of dis- tinct artifacts, which can, for example, be presented in a dissemination or handed over to third parties. Before the first phase, focus groups of stakeholders (poten- tial users, domain experts, etc.) should be set up that accompany the design process. This article presents a design methodology for argumentation-based health information systems that can facilitate stronger applied research in this domain in the future. The design methodology is informed by our own research experiences, and in particular, the lessons we have learned during the past 15 years of research at the intersection of formal argumentation and artificial intelligence for healthcare. Table 1 pro- vides an overview of some application scenarios of argumentation to the healthcare domain, during which the methodology was defined and refined, and highlights some design aspects (which are explained in this article) for each use case. The methodology can Use Case and Architecture Identification The first phase is concerned with use case identifica- tion and high-level application architecture definition. Argumentation-Based Health Information Systems: A Design Methodology Note that in the context of formal argumentation, arguments can model any type of knowledge, and are not necessarily based on natu- ral language. To solve the framework in Figure 1, we first need to answer some (potentially use case- specific) questions, for instance: 1) Are some arguments stronger than others, for example because they are more likely to be true or come from a more authoritative source? 2) What medical knowledge should inform the inter- nal structure of an argument, and how should its relation to other arguments be generated? This work is licensed under a Creative Commons Attribution 4.0 License. For more information, see https:// creativecommons.org/licenses/by/4.0/ Digital Object Identifier 10.1109/MIS.2020.3044944 Date of publication 15 December 2020; date of current version 14 May 2021. 3) How should cycles of arguments that can arise from dependencies between different sources of information (e.g., medical guidelines from differ- ent organizations, diverging opinions of medical practitioners) be resolved? Published by the IEEE Computer Society 72 IEEE Intelligent Systems March/April 2021 SPECIAL ISSUE ON ARGUMENTATION-BASED REASONING SPECIAL ISSUE ON ARGUMENTATION-BASED REASONING FIGURE 1. Example of an argument graph. Treatment c implies abstaining from the intake of medicine a, whereas treatment d implies not taking b. Hence, only one of the treatment options can be chosen. be considered a complementary, argumentation-cen- tered perspective on a software development process that assumes a somewhat agile, iterative approach to software development,5 which we consider a reason- able assumption in a research-intense development context; however, it can be adjusted to better inte- grate with other, noniterative software development approaches. be considered a complementary, argumentation-cen- tered perspective on a software development process that assumes a somewhat agile, iterative approach to software development,5 which we consider a reason- able assumption in a research-intense development context; however, it can be adjusted to better inte- grate with other, noniterative software development approaches. FIGURE 1. Example of an argument graph. Treatment c implies abstaining from the intake of medicine a, whereas treatment d implies not taking b. Hence, only one of the treatment options can be chosen. FIGURE 1. Example of an argument graph. Treatment c implies Define high-level architecture After the use case has been defined, high-level requirements should be specified, and the preliminary architecture should be designed. It is important that the architecture primarily serves the use case; the alignment with a basic research purpose should merely be a desirable side effect. At this point, it is typ- ically sufficient to model the architecture using gen- eral graphical diagramming tools; more detailed specifications in standardized modeling notations such as the Unified Modeling Language or ArchiMate 9 can follow later. Cocreate interactive prototypes In parallel to building the knowledge model, interac- tive prototypes are created; again, domain experts should be involved, and in addition, potential nonex- pert stakeholders like patients. Knowledge model and user interface depend on each other. On the one hand, the user interface provides abstractions of the knowledge model that compromises between accu- racy and conciseness. On the other hand, the knowl- edge model needs to consider user interaction needs. During the initial prototype design phase, knowledge model and interactive prototype should be only loosely coupled, to ensure that user interaction needs are treated as first-class citizens. Tools that allow for the rapid creation of prototypes can already be used in this phase. However, the employment of such tools at a too early stage can hamper creativity, as a rather strict frame for the system’s user interface (UI) design is dictated by these platforms. Inflection point. After this phase, it should be clear whether an argumentation-based system can, indeed, serve the use case at hand. If this is not the case, a dif- ferent type of system (for example: a simple rule- based system or a machine learning classifier) can be implemented, or—if this is not feasible—the project can be abandoned. In particular, the application of for- mal argumentation as an agreement technology implies that the use case requires the management of potentially inconsistent or uncertain information from multiple sources in decision processes. Artifacts. Results of this phase are a preliminary feasibility analy- sis, a high-level architecture and requirements specifi- cation, and an activity analysis specifying the work and decision-making processes that are to be supported. Identify use case Right from the start, the system’s use case should be defined in close collaboration with relevant medical experts. Adopting an activity-centric perspective is important to specify the type of support the system should provide in the application scenario, as well as the way the system and its users are supposed to TABLE 1. Medical use case examples, their use case types, knowledge bases, conflict types, and argumentation approaches. Medical Use Case Use Case Type Knowledge Base Conflict Types Argumentation Approach Reference Decision-support for dementia diagnosis and treatment Clinical decision- support system Possibilistic answer-set programs Guideline inconsistencies Inquiry dialogues Yan et al.6 Self-management application for mental/social well-being Patient-facing system Extended logic programs Human goal conflicts Abstract argumentation Guerrero et al. 7 Fall prevention application for the elderly Patient-facing system Extended logic programs Inconsistencies in human activities Abstract argumentation Guerrero et al. 8 March/April 2021 IEEE Intelligent Systems xamples, their use case types, knowledge bases, conflict types, and argumentation approaches. TABLE 1. Medical use case examples, their use case types, knowledge bases, conflict types, and argumentati 73 IEEE Intelligent Systems March/April 2021 SPECIAL ISSUE ON ARGUMENTATION-BASED REASONING collaborate to work toward the users’ and the health- care providing organization’s goals. always given. Indeed, in this fact lies strength of the argumentation-based approach: Inconsistencies bet- ween ideal standards and local realities can be exp- licitly modeled and resolved at run-time. Another important aspect when designing the knowledge model is the knowledge modeling language. Because medical professionals are not necessarily well-versed in knowledge modeling languages like the Web Ontol- ogy Language, it is important to use a high-level, potentially informal language to specify the rough model, and, when agreement on the most important aspects is reached, iteratively refine details. Elicit arguments The second phase implements a system prototype that is based on the artifacts that result from the previous phase. The implementation is conducted iter- atively in collaboration with relevant stakeholders. After the general knowledge model and UI have been designed, argument elicitation can begin. As the first step in this activity, one needs to distinguish between elicitation at design-time and elicitation at run-time. IEEE Intelligent Systems SPECIAL ISSUE ON ARGUMENTATION-BASED REASONING regards to argument strength and dialogue type, the structure of the arguments should be defined. The structure is dependent on the knowledge model, i.e., on the nature and structure of the knowledge base(s) from which conclusions are to be derived. For this, one can rely—to some extent—on the argument inter- change format,15 which is an early stage effort to pro- vide guidelines and best practices for the elicitation of arguments from knowledge bases, as well as for argu- ment exchange. In conjunction with the design of argument structure and argument strength, the argu- mentation semantics that determines how an argu- mentation graph is resolved needs to be defined; in this context, the argumentation principles16 that a specific semantics fulfills should be considered. In par- ticular, principles that are aligned with the require- ments of the application scenario should be identified. To construct arguments and detect conflicts, the formal logic that formalizes the semantics of the knowl- edge specification language should provide polynomial- time inference operators. This means it is feasible to define efficient algorithms for constructing arguments and detecting attacks between arguments in a medical knowledge base. To mine arguments from unstructured or poorly structured data, machine learning techniques can be applied.11 During elicitation, it is recommendable to assign each argument to one or several groups; for example, one argument can be assigned to the group end-user preference, whereas another one is assigned to the groups expert diagnosis and International Classi- fication of Diseases. No matter whether an argument is elicited at run-time or design-time, the argument’s strength needs to be considered. The strength can be derived from the groups the argument is in, or be inferred from data. For instance, in case a diagnosis-sup- port system is implemented, expert opinions might be considered stronger than end-user self-assessments, because the latter can be considered as less reliable.6 However, when designing a self-management mobile application, end-user preferences can be considered more important than expert opinions in some contexts, for example with regards to the configuration of daily schedules and motivational recommendations.7 In for- mal argumentation, strength can be modeled qualita- tively, for example by constructing preference orders over arguments,12 or quantitatively, for example, using probabilistic approaches.13 An important concern in argumentation seman- tics is their computational complexity. Design knowledge model 1) At design-time, arguments are manually curated, mined from an unstructured dataset, or auto- matically transferred from another already well- structured knowledge base. Arguments and argumentation frameworks can be refined and sanity-checked before deployment, which places less strict requirements on the algorithms for argument generation. A domain-specific knowledge model should be devised, again in collaboration with domain experts. The knowledge model should be based on existing models of the corresponding domain, for example, on standardized data models like Health Level Seven (HL7),10 clinical paths that have been specified by the relevant authorities, or international disease classifi- cation standards. However, it is important to consider that local realities might diverge from the standard- ized specifications. For example, the information scheme that a specific electronic health record sys- tem uses might not be standard-compliant, and even if it is, information-completeness is practically not 2) At run-time, arguments can be derived directly from user-interactions, or from additional data that is uploaded to the system; i.e., in this case, algo- rithms for the autogeneration of arguments need to be defined and properly tested to ensure they perform as intended when the system is deployed. 74 March/April 2021 March/April 2021 SPECIAL ISSUE ON ARGUMENTATION-BASED REASONING The deci- sion problems of the well-accepted argumentation semantics range from NP-complete to PðpÞ 2 -com- plete.17 In this context, tradeoffs may need to be made; for instance, argumentation semantics like the grounded semantics that are of comparably low computational complexity do not allow for particu- larly nuanced conflict resolution (colloquially speak- ing). To enhance computational performance, some applications of formal argumentation place condi- tions on the structure or size of the argumentation graph and, for example, only construct noncyclic argumentation graphs.18 Hence, use case-specific running examples should be constructed to evaluate if a semantics’ output is reasonable from a compu- tational complexity and a subject-matter expert perspective. Also, it is crucial to consider the type(s) of argu- mentation dialogues the system should support; fol- lowing are the examples.14 1) Inquiry dialogues: The system uses (multiagent) argumentation for knowledge-seeking purposes, for example, to derive new conclusions by elicit- ing arguments from distributed and potentially inconsistent knowledge bases. Evaluate qualitatively To assess whether the system is ready to be deployed for a long-running empirical study, a preliminary, qualita- tive evaluation should be conducted. In contrast to the previous stages of the iterative design process, this evaluation should be conducted using a deployed, run- ning, and stable system instance. Ideally, a new set of experts and end-users is involved in the evaluation. This avoids biased feedback from persons who are already invested from a design perspective. As a first evaluation step, the initial knowledge base should be validated. For example, the system’s outputs can be compared to clini- cal guidelines and protocols, and be evaluated by medi- cal experts and patients. Additional feedback can be solicited on the system itself and the conclusions derived from it in example scenarios, and by conducting trial runs of the system in a carefully controlled real-life environment. Also, observations should be made on how the use of the system affects decision-making and human behavior. For example, given a decision support system, it should be documented to what extent users follow the system’s recommendations. Inflection point. When the empirical evaluation has been concluded, further steps can be planned based on the implications of the results. In particular, follow- up studies can be conducted, for which the system might need to be customized to fit a new context (for example, to the needs of medical practitioners in a dif- ferent country). In successful cases, the hand-over of the system to parties that can ensure long-term oper- ations can be initiated; for instance, local health authorities or other healthcare-providing organiza- tions may have an interest in taking over the mainte- nance and operations of a system that is evidently useful in a particular medical context. Artifacts. The resulting artifact of this phase is an analysis document that contains an empirical evaluation of the system. Figure 2 shows the process diagram of the design methodology. Inflection point. When an implementation and design cycle has been absolved, the system designers should decide whether 1) further iterations are neces- sary, 2) the system is sufficiently mature for an empirical study, or 3) the system prototype should be discontin- ued without an empirical evaluation being feasible. Arti- facts. The resulting artifacts of this phase are a detailed system specification, a system prototype, and a prelimi- nary qualitative evaluation of the system. SPECIAL ISSUE ON ARGUMENTATION-BASED REASONING SPECIAL ISSUE ON ARGUMENTATION-BASED REASONING the existing knowledge base, and what actions should be triggered by such user feedback? Empirical Evaluation In the final phase, the system is empirically evaluated with the objective to provide a strong assessment of the system’s usefulness in real-world medical applica- tion scenarios. As a rule of thumb, the level of detail the user inter- face provides in terms of both data view abstractions and data input and feedback opportunities should be more fine-grained for medical expert users than for patients. Evaluate empirically To assess the medical efficacy of the developed sys- tem, an empirical evaluation in practice is necessary, if possible in a randomized-controlled trial setting. Ide- ally, the medical practitioners who participate in the empirical evaluation of the system are not (at least not exclusively) the ones who have helped design it. Otherwise, there is a risk that the participating medi- cal experts are 1) biased toward the efficacy of the sys- tem and 2) have a level of expertise in working with the system that is hard to obtain by third parties. Implement system prototype In parallel to the previous four design steps, the system prototype should be iteratively implemented. As men- tioned earlier, rapid prototyping tools can facilitate the implementation. In particular, data schemes that underlie the system should ideally be (auto)generated directly from the models that have been defined in col- laboration with the focus group(s). Given the safety- critical nature of medical information systems, even for the prototype implementation, quality assurance best practices like test-driven development and continuous integration should be followed. Expert-validated run- ning examples can serve as test cases. To enable a real-world study, the system needs to be integrated into the corresponding healthcare process or clinical pathway. If the study is conducted across regions with different local routines, the study should ideally be detached from these local routine variants to ensure comparability across regions. In the empirical evaluation, a mixed-method approach is recom- mended. Generally, the system’s recommendations or decisions can be quantitatively evaluated. Unexpected system behavior can be analyzed qualitatively to find the reason for the deviation and to determine whether the unexpected behavior is indeed undesirable. Design knowledge model UI Design knowledge model UI In parallel to the argument elicitation step, the abstraction the user interface provides on system data and processes, i.e., on argumentation frame- works, knowledge bases, and reasoning methods, should be designed. In particular, the following ques- tions should be answered. 2) Deliberation dialogues: The system facilitates tradeoffs between the positions of different par- ties, for example, by consolidating conflicting opinions of experts or conflicts between medical guidelines on regional and national level. 1) What abstractions should the user interface pro- vide on the knowledge base and how detailed should these abstractions be? 3) Persuasion dialogues: The system uses formal argumentation to persuade a user, for example, by exchanging arguments with a patient to moti- vate them to work toward a specific goal. 2) Are there data that should under no circumstan- ces be exposed to a user, for example, for data- privacy reasons? The selection of dialogue type is done based on the activity analysis that specifies phases and deci- sion points in the clinical pathway and decision pro- cesses. Alongside with making design decisions with 3) When should the user be able to add additional knowledge as a means of providing feedback, how should the knowledge be integrated into IEEE Intelligent Systems 75 IEEE Intelligent Systems Clinical Decision-Support Systems Clinical Decision-Support Systems Clinical Decision-Support Systems In recent years, it has been acknowledged that the han- dling of inconsistent knowledge, for example, of diverg- ing expert opinions, is crucial in many medical decision- support scenarios. However, existing, industry-scale products do not provide first-class abstractions for managing inconsistent or conflicting knowledge. Argu- mentation-based clinical decision-support systems can help facilitate decision-making by enabling the manage- ment of these inconsistencies that are common in med- ical decision-making, and indeed often part of the medical process by design, e.g., when the opinions of several medical experts are solicited with regards to a specific case. An instance of an argumentation-based decision support system is a dementia diagnosis and management support application as presented by Yan et al.6 In a decision-support context, it is important that arguments from different types of sources are marked according to the corresponding source type’s strength. For instance, the argument strength assignment can reflect that the opinion of a single practitioner can- not invalidate well-established domain knowledge as specified in national or international guidelines. A potential hierarchy of arguments could be as follows (assuming a total ordering), but is most certainly scenario-dependent. FIGURE 2. Design methodology process diagram. 1) Arguments derived from global guidelines and standards take precedence over all other arguments. 2) Local guidelines define aspects that have not been specified in sufficient detail by global guidelines and standards, but arguments derived from local guidelines are weaker than arguments derived from global guidelines. 3) Arguments derived from practitioner opinions can inform decision-making on individual cases but are weaker than local and global guidelines. From a formal perspective, a plethora of potential argumentation strength design mechanism exist19; so far, there are no best practices as to when to choose which mechanism. March/April 2021 APPLICATION SUBDOMAINS The context in which a health information system is applied should self-evidently inform its design. A par- ticularly important distinction is whether the system is primarily used by medical experts, by patients, or by administration staff. 76 IEEE Intelligent Systems March/April 2021 SPECIAL ISSUE ON ARGUMENTATION-BASED REASONING CHALLENGES AND RESEARCH STRATEGIES The design methodology may be considered an ideal- ized approach that cannot always be complied with in reality, for example, because of constraints in time, budget, or expertise. Indeed, when examining existing literature on the topic, we observe a set of shortcom- ings, from which we derive new research strategies. Lack of integration with theoretical foundations A large part of the theoretical research results on for- mal argumentation has not been examined from an application perspective. This applies in particular to the broad range of works that exist on argument strength19 and argumentation principles.16 When deciding on the knowledge model, argument structure and strength, as well as on the argumenta- tion reasoner, works that provide concise overviews of the corresponding basic research foundations should be considered in an as systematic manner as possible; also, the practical considerations that guide this process step in particular instances should inform theoretical research going forward and not only vice versa. Patient-Facing Systems When implementing patient-facing health information systems, it is crucial that tradeoffs are made between the advice and recommendations that the system provides based on existing medical knowledge and patient data, and the patient’s self-assessment and personal preferences. To facilitate these compro- mises, formal argumentation methods can be consid- ered a natural fit. A particularity of patient-facing FIGURE 2. Design methodology process diagram. systems is that the recommendations provided and the actions executed by these systems must not only fulfill the quality standards of the healthcare domain, but also allow for informed dissent if the assumptions systems is that the recommendations provided and the actions executed by these systems must not only fulfill the quality standards of the healthcare domain, but also allow for informed dissent if the assumptions 77 IEEE Intelligent Systems March/April 2021 SPECIAL ISSUE ON ARGUMENTATION-BASED REASONING the system makes are not aligned with a patient’s per- sonal preferences or life circumstances. An instance of this system type is an argumentation-enabled behavior change management personal assistant.7 If the assistant recommends a user to do more physical exercise, the user should have the possibility to pro- vide the feedback to the system that in the current sit- uation, family responsibilities do not allow to act according to the recommendation. The possibility for dissent is not only important from the perspective of medical efficacy, but also for the sake of personal autonomy: A user should be empowered to make tradeoffs between an optimally healthy lifestyle and other aspects of life, and not have a system dictate all details. From the perspective of argumentation, this means that users should be able to add new argu- ments to the system that defeat the conclusions the system provides; i.e., the system should be able to provide alternative conclusions based on the new arguments while maintaining the evidence-based knowledge foundation. This behavior allows for verifi- able compliance with guidelines and regulations, which is, for example, a requirement for regulatory approvals like CE-marking. In this regard, the testabil- ity and verifiability of argumentation-based systems is a key advantage, for example, in comparison to machine learning-based systems. Besides the aforementioned peculiarities, the imple- mentation of argumentation-based healthcare admin- istration systems can have similar implications as the implementation of systems for medical experts in that conflicts between global (e.g., governmental) guidelines, local implementation details, and diverging expert assessments need to be made. Systems y A third type of healthcare information system in which formal argumentation can be applied are administra- tive systems, for example, enterprise resource plan- ning systems that are used in the healthcare domain. To our knowledge, little research exists on the applica- tion of argumentation-based methods for this health- care information system type; one exception is an argumentation-based scheduling tool for nurses.20 A possible reason for the sparsity of related research is that administrative systems are tightly integrated into the information system landscape of an organiza- tion; this increases the engineering effort and organi- zational overhead of implementing prototypes that are sufficiently mature for pilot studies. However, from a use-case perspective, the management of conflict- ing information may be considered useful from an administrative planning perspective; for example, in the wake of the COVID-19 crisis, decisions on how to distribute medical equipment to different administra- tive regions could be informed by infection spread prognoses that are provided by disagreeing epidemiol- ogy experts or based on conflicting base scenarios. IEEE Intelligent Systems Short-lived software artifacts Short-lived software artifacts Short-lived software artifacts CONCLUSION 7. E. Guerrero, J. C. Nieves, and H. Lindgren, “An activity-centric argumentation framework for assistive technology aimed at improving health,” Argument Comput., vol. 7, no. 1, pp. 5–33, 2016. The presented design methodology is a point of de- parture toward a more structured and rigorous application of argumentation-based approaches for intelligent health information systems. The methodol- ogy is an important tool to bridge the divide between the communities that advance the theoretical and engineering foundations of formal argumentation, and researchers who study the application of intelligent systems in the healthcare domain. We expect that the methodology will further mature as the body of research that studies the application of formal argu- mentation to health information systems growths. Also, the presented methodology can be transformed into a domain-independent model, or be adjusted to other system types. 8. E. Guerrero, J. C. Nieves, M. Sandlund, and H. Lindgren, “Activity qualifiers using an argument- based construction,” Knowl. Inf. Syst., vol. 54, no. 3, pp. 633–658, 2018. 9. A. Q. Gill, “Agile enterprise architecture modelling: Evaluating the applicability and integration of six modelling standards,” Inf. Softw. Technol., vol. 67, pp. 196–206, 2015. 10. B. G. M. E. Blobel, K. Engel, and P. Pharow, “ Semantic interoperability—Hl7 Version 3 compared to advanced architecture standards,” Methods Inf. Med., vol. 45, no. 4, pp. 343–353, 2006. 11. M. Lippi and P. Torroni, “Argument mining: A machine learning perspective,” in Theory and Applications of Formal Argumentation, E. Black, S. Modgil, and N. Oren, Eds. Cham, Switzerland: Springer, 2015, pp. 163–176. Lack of strong empirical research Research on argumentation-based health information systems lacks strong empirical evaluation results. This is in contrast to research on traditional decision- support systems, but can potentially be explained by the relative novelty of the argumentation-based approach. Another possible reason for the lack of strong empirical evaluations of argumentation-based health information systems is the fact that the artifi- cial intelligence researchers who are most engaged with the design and implementation of these systems typically lack experience in running long-term empiri- cal assessments of complex sociotechnical systems. Hence, before the last stage of the design process, a handover of the developed system prototypes from artificial intelligence researchers to research groups that focus on the evaluation of information systems can be considered recommendable. 78 IEEE Intelligent Systems March/April 2021 SPECIAL ISSUE ON ARGUMENTATION-BASED REASONING REFERENCES A related problem is the lack of openly shared and well-documented software artifacts (as well as of commercial systems) that emerge from research on the topic. This hinders the development of systems as joined community efforts, and the study and applica- tion of the systems by information systems research- ers who do not have low-level engineering expertise. To increase the chances that the developed systems’ lifetimes do not end when the results are dissemi- nated, a technology transfer plan should be integrated into the initial research outline. Such a plan can, for example, entail the handover of the artifact to health- care authorities, or the extraction of generically useful system components as open-source libraries and frameworks. Realities in the healthcare sector that are prone to limit the adoption of new technology must be acknowledged, i.e., limited resources and health- care professionals’ tight schedules. Also, healthcare professionals may be skeptical when technology is introduced as a replacement and not an augmenta- tion of human care. A related problem is the lack of openly shared and well-documented software artifacts (as well as of commercial systems) that emerge from research on the topic. This hinders the development of systems as joined community efforts, and the study and applica- tion of the systems by information systems research- ers who do not have low-level engineering expertise. 1. P. Baroni, D. M. Gabbay, M. Giacomin, and L. van der Torre, Handbook of Formal Argumentation. London, U.K.: College Publications, 2018. 2. L. Longo, B. Kane, and L. Hederman, “Argumentation theory in health care,” in Proc. 25th IEEE Int. Symp. Comput.-Based Med. Syst., 2012, pp. 1–6. 3. P. M. Dung, “On the acceptability of arguments and its fundamental role in nonmonotonic reasoning, logic programming and n-person games,” Artif. Intell., vol. 77, no. 2, pp. 321–357, 1995. 4. J. Fox et al., “Delivering clinical decision support services: There is nothing as practical as a good theory,” J. Biomed. Informat., vol. 43, no. 5, pp. 831–843, 2010. 5. R. Hoda, N. Salleh, and J. Grundy, “The rise and evolution of agile software development,” IEEE Software, vol. 35, no. 5, pp. 58–63, Sep./Oct. 2018. 6. C. Yan, H. Lindgren, and J. C. Nieves, “A dialogue- based approach for dealing with uncertain and conflicting information in medical diagnosis,” Auton. Agents Multi-Agent Syst., vol. 32, no. 6, pp. 861–885, 2018. [Online]. Available: https://doi.org/10.1007/ s10458-018-9396-x ACKNOWLEDGEMENTS The authors thank the anonymous referees. This work was supported by the Wallenberg AI, Autonomous Systems and Software Program (WASP), funded by the Knut and Alice Wallenberg Foundation and by the European Union’s Horizon 2020 programme, under Grant 825619. 12. L. Amgoud and C. Cayrol, “Inferring from inconsistency in preference-based argumentation frameworks,” J. Automated Reasoning, vol. 29, no. 2, pp. 125–169, 2002. 79 March/April 2021 IEEE Intelligent Systems SPECIAL ISSUE ON ARGUMENTATION-BASED REASONING 13. H. Li, N. Oren, and T. J. Norman, “Probabilistic argumentation frameworks,” in Theory and Applications of Formal Argumentation, S. Modgil, N. Oren, and F. Toni, Eds. Berlin, Germany: Springer, 2012, pp. 1–16. received the Nordea Scientific Award 2018. She serves in committees of several large-scale research programs on AI. Contact her at helena@cs.umu.se. TIMOTHEUS KAMPIK is currently working toward the Ph.D. degree with Umea  University, Umea , Sweden. His research interests are the intersection of non-monotonic reasoning and decision theory, and the engineering of multi-agent sys- tems. He is an avid open-source software contributor and has a decade of experience in the enterprise software indus- try. Contact him at tkampik@cs.umu.se. 14. D. Walton and E. C. W. Krabbe, Commitment in Dialogue: Basic Concepts of Interpersonal Reasoning. Albany, NY, USA: State Univ. New York Press, 1995. 15. I. Rahwan and C. Reed, The Argument Interchange Format. Boston, MA, USA: Springer, 2009, pp. 383–402. 16. L. van der Torre and S. Vesic, “The principle-based approach to abstract argumentation semantics,” IfCoLog J. Logics Appl., vol. 4, no. 8, pp. 2735–2778, Oct. 2017. ESTEBAN GUERRERO ROSERO is currently a Researcher with Umea  University, Umea , Sweden. His research interests include the theory and application of formal argumentation and multiagent systems. He received the Ph.D. degree from Umea  University in 2016. Contact him at esteban@cs.umu.se. 17. P. E. Dunne and M. Wooldridge, Complexity of Abstract Argumentation. Boston, MA, USA: Springer, 2009, pp. 85–104. [Online]. Available: https://doi.org/ 10.1007/978-0-387-98197-0_5 18. A. J. Garcõa and G. R. Simari, “Defeasible logic programming: An argumentative approach,” Theory Pract. Logic Program., vol. 4, no. 1/2, pp. 95–138, 2004. MADELEINE BLUSI is currently a Researcher with the Depart- ment of Community Medicine and Rehabilitation and an Asso- ciate Professor with the Department of Nursing, Umea  University, Umea , Sweden. Her research interests include intelli- gent systems and patient-centered healthcare. She received the Ph.D. degree from Mid Sweden University, €Ostersund, Swe- den, in 2014. ACKNOWLEDGEMENTS Contact her at madeleine.blusi@umu.se. 19. M. Beirlaen, J. Heyninck, P. Pardo, and C. Straûer, “Argument strength in formal argumentation,” J. Appl. Logics—IfCoLog J. Logics Appl., vol. 5, no. 3, pp. 629–675, 2018. 20. K. Cyras, D. Letsios, R. Misener, and F. Toni, “Argumentation for explainable scheduling,” in Proc. AAAI Conf. Artif. Intell., vol. 33, 2019, pp. 2752–2759. JUAN CARLOS NIEVES is currently an Associate Professor with Umea  University, Umea , Sweden. He received the Ph.D. degree from Universitat Politecnica de Catalunya—Barcelona Tech (UPC), Barcelona, Spain, in 2008. His research interests include the theory and application of nonmonotonic reasoning and mul- tiagent systems. Contact him at juan.carlos.nieves@umu.se. HELENA LINDGREN is currently a Professor with Umea  HELENA LINDGREN is currently a Professor with Umea  HELENA LINDGREN is currently a Professor with Umea  University, Umea , Sweden. She received the Ph.D. degree from Umea  University in 2007. She has combined clinical expertise with artificial intelligence research, for which she University, Umea , Sweden. She received the Ph.D. degree from Umea  University in 2007. She has combined clinical expertise with artificial intelligence research, for which she 80 March/April 2021 IEEE Intelligent Systems March/April 2021
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ERICA: prevalence of healthy eating habits among Brazilian adolescents
Revista de saúde pública/Revista de Saúde Pública
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Correspondence: Laura Augusta Barufaldi Edifício Premium Térreo, sala 15 SAF Sul, Trecho 02 Lotes 05/06 Bloco “F” Torre 1 70070-600 Brasília, DF, Brasil E-mail: laurabarufaldi@yahoo.com.br http://www.rsp.fsp.usp.br/ Supplement ERICA Original Article Supplement ERICA Original Article Rev Saúde Pública 2016;50(suppl 1):6s ERICA: prevalence of healthy eating habits among Brazilian adolescents Laura Augusta BarufaldiI, Gabriela de Azevedo AbreuII, Juliana Souza OliveiraIII, Debora França dos SantosII, Elizabeth FujimoriIV, Sandra Mary Lima VasconcelosV, Francisco de Assis Guedes de VasconcelosVI, Bruno Mendes TavaresVII I Departamento de Vigilância de Doenças e Agravos Não Transmissíveis e Promoção da Saúde. Secretaria de Vigilância em Saúde. Ministério da Saúde. Brasília, DF, Brasil g II Instituto Medicina Social. Universidade do Estado do Rio de Janeiro. Rio de Janeiro, RJ, Brasil III Núcleo de Nutrição. Centro Acadêmico de Vitória. Universidade Federal de Pernambuco. Vitória de Santo Antão, PE, Brasil II Instituto Medicina Social. Universidade do Estado do Rio de Janeiro. Rio de Janeiro, RJ, Brasil III Núcleo de Nutrição. Centro Acadêmico de Vitória. Universidade Federal de Pernambuco. Vitória de San Antão, PE, Brasil , , IV Escola de Enfermagem. Universidade de São Paulo. São Paulo, SP, Brasil VI Departamento de Nutrição. Universidade Federal de Santa Catarina. Florianópolis, SC, Brasil VII Instituto de Saúde e Biotecnologia Universidade Federal do Amazonas Coari AM Brasil VI Departamento de Nutrição. Universidade Federal de Santa Catarina. Florianópolis, SC, Brasil VII Instituto de Saúde e Biotecnologia. Universidade Federal do Amazonas. Coari, AM, Brasil VI Departamento de Nutrição. Universidade Federal de Santa Catarina. Florianópolis, SC, Brasil VII Instituto de Saúde e Biotecnologia. Universidade Federal do Amazonas. Coari, AM, Brasil How to cite: Barufaldi LA, Abreu GZ, Oliveira JS, Santos DF, Fujimori E, Vasconcelos SML et al. ERICA: prevalence of healthy eating habits among Brazilian adolescents. Rev Saude Publica. 2016;50(suppl 1):6s. INTRODUCTION Adolescence is a period of intense change, in which individuals suffer influences from socioeconomic conditions, family habits, values, and social and cultural rules. Habits and knowledges acquired during this period influence many aspects of adult life related to food, health, and psychosocial development preferences, among others. Thus, healthy eating habits during adolescence are crucial to a healthy productive and reproductive life and for the prevention of noncommunicable chronic diseases in adult lifea. For Matias et al.14, eating habits comprise a set of actions related to food, starting with the decision, availability, preparation, utensils, eating-schedule and meals division, and finishing with consumption. Having meals with family members, consuming breakfast, and drinking the recommended amount of water are considered healthy habits. Studies suggest a positive association between having meals with family members and ingesting healthy foods, and inverse association between such habit and obesity occurence2,11. The National Survey of School Health (PeNSE)12, performed with Brazilian students of the ninth grade of elementary school, evaluated these children’s habits of having meals in the presence of their mothers or legal guardians, among other variables of food consumption. Two-thirds of the students frequently had at least one of the main meals in the presence of the mother or legal guardian (for five or more days a week), even though about a quarter of them rarely or had never done this12. According to the Dietary Guidelines for the Brazilian Population, breakfast is one of the three main meals of the dayb. Compared to snacks, breakfast provides a greater intake of vitamins and minerals and a lower intake of fat and cholesterol22. Studies suggest a positive relationship between breakfast consumption and a healthy lifestyle19. Scientific evidences relate the frequent consumption of breakfast to lower risks of overweight and abdominal obesity16. On the other hand, the absence of this meal may contribute to calcium deficiency, considering that, generally, breakfast concentrates the highest daily intake of milk and dairy products, sources of the mineral24. Studies conducted in different countries indicate that the proportion of adolescents that consume breakfast ranges from 45.0% to 95.0%24. With the increase in the number of individuals living alone and the lack of time to have meals, the way of life of contemporary society have been associated to the decline of breakfast consumption19. Proper water intake is vital, preventing dehydration and its adverse effects, such as headaches, urinary lithiasis, and prejudices in cognition, among others18. a World Health Organization. Nutrition in adolescence: issues and challenges for the health sector: issues in adolescent health and development. Geneva; 2005 [cited 2015 Jul 31]. (WHO discussion paper on adolescence). Available from: http://apps.who. int/iris/bitstream/10665/43342/1/ 9241593660_eng.pdf b Ministério da Saúde, Secretaria de Atenção à Saúde, Departamento de Atenção Básica. Guia alimentar para a população brasileira. 2.ed. Brasília (DF); 2014. INTRODUCTION Drinking water before meals and in the place of sugary drinks decrease the consumption of energy and prevent obesity and dental caries18,23. Despite the importance of healthy eating habits, studies approaching adolescents are still scarce, which justifies the importance and relevance of this article, whose data represent adolescent students nationally. The aim of this study is to describe the prevalence of healthy eating habits in adolescents according to sex, age, education level of the mother, school type, session of study, and geographic region. ABSTRACT OBJECTIVE: To describe the prevalence of eating habits considered healthy in adolescents according to sex, age, education level of the mother, school type, session of study, and geographic region. METHODS: The assessed data come from the Study of Cardiovascular Risks in Adolescents (ERICA), a cross-sectional, national and school-based study. Adolescents of 1,247 schools of 124 Brazilian municipalities were evaluated using a self-administered questionnaire with a section on aspects related to eating behaviors. The following eating behaviors were considered healthy: consuming breakfast, drinking water, and having meals accompanied by parents or legal guardians. All prevalence estimates were presented proportionally, with their respective 95% confidence intervals. The Chi-square test was used to evaluate the differences in healthy eating habits prevalences according to other variables. The module survey of the Stata program version 13.0 was used to analyze complex data. RESULTS: We evaluated 74,589 adolescents (72.9% of the eligible students). Of these, 55.2% were female, average age being 14.6 years (SD = 1.6). Among Brazilian adolescents, approximately half of them showed healthy eating habits when consuming breakfast, drinking five or more glasses of water a day, and having meals with parents or legal guardians. All analyzed healthy eating habits showed statistically significant differences by sex, age, type of school, session of study, or geographic region. Received: 9 Set 2015 Approved: 4 Nov 2015 How to cite: Barufaldi LA, Abreu GZ, Oliveira JS, Santos DF, Fujimori E, Vasconcelos SML et al. ERICA: prevalence of healthy eating habits among Brazilian adolescents. Rev Saude Publica. 2016;50(suppl 1):6s. CONCLUSIONS: We suggest that specific actions of intersectoral approach are implemented for the dissemination of the benefits of healthy eating habits. Older female adolescents (15 to 17 years old) who studied in public schools, resided in the Southeast region, and whose mothers had lower education levels, should be the focus of these actions since they present lower frequencies concerning the evaluated healthy habits. Copyright: 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 that the original author and source are credited. DESCRIPTORS: Adolescent. Food Habits. Food Preferences. Health Behavior. Cross-Sectional Studies. 1s DOI:10.1590/S01518-8787.2016050006678 1s 1 DOI:10.1590/S01518-8787.2016050006678 1s Healthy eating habits among adolescents Barufaldi LA et al INTRODUCTION METHODS This is an analysis of data from the Study of Cardiovascular Risks in Adolescents (ERICA). The ERICA is a cross-sectional, national and school-based study that aimed to estimate the prevalence of diabetes mellitus, obesity, cardiovascular risk factors, and inflammatory and insulin resistance markers in adolescents aged from 12 to 17 years, enrolled in public and private schools of Brazilian municipalities with more than 100,000 inhabitants. 2s Healthy eating habits among adolescents Barufaldi LA et al Data collection took place between March 2013 and December 2014. About 74,589 adolescents of 1,247 schools from 124 Brazilian municipalities were evaluated. The population under research was stratified in 32 strata comprising 27 capitals and five sets of municipalities with more than 100,000 inhabitants in each of the five geographical macro-regions of the Country. For each geographic stratum, schools were selected with probability proportional to size and inversely proportional to the distance from the capital. The sample is representative for medium and large municipalities (> 100,000 inhabitants) at national and regional level, and also for all Brazilian capitals. More details on the sampling process can be found in the publication by Vasconcellos et al.25 Data collection took place between March 2013 and December 2014. About 74,589 adolescents of 1,247 schools from 124 Brazilian municipalities were evaluated. The population under research was stratified in 32 strata comprising 27 capitals and five sets of municipalities with more than 100,000 inhabitants in each of the five geographical macro-regions of the Country. For each geographic stratum, schools were selected with probability proportional to size and inversely proportional to the distance from the capital. The sample is representative for medium and large municipalities (> 100,000 inhabitants) at national and regional level, and also for all Brazilian capitals. More details on the sampling process can be found in the publication by Vasconcellos et al.25 All students of the selected classes that signed the assent form were interviewed and examined. Adolescents that did not belong to the age group of 12 to 17 years, pregnant girls, and individuals with physical or mental disabilities (temporary or permanent) were considered not eligible and were excluded from the analysis. This study was approved by the Research Ethics Committee of the central coordinating institution of study (IESC/UFRJ) and of each Brazilian State. Bloch et al.3 presents details concerning the protocol of this study. METHODS The self-administered questionnaire used was applied with the use of an electronic data collector, the personal digital assistant (PDA). It contained approximately 100 questions divided into 11 sections: sociodemographic aspects, occupational activities, physical practices, eating habits, smoking habits, use of alcoholic beverages, reproductive health, oral health, referred morbidity, sleeping hours, and common mental disorders. The following eating habits were considered healthy: consuming breakfast, drinking water, and having meals with parents or legal guardians. The section about eating habits included questions on breakfast and on the company of parents or legal guardians during meals as lunch and dinner, with the following answer options: “no”, “sometimes”, “almost every day” and “every day”. For the analysis, we grouped the responses to “almost every day” and “every day”, thus obtaining a variable with the options: “I do not consume it”; “sometimes I consume it” and “I consume it almost/every day”. We created the variable “meals with parents or legal guardians” by combining the variables “I never have lunch or dinner with my parents or legal guardians” presenting the following answer options: “I never have meals with my parents or legal guardians”; “sometimes I have one or two meals with my parents or legal guardians” and “I often (or always) have at least one meal with my parents or legal guardians”. Another habit evaluated in the same questionnaire was daily water intake, by considering as answer options: “I do not drink water”, “I drink from one to two glasses”, “I drink from three to four glasses” and “I drink at least five glasses per day”. The prevalence of healthy habits related to food was analyzed according to the following variables: geographic region (North, Northeast, Midwest, Southeast, and South), sex (male or female), age (in years, analyzed categorically: from 12 and 14, and from 15 to 17), education level of the mother (analyzed categorically: incomplete high school, complete high school or more), session of study (morning or afternoon sessions), and type of school (public or private). Data analyses were performed in the Strata software, version 13.0, using the module survey for the analysis of complex sample data, in which prevalence estimates were presented proportionally (%), with their respective 95% confidence intervals (95%CI). The Chi-square test was used to evaluate the differences in healthy eating habits prevalences according to other variables. 3s DOI:10.1590/S01518-8787.2016050006678 RESULTS Of the 102,327 eligible students, we evaluated about 74,589, which represent 72.9% of the eligible students enrolled in schools and classes previously selected during the sampling process. The study coverage was higher in females (75.8%) than in males (69.6%), and also Of the 102,327 eligible students, we evaluated about 74,589, which represent 72.9% of the eligible students enrolled in schools and classes previously selected during the sampling process. The study coverage was higher in females (75.8%) than in males (69.6%), and also 3s Healthy eating habits among adolescents Barufaldi LA et al higher in younger adolescents (77.6%) when compared to those of a higher age group (66.4%). The geographic region where coverage was the highest was the South (81.0%), while the region with the smallest coverage was the Midwest (68.3%). Of the evaluated adolescents, 55.2% were female with average age being 14.6 years (SD = 1.6). Regarding the type of school, 78.7% were public and 71.5% of the students studied in the morning session. About 31.0% of the adolescents evaluated resided in the Northeast, 22.9% in the Southeast, 20.2% in the North, 13.0% in the Midwest, and 12.8% in the South. More than two-thirds of adolescents (68.0%) “often (or always)” had meals with their parents or legal guardians. However, almost 25.0% had meals in the company of their parents or legal guardians only “sometimes”, while 7.4% declared to “never” do it (Table 1). The prevalence of such habit presented statistically significant difference for all the variables studied, except for study session. We observed higher prevalences of consumption of meals in the presence of parents or legal guardians among younger male adolescents that studied in private schools, resided in the South and Midwest regions, and whose mothers had higher education level (Table 1). Table 1. Prevalence of meals with their parents or legal guardians in Brazilian adolescents aged between 12 and 17 years, considering complex sampling. ERICA, 2013-2014. * Statistically significant difference – Chi-square test. RESULTS (N = 74,589) Variable Prevalences Never Sometimes Often/always % 95%CI % 95%CI % 95%CI Sex* Female 7.9 7.3-8.5 26.7 25.6-27.8 65.4 64.2-66.6 Male 6.9 6.3-7.6 22.4 21.5-23.5 70.6 69.3-71.9 Age (years)* 12-14 5.7 5.1-6.4 21.6 20.7-22.5 72.7 71.3-74.0 15-17 9.3 8.6-9.9 27.9 26.8-28.9 62.9 61.6-64.1 Education level of the mother Incomplete high school 7.5 6.6-8.5 25.5 24.1-27.0 67.0 64.9-69.0 Complete high school or more 7.2 6.4-7.9 22.4 21.2-23.7 70.5 68.8-72.0 Type of school* Public 7.5 7.0-8.1 25.3 24.5-26.1 67.2 66.1-68.2 Private 6.7 5.3-8.4 21.1 19.2-23.2 72.2 69.1-75.1 Session of study Morning 7.4 6.8-8.1 24.6 23.7-25.7 67.9 66.5-69.3 Afternoon 7.3 6.6-8.1 24.3 22.9-25.8 68.3 66.6-70.0 Geographical region* North 6.9 6.4-7.4 25.3 24.3-26.3 67.8 66.7-68.9 Northeast 8.6 7.7-9.5 29.2 27.8-30.6 62.3 60.6-63.9 Midwest 7.0 6.1-8.1 21.2 19.8-22.5 71.8 70.2-73.1 Southeast 7.4 6.6-8.3 24.8 23.5-26.2 67.8 65.9-69.6 South 5.8 4.9-6.7 16.9 15.6-18.2 77.3 75.9-78.6 Total 7.4 6.9-7.9 24.6 23.8-25.3 68.0 67.0-69.0 * Statistically significant difference – Chi-square test. Table 1. Prevalence of meals with their parents or legal guardians in Brazilian adolescents aged between 12 and 17 years, considering complex sampling. ERICA, 2013-2014. (N = 74,589) Healthy eating habits among adolescents Barufaldi LA et al As for the consumption of breakfast, nearly half of the adolescents (48.5%) reported consuming breakfast frequently (or always), but more than one-fifth (21.9%) usually do not eat this meal (Table 2). We observed a statistically significant difference in the prevalence of breakfast consumption for all the variables analyzed: the greatest prevalences were among younger male adolescents (12 to 14 years), who studied in private schools during the afternoon session, resided in the North and Northeast regions, and whose mothers had higher education (Table 2). Regarding water intake, about half of the adolescents (48.2%) reported drinking five or more glasses of water per day, while 18.9% only drink from one to two glasses, and 1.6% reported not drinking water at all (Table 3). Prevalences of such habit presented significant differences according to sex, age, and geographic region. Younger adolescent boys residing in the Northern region presented the habit of consuming more water daily. Table 2. Prevalence of breakfast consumption in Brazilian adolescents aged between 12 and 17 years, considering complex sampling. ERICA, 2013-2014. RESULTS (N = 74,589) Variable Prevalence Never Sometimes Often/Always % 95%CI % 95%CI % 95%CI Sex* Female 25.8 24.5-27.2 31.8 30.7-33.0 42.3 40.6-44.0 Male 18.2 17.0-19.4 27.2 26.2-28.3 54.6 53.1-56.1 Age (years)* 12-14 20.1 18.8-21.6 28.3 27.0-29.5 51.6 50.0-53.1 15-17 24.0 22.5-25.7 30.9 29.6-32.3 45.0 42.7-47.4 Education level of the mother Incomplete high school 21.9 20.6-23.3 30.3 28.8-31.8 47.8 45.7-49.9 Complete high school or more 20.9 19.8-21.9 27.7 26.4-29.0 51.5 50.1-52.8 Type of school* Public 21.9 20.7-23.1 30.9 30.0-31.9 47.2 45.5-48.9 Private 22.5 20.4-24.9 22.8 20.7-25.1 54.6 51.9-57.3 Session of study Morning 26.4 25.3-27.7 30.3 29.0-31.7 43.2 41.3-45.1 Afternoon 12.2 11.2-13.3 27.7 26.6-28.9 60.0 58.2-61.8 Geographical region* North 13.7 12.6-15.0 25.5 24.2-26.8 60.8 58.6-62.9 Northeast 18.2 16.3-20.2 27.2 25.7-28.6 54.7 52.5-56.8 Midwest 22.3 20.2-24.6 35.2 32.9-37.5 42.6 40.1-45.1 Southeast 23.2 21.4-25.2 30.0 28.5-31.6 46.7 44.2-49.2 South 29.1 26.9-31.4 30.8 28.6-33.0 40.1 36.7-43.6 Total 21.9 20.9-23.1 29.5 28.6-30.4 48.5 47.0-49.9 * Statistically significant difference – Chi-square Test. Table 2. Prevalence of breakfast consumption in Brazilian adolescents aged between 12 and 17 years, considering complex sampling. ERICA, 2013-2014. (N = 74,589) Healthy eating habits among adolescents Barufaldi LA et al Table 3. Prevalence of daily water intake in Brazilian adolescents aged between 12 and 17 years, considering complex sampling. ERICA, 2013-2014. RESULTS (N = 74,589) Variable Prevalence I do not drink water 1 to 2 glasses 3 to 4 glasses 5 or more glasses % 95%CI % 95%CI % 95%CI % 95%CI Sex* Female 2.3 1.9-2.6 24.0 22.8-25.3 33.1 31.9-34.2 40.6 39.2-42.0 Male 0.9 0.7-1.0 13.9 12.8-15.0 29.5 28.2-30.8 55.7 53.9-57.5 Age (years)* 12-14 1.4 1.1-1.6 17.8 16.9-18.6 30.7 29.7-31.7 50.1 48.9-51.4 15-17 1.8 1.5-2.1 20.2 18.9-21.6 31.9 30.6-33.3 46.0 44.2-47.9 Education level of the mother Incomplete high school 1.5 1.2-1.8 18.9 17.4-20.5 32.0 30.7-33.3 47.6 45.9-49.5 Complete high school or more 1.4 1.2-1.8 17.2 16.1-18.3 30.9 30.5-33.2 49.5 47.8-51.2 Type of school* Public 1.6 1.4-1.8 19.3 18.3-20.3 30.8 29.8-31.8 48.4 47.0-49.7 Private 1.5 0.99-2.2 17.4 15.6-19.4 33.7 31.7-35.8 47.4 44.1-50.7 Session of study Morning 1.7 1.4-1.9 19.2 18.2-20.3 31.7 30.6-32.9 47.4 45.9-48.8 Afternoon 1.4 1.1-1.7 18.3 16.9-19.8 30.3 28.8-31.7 50.0 47.8-52.2 Geographical region* North 0.3 0.2-0.4 8.9 8.3-9.7 25.1 24.1-26.1 65.7 64.6-66.8 Northeast 0.5 0.3-0.6 12.7 11.2-14.3 29.8 27.5-32.2 57.0 53.7-60.3 Midwest 0.9 0.6-1.2 1.9 14.1-17.3 31.0 29.7-32.4 52.5 50.4-54.6 Southeast 1.8 1.5-2.1 21.2 19.8-22.6 33.1 31.7-34.5 43.9 42.1-45.8 South 4.1 3.2-5.2 30.0 27.8-32.3 30.7 28.9-32.6 35.1 33.5-36.8 Total 1.6 1.4-1.8 18.9 18.1-19.8 31.3 30.4-32.2 48.2 47.0-49.4 ll fi d ff h Table 3. Prevalence of daily water intake in Brazilian adolescents aged between 12 and 17 years, considering complex sampling. ERICA, 2013-2014 (N = 74,589) Table 3. Prevalence of daily water intake in Brazilian adolescents aged between 12 and 17 years, considering complex sampling. ERICA, 2013-2014. (N = 74,589) DISCUSSION Among Brazilian adolescents, approximately half of them showed healthy eating habits when it comes to having meals with their parents or legal guardians, consuming breakfast, and drinking five or more glasses of water per day. However, a significant percentage reported never (or only sometimes) having meals with their parents, never consuming breakfast, and not drinking water or just drinking from one to two glasses daily. All the analyzed healthy eating habits showed statistically significant differences according to sex, age, type of school, session of study, or geographic region. The results showed that younger male adolescents who studied in private schools, resided in the South and Midwest regions, and whose mothers had higher education, had more meals with their parents or legal guardians. We observed that the greatest prevalences of breakfast consumption, by its turn, were among younger male adolescents who studied in private schools, during the afternoon session, resided in the North and Northeast regions of Brazil, and whose mothers 6s DOI:10.1590/S01518-8787.2016050006678 Healthy eating habits among adolescents Barufaldi LA et al had higher education level. Water intake was higher among younger male adolescents who resided in the North region. Having family meals is an important aspect of the familiar environment that promotes healthy eating habits in adolescence and its maintenance in adulthood. It also contributes to the reduction of unhealthy eating practices, influencing positively increased consumption of fruits, vegetables, and dairy products and decreased consumption of sugary drinks7. Despite not having a consensus on the subject, some records consider family meals as contributors to the prevention of obesity. Results of the meta-analyses showed that children and adolescents that have regular family meals were less likely to be overweight7. In addition to healthy eating habits and obesity prevention, family meals can help to reduce violence, sexual activity, mental health problems, and self-injury among children and adolescents6,21. They can also promote positive family interactions, including greater communication, socialization, and transmission of values and culture10. The frequency of meals with parents or legal guardians observed in this study (68.0%) is similar to that found by PeNSE12, which found that 62.6% of adolescents had (at least) lunch or dinner with the mother or legal guardian. DISCUSSION The association with the variable sex also showed to be similar to the results from PeNSE, which observed a higher prevalence of this habit among males, but without distinction between public and private school students12, differently to what we observed in this study. The greatest prevalence of breakfast by male adolescents reaffirms the results found in other studies9,20. To Keski-Rahkonen et al.9, the smallest prevalence among girls may be due to body dissatisfaction and the attempt to lose weight; these authors found associations between having breakfast and having breakfast accompanied by the parents and not having this meal with low education levels at age 16, smoking habits, frequent use of alcohol beverages, little exercise and high body mass indices. The inverse relation we found in this study between breakfast and age is similar to the observed in other studies. Breakfast consumption seems to increase with age when it comes to adults (between 18 and 60 years)4 and decrease with age in children and adolescents (between four and 18 years)19. The main excuses that adolescents who do not have breakfast give are: “lack of time”, “lack of hunger”, “intention of dieting” and “preference for sleeping”24. Adolescents from private schools and children of mothers with higher education (complete high school or more) consume breakfast and meals with parents or legal guardians with greater frequency. This indicates that socioeconomic status may be related to these eating habits, i.e., adolescents from higher socioeconomic levels present healthier behaviors. Although water balance involves complex mechanisms, an adequate supply of water represents a health practice as important as the adoption of healthy eating (being also considered part of it)8. Moreover, drinking water can help controlling weight15,23 and the consumption of sugary drinks; consequently, it prevents problems associated with the consumption of these drinks, such as dental caries1, obesity5, and type 2 diabetes13. Park et al.17 observed that 54.0% of high school students from the United States reported drinking water less than three times a day. The authors also found a significant association between low intakes of pure water and the following factors: lower consumption of milk, fruits (including 100% fruit juices), and vegetables; higher consumption of soft drinks, other sugary drinks, and fast food; and physical inactivity. Despite quantifying water intake differently, the results of this study suggest that Brazilian adolescents present a healthier level of water intake than American adolescents. 7s DOI:10.1590/S01518-8787.2016050006678 REFERENCES 1. Armfield JM, Spencer AJ, Roberts-Thomson KF, Plastow K. Water fluoridation and the association of sugar-sweetened beverage consumption and dental caries in Australian children. Am J Public Health. 2013;103(3):494-500. DOI:10.2105/AJPH.2012.300889 2. Berge JM, Jin SW, Hannan P, Neumark-Sztainer D. Structural and interpersonal characteristics of family meals: associations with adolescent body mass index and dietary patterns. J Acad Nutr Diet. 2013;113(6):816-822. DOI:10.1016/j.jand.2013.02.004 3. Bloch KV, Szklo M, Kuschnir MCC, Abreu GA, Barufaldi LA, Klein CH et al. The study of cardiovascular risk in adolescents - ERICA: rationale, design and sample characteristics of a national survey examining cardiovascular risk factor profile in Brazilian adolescents. BMC Public Health. 2015;15:94-103. DOI:10.1186/s12889-015-1442-x 4. Carson TA, Siega-Riz AM, Popkin BM. The importance of breakfast meal type to daily nutrient intake: differences by age and ethnicity. Cereal Foods World. 1999;44(6):414- 22. 5. Ebbeling CB, Feldman HA, Chomitz VR, Antonelli TA, Gortmaker SL, Osganian SK et al. A randomized trial of sugar-sweetened beverages and adolescent body weight. N Engl J Med. 2012;367:1407-16. DOI:10.1056/NEJMoa1203388 6. Goldfarb S, Tarver WL, Sen B. Family structure and risk behaviors: the role of the family meal in assessing likelihood of adolescent risk behaviors. Psychol Res Behav Manag. 2014;7:53-66. DOI:10.2147/PRBM.S40461 7. Hammons AJ, Fiese BH. Is frequency of shared family meals related to the nutritional health of children and adolescents? Pediatrics. 2011;127(6):e1565–74. DOI:10.1542/peds.2010-1440 8. Jéquier E, Constant F. Water as an essential nutrient: the physiological basis of hydration. Eur J Clin Nutr. 2010;64(2):115-23. DOI:10.1038/ejcn.2009.111 9. Keski-Rahkonen A, Kaprio J, Rissanen A, Virkkunen M, Rose RJ. Breakfast skipping and health- compromising behaviors in adolescents and adults. Eur J Clin Nutr. 2003;57(7):842-53. DOI:10.1038/sj.ejcn.1601618 10. Larson RW, Branscomb KR, Wiley AR. Forms and functions of family mealtimes: multidisciplinary perspectives. New Dir Child Adolesc Dev. 2006;2006(111):1-15. DOI:10.1002/cd.152 11. Larson N, Mac Lehose R, Fulkerson JA, Berge JM, Story M, Neumark-Sztainer D. Eating breakfast and dinner together as a family: associations with sociodemographic characteristics and implications for diet quality and weight status. J Acad Nutr Diet. 2013;113(12):1601-9. DOI:10.1016/j.jand.2013.08.011 12. Levy RB, Castro IRR, Cardoso LO, Tavares LF, Sardinha LMV, Gomes FS et al. Consumo e comportamento alimentar entre adolescentes brasileiros: Pesquisa Nacional de Saúde do Escolar (PeNSE), 2009. Cienc Saude Coletiva. 2010;15 Supl 2:3085-97. DOI:10.1590/S1413-81232010000800013 13. Malik VS, Popkin BM, Bray GA, Després JP, Willett WC, Hu FB. Sugar-sweetened beverages and risk of metabolic syndrome and type 2 diabetes: a meta-analysis. Diabetes Care. 2010;33(11):2477-83. DOI:10.2337/dc10-1079 14. DISCUSSION Considering the size of the sample and the representativeness of the study, ERICA brings important contributions to the mapping of healthy eating habits among adolescents. However, for being a cross-sectional study, it makes possible extrapolations on causal factors. Whereas adolescence represents a chance for the prevention of chronic diseases related to nutrition in adulthooda, the results of this study suggest that specific actions of intersectoral approach are necessary. They should be implemented to spread the benefits of having meals Healthy eating habits among adolescents Barufaldi LA et al with the family, consuming all meals (especially breakfast), and drinking water, thus building healthy eating habits that tend to remain during adulthood. Older female adolescents (15 to 17 years) who studied in public schools, resided in the Southeast region, whose mothers had lower education levels, should be the focus of these actions since they present lower frequencies concerning the evaluated healthy habits. We recommend future investigations to assess the relationship between healthy eating habits employing anthropometric, biochemical, and food consumption data. REFERENCES Matias CT, Fiore EG. Mudanças no comportamento alimentar de estudantes do curso de nutrição em uma instituição particular de ensino superior. Nutrire Rev Soc Bras Aliment Nutr. 2010;35(2):53-66. 15. Muckelbauer R, Barbosa CL, Mittag T, Burkhardt K, Mikelaishvili N, Müller-Nordhorn J. Association between water consumption and body weight outcomes in children and adolescents: a systematic review. Obesity (Silver Spring). 2014;22(12):2462-75. DOI:10.1002/oby.20911 8 DOI:10.1590/S01518-8787.2016050006678 8s Healthy eating habits among adolescents Barufaldi LA et al 16. Nurul-Fadhilah A, Teo PS, Huybrechts I, Foo LH. Infrequent breakfast consumption associated with higher body adiposity and abdominal obesity in Malaysian school-aged adolescents. PLoS ONE. 2013;8(3): e59297. DOI:10.1371/journal.pone.0059297 16. Nurul-Fadhilah A, Teo PS, Huybrechts I, Foo LH. Infrequent breakfast consumption associated with higher body adiposity and abdominal obesity in Malaysian school-aged adolescents. PLoS ONE. 2013;8(3): e59297. DOI:10.1371/journal.pone.0059297 17. Park S, Blanck HM, Sherry B, Brener N, O’Toole T. Factors associated with low water intake among US high school students - National Youth Physical Activity and Nutrition Study, 2010. J Acad Nutr Diet. 2012;112(9):1421-7. DOI:10.1016/j.jand.2012.04.014 18. Popkin B, D’Anci K, Rosenberg I. Water, hydration and health. Nutr Rev. 2010;68(8):439-58. DOI:10.1111/j.1753-4887.2010.00304.x 19. Rampersaud GC, Pereira MA, Girard BL, Adams J, Metzl JD. Breakfast habits, nutritional status, bodyweight, and academic performance in children and adolescents. J Am Diet Assoc. 2005;105(5):743-60;quiz 61-2. DOI:10.1016/j.jada.2005.02.007 20. Rampersaud GC. Benefits of breakfast for children and adolescents: update and recommendations for practitioners. Am J Lifestyle Med. 2009;3(2):86-103. DOI:10.1177/1559827608327219 21. Skeer MR, Ballard EL. Are family meals as good for youth as we think they are? A review of the literature on family meals as they pertain to adolescent risk prevention. J Youth Adolesc. 2013;42(7):943-63. DOI:10.1007/s10964-013-9963-z 22. Sugiyama S, Okuda M, Sasaki S, Kunitsugu I, Hobara T. Breakfast habits among adolescents and their association with daily energy and fish, vegetable, and fruit intake: a community-based cross-sectional study. Environ Health Prev Med. 2012;17(5):408-14. DOI:10.1007/s12199-012-0270-1 23. Tate DF, Turner-McGrievy G, Lyons E, Stevens J, Erickson K, Polzien K et al. Replacing caloric beverages with water or diet beverages for weight loss in adults: main results of the Choose Healthy Options Consciously Everyday (CHOICE) randomized clinical trial. Am J Clin Nutr. 2012;95(3):555-63. DOI:10.3945/ajcn.111.026278 24. Trancoso SC, Cavalli SB, Proença RPC. Café da manhã: caracterização, consumo e importância para a saúde. Rev Nutr. 2010;23(5):859-69. DOI:10.1590/S1415-52732010000500016 25. Vasconcellos MTL, Silva PLN, Szklo M, Kuschnir MCC, Klein CH, Abreu GA et al. REFERENCES Desenho da amostra do Estudo do Risco Cardiovascular em Adolescentes (ERICA). Cad Saude Publica. 2015;31(5):921-30. DOI:10.1590/0102-311X00043214 Funding: Department of Science and Technology of the Secretary of Science, Technology and Strategic Inputs of the Brazilian Ministry of Health (Decit/SCTIE/MS); Health Sector Fund (CT-Saúde) of the Brazilian Ministry of Science, Technology and Innovation (MCTI) (FINEP Protocol: 01090421 and CNPq 565037/2010-2 and 405.009/2012-7). The author Santos DF is a fellow of CNPq (Process 444138 / 2014-5). Author’s Contribution: LAB participated in the design and planning of the study, in the analysis and interpretation of data, and in the drafting and approval of the final version of the manuscript. GAA participated in the design and planning of the study and of the drafting and approval of the final version of the unpublished study. JSO, DFS, EF, SMLV, FAGV, and BMT joined the drafting and the approval of the unpublished study. Acknowledgements: To the Brazilian Institute of Geography and Statistics (IBGE) for the disposal of electronic data collectors, the PDA (personal digital assistant), and to the Fundação Universitária José Bonifácio (FUJB) for managing the resources of this study. Conflict of Interest: The authors declare no conflict of interest.
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Barcoding Chrysomelidae: a resource for taxonomy and biodiversity conservation in the Mediterranean Region
ZooKeys
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http://zoobank.org/4D7CCA18-26C4-47B0-9239-42C5F75E5F42 Citation: Magoga G, Sassi D, Daccordi M, Leonardi C, Mirzaei M, Regalin R, Lozzia G, Montagna M (2016) Barcoding Chrysomelidae: a resource for taxonomy and biodiversity conservation in the Mediterranean Region. In: Jolivet P, Santiago-Blay J, Schmitt M (Eds) Research on Chrysomelidae 6. ZooKeys 597: 27–38. doi: 10.3897/ zookeys.597.7241 * these authors contributed equally Copyright Giulia Magoga et al. This is an open access article distributed under the terms of the Creative Commons Attribution License (CC BY 4.0), which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited. onservation... 27 Launched to accelerate biodiversity research A peer-reviewed open-access journal onservation... 27 Launched to accelerate biodiversity research A peer-reviewed open-access journal Barcoding Chr ZooKeys 597: 27–38 (2016) doi: 10.3897/zookeys.597.7241 http://zookeys.pensoft.net Barcoding Chry ZooKeys 597: 27–38 (2016) doi: 10.3897/zookeys.597.7241 http://zookeys.pensoft.net esource for taxonomy a RESEARCH ARTICLE Barcoding Chrysomelidae: a resource for ta and biodiversity conservation in the Mediterranean Region Giulia Magoga1,*, Davide Sassi2, Mauro Daccordi3, Carlo Leonardi4, Mostafa Mirzaei5, Renato Regalin6, Giuseppe Lozzia7, Matteo Montagna7,* Giulia Magoga1,*, Davide Sassi2, Mauro Daccordi3, Carlo Leonardi4, Mostafa Mirzaei5, Renato Regalin6, Giuseppe Lozzia7, Matteo Montagna7,* 1 Via Ronche di Sopra 21, 31046 Oderzo, Italy 2 Centro di Entomologia Alpina–Università degli Studi di Milano, Via Celoria 2, 20133 Milano, Italy 3 Museo Civico di Storia Naturale di Verona, lungadige Porta Vittoria 9, 37129 Verona, Italy 4 Museo di Storia Naturale di Milano, Corso Venezia 55, 20121 Milano, Italy 5 Department of Plant Protection, College of Agriculture and Natural Resources–University of Tehran, Karaj, Iran 6 Dipartimento di Scienze per gli Alimenti, la Nutrizione e l’Ambiente–Università degli Studi di Milano, Via Celoria 2, 20133 Milano, Italy 7 Dipartimento di Scienze Agrarie e Ambientali–Università degli Studi di Milano, Via Celoria 2, 20133 Milano, Italy Corresponding authors: Matteo Montagna (matteo.montagna@unimi.it) Corresponding authors: Matteo Montagna (matteo.montagna@unimi.it) Corresponding authors: Matteo Montagna (matteo.montagna@unimi.it) cademic editor: J. Santiago-Blay  |  Received 20 November 2015  |  Accepted 30 January 2016  |  Published 9 June Academic editor: J. Santiago-Blay  |  Received 20 November 2015  |  Accepted 30 January 2016  |  Published 9 Ju http://zoobank.org/4D7CCA18-26C4-47B0-9239-42C5F75E5F42 Keywords f b l y eaf beetles, molecular taxonomy, DNA barcoding, Cytochrome c oxidase subunit 1, C-bar project Leaf beetles, molecular taxonomy, DNA barcoding, Cytochrome c oxidase subunit 1, C-bar project Abstracth The Mediterranean Region is one of the world’s biodiversity hot-spots, which is also characterized by high level of endemism. Approximately 2100 species of leaf beetle (Coleoptera; Chrysomelidae) are known from this area, a number that increases year after year and represents 5/6% of the known species. These features, associated with the urgent need to develop a DNA-based species identification approach for a broad spectrum of leaf beetle species, prompted us to develop a database of nucleotide sequences, with a solid taxonomic background, for all the Chrysomelidae Latreille, 1802 sensu latu inhabiting the Mediter­ ranean region. The Mediterranean Chrysomelidae Barcoding project, which has started in 2009, involves more than fifty entomologists and molecular biologists from different European countries. Numerous collecting campaigns have been organized during the first seven years of the project, which led to the col­ Giulia Magoga et al. / ZooKeys 597: 27–38 (2016) 28 lection of more than 5000 leaf beetle specimens. In addition, during these collecting campaigns two new allochthonous species for Europe, namely Ophraella communa LeSage, 1986 and Colasposoma dauricum Mannerheim, 1849, were intercepted and some species new to science were discovered (e.g., Pachybrachis sassii Montagna, 2011 and Pachybrachis holerorum Montagna et al., 2013). DNA was extracted from 1006 specimens (~13% of the species inhabiting the Mediterranean region) and a total of 910 cox1 gene sequences were obtained (PCR amplification efficiency of 93.8%). Here we report the list of the bar­ coded subfamilies, genera and the number of species for which cox1 gene sequences were obtained; the metadata associated with each specimen and a list of problematic species for which marker amplification failed. In addition, the nucleotide divergence within and between species and genera was estimated and values of intraspecific nucleotide divergence greater than the average have been discussed. Cryptocephalus quadripunctatus G. A. Olivier, 1808, Cryptocephalus rugicollis G. A. Olivier, 1791 and Exosoma lusitanicum Linnaeus, 1767) are representatives of these cases. Introduction In the last decades we have witnessed what has been defined as the “taxonomy impedi­ ment” (Rodman and Cody 2003) indicating the crisis in taxonomic studies due primar­ ily to a shortage of time and taxonomists (Wheeler 2004, Wheeler et al. 2004, Wilson 2004), a situation that is made even more critical due to the decrease in the funding of natural history studies. The causes of the taxonomy crisis are many and complex, and a comprehensive analysis of this situation is beyond our purpose (see as example Boero 2001, Tautz et al. 2003). In our view, the causes can be described by the sentence …a lack of prestige and resources that is crippling the continuing cataloguing of biodiversity (God­ fray 2002). If we consider the increased rate of species extinction (Thomas et al. 2004) amplified by climate change and habitat erosion due to exploitation by human beings the situation is worsened. A DNA-based strategy, which plays a central role in modern taxo­ nomic studies, has been proposed by different authors as a methodology to overcome the identified problems (Tautz et al. 2002, Tautz et al. 2003, Hebert et al. 2004, Goldstein and DeSalle 2010) whilst maintaining the importance of a traditional approach mainly based on morphology. Interestingly, in a survey conducted among Coleopteran taxono­ mists, taxonomic initiatives based on DNA have been regarded of potential utility in solving the “taxonomy impediment”, even if a few consider it absolutely useless (Löbl 2005). Currently, in the scientific world, an agreement on the correct approach to be adopted has not yet been reached. The “gold standard” for species identification studies based on molecular markers (e.g. mitochondrial cytochrome oxidase subunit I–cox1, or the nuclear small ribosomal subunit–SSU 18S rRNA) is to develop sequence databases used as a reference, beginning with DNA extracted from type and type series specimens preserved in Museum dry collections. The main problem with this strategy is related to the conservation status of the old dry specimens; 18th and 19th century specimens have fragmented DNA (not easily amplified through standard PCR approaches targeting Barcoding Chrysomelidae: a resource for taxonomy and biodiversity conservation... 29 fragments of 500-700 bp) and are often infested by fungal hypha, which contaminate the insect’s genomic DNA. Even with the advent of high-throughput sequencing tech­ nologies to solve the problem of fragmented sequences, the contamination due to fungal DNA remains. Introduction Developing strategies for the acquisition and storage of molecular data to address molecular taxonomy purposes, we face another problem, which affects the DNA sequences deposited in publicly available databases, i.e. the accuracy of specimen identi­ fication. In light of these issues, an alternative strategy has been adopted in the Mediter­ ranean Chrysomelidae Barcoding project (C-Bar). The aim of the C-Bar project is to develop a reference database of cox1 gene sequences for all the Chrysomelidae (excluding Bruchinae Latreille, 1802), the Megalopodidae Latreille, 1802 and the Orsodacnidae Thomson, 1859 (hereafter indicated as Chrysomelidae or leaf beetles sensu latu – s. l.) inhabiting the Mediterranean region. The study area of C-Bar includes all the states that possess coastline on the Mediterranean Sea or territories characterized by Mediterranean- type habitat plus Romania and Switzerland (Figure 1). Starting from the Catalogue of Palaearctic Coleoptera (Löbl and Smetana 2010), about 2100 species of Chrysomelidae s. l. (corresponding to an estimated 5/6% of all described species) are present in this area. The Mediterranean Region is one of the world’s biodiversity “hot-spots” (Myers et al. 2000, Cuttelod et al. 2008), which is characterized by exceptional concentrations of species with high levels of endemism that inhabit one of the most populated areas. The assumption of high levels of endemic species inhabiting the Mediterranean Region is also valid for leaf beetles (Biondi et al. 2013, Sassi 2006). Although the Mediterranean region has been the subject of investigation by generations of entomologists, knowledge of Chrysomelidae inhabiting this area is far from being fully known. The number of leaf beetle species new to science described from the Mediterranean region in the last dec­ ades, associated with the fact that they are widespread among different genera, confirms the need to increase the effort in biodiversity-based studies (e.g. Cryptocephalus O.F. Muller, 1764, Chrysolina Motschulsky, 1860, Gonioctena Motschulsky, 1860, Longitar­ sus Berthold, 1827, Psylliodes Berthold, 1827, Colaspidea Laporte de Castelnau, 1833; Bastazo 1997, Biondi 1997, Sassi 2001, Leonardi 2007, Daccordi and Ruffo 2005, Bavi­ era 2007, Vela and Bastazo 2012, Zoia 2014). In this project are involved taxonomists, specialized in different leaf beetle clades, in order to guarantee the accurate specimen identification. Introduction In our view, the adoption of this strategy is a way to bring together traditional (intended as based on morphology) and molecular taxonomy in order to tentatively overcome the “taxonomy impedi­ ment” (Rodman and Cody 2003).h The purpose of this paper is to report the preliminary results achieved during the first seven years of the project in order to show the potential of a cooperation between molecular biologists and traditional taxonomists. In particular, we report: i) the meth­ od adopted and issues arisen in the development of the sequence dataset; ii) the list of subfamilies, genera and the number of species for which cox1 gene sequences were obtained; iii) the metadata associated with the processed organisms; iv) mean values of intraspecific and interspecific nucleotide divergence v) the new species described and the important faunistic findings. Giulia Magoga et al. / ZooKeys 597: 27–38 (2016) 30 Figure 1. Area investigated by the Chrysomelidae Barcoding project. The countries in which were per­ formed the collecting campaigns are reported in dark grey. The percentage of the total processed speci­ mens is reported for each country. Figure 1. Area investigated by the Chrysomelidae Barcoding project. The countries in which were per­ formed the collecting campaigns are reported in dark grey. The percentage of the total processed speci­ mens is reported for each country. Specimen collection and identification More than 50 entomologists, from different European Countries, have joined the C- Bar project and have actively participated in samples collection. During the first seven years of the project (from 2009 to 2015) numerous collecting campaigns were organ­ ized from March to September of each year. The specimens were collected using dif­ ferent methods: from the vegetation by sweep net or by beating sheet, and directly by hand in specific habitats (e.g. under stones or digging the host plant roots). All the collected specimens were placed in 5 ml vials filled with absolute ethanol in order to preserve the genomic DNA. Within an hour of specimen collection, the mixture in the vials was replaced with fresh absolute ethanol in order to obtain better sample dehydra­ tion and preservation for long-term storage. Each vial was preserved at -20°C and was labeled by a unique identifier plus other metadata related to the sampling locality (i.e. Country, Province, Region, exact site, latitude, longitude and elevation), the date of collection, the collector/s and other ecological information related to the specimens. Specimen manipulation and dissection (when necessary) were completed with the auxiliary use of a stereomicroscope. Images of the specimen habitus were acquired by a reflex camera (Canon EOS 450D, macro objective 60 mm or 100 mm with a set of Barcoding Chrysomelidae: a resource for taxonomy and biodiversity conservation... 31 macro extension tubes) or with Axiocam 506 mounted on Zeiss Axio Zoom V16. The specimens were morphologically identified by Italian taxonomists expert in different leaf beetle clades (most of them are listed among the authors of the present article). The nomenclature adopted in the C-bar project follows the work of Bouchard et al. (2011) at the levels of family and subfamily, while at the levels of genus and species was adopted the recently published Catalogue of Palaearctic Coleoptera–Chrysomeloidea (Löbl and Smetana 2010). DNA extraction, PCRs and sequence quality control Giulia Magoga et al. / ZooKeys 597: 27–38 (2016) 32 DNA extraction, PCRs and sequence quality control DNA extraction was performed in two different ways since it took place in different laboratories (Biodiversity Institute of Ontario, University of Guelph and Department of Agricultural and Environmental Sciences, Università degli Studi di Milano): for 950 samples the DNA was extracted from one hind leg while for the 56 remaining samples the DNA was extracted from the whole specimen, after the removal of the abdomen. The latter procedure ensures to keep specimen morphology intact. In both cases, DNA was purified using the Qiagen DNeasy Blood and Tissue Kit (Qiagen, Hilden, Germany). Here we describe the adopted non-destructive procedure: the specimen was taken off from absolute ethanol and dried in single 1.5 ml vials for 45 minutes at 30°C; after the removal of the abdomen with the use of sterile pins and tweezers the specimen was placed in 180 µL of ATL lysis buffer (Qiagen) with 200 ng/mL proteinase K (Sigma Aldrich, St. Louis, MO, USA) at 56°C for 12 hours. The following steps of the DNA extraction were performed according to the manufac­ turer’s instructions of Qiagen DNeasy Blood and Tissue Kit. After DNA extraction, the specimens were dry mounted on pins together with genitalia and kept for future reference. A quote of the extracted DNA was preserved in the C-bar DNA library at -80°C for long term storage and a rate was preserved at -20° in order to perform the following amplifications. A fragment of 658 bp at the 5’-end of the mitochondrial cytochrome c oxidase subunit 1 gene (cox1) was amplified with primers LCO1490 5’-GGT CAA CAA ATC ATA AAG ATA TTG G / HCO2198 5’-TAA ACT TCA GGG TGA CCA AAA AAT CA (Folmer et al. 1994). When this pair of primers resulted in unsuccessful amplification of the target marker, other primers amplify­ ing the same gene region were used, i.e. LepF1 5’-ATT CAA CCA ATC ATA AAG ATA TTG G / LepR1 5’-TAA ACT TCT GGA TGT CCA AAA AAT CA (Hebert et al. 2004). Successful amplifications were determined by gel electrophoresis. PCR products were directly sequenced on both strands using the marker-specific primers from ABI technology (Applied Biosystems, Foster City, CA, USA). The obtained sequences were edited using Geneious R8 (Biomatters Ltd., Auckland, New Zealand) and primers, pseudogenes and contaminations removed. Finally, they were deposited in the Bold Systems (Ratnasingham and Hebert 2007) and in the European Nucleo­ tide Archive (Montagna et al. under revision). Intraspecific and intrageneric nucleotide divergence The obtained cox1 gene sequences were aligned at codon level using MUSCLE (Edgar 2004) with default parameters. A pairwise nucleotide distance matrix was estimated starting from the aligned sequences implementing the Kimura-two-parameter (K2P) model (Kimura 1980), considered as an adequate evolutionary nucleotide model when p-distances between sequences are low (Nei and Kumar 2000). The nucleotide distance matrix was used for the calculation of the mean intraspecific and interspecific nu­ cleotide distances and for the calculation of mean intrageneric distance; these analyses were performed using the R package Spider (Brown et al. 2012). We also calculated nucleotide intraspecific distances for some species with a wide range of distribution. Results and discussion Until now, C-Bar collecting campaigns have investigated some areas of Bulgaria, France, Greece, Italy, Morocco, Romania, Spain, Switzerland, Turkey and Tunisia (Figure 1). The sampling efforts that have been accomplished until now led to the collection of more than 5000 Chrysomelidae specimens. During the identification process, some specimens of previously unknown species were recognized, these samples were used for the description of the following species: Pachybrachis sassii (Montagna 2011) from the Giglio Island in the Tuscan Archipelago; Pachybrachis holerorum (Montagna et al. 2013) from the Northern Apennines and Oulema mauroi Bezděk & Baselga, 2015, from Northen Italy. Other samples collected during the C-Bar collecting campaigns were used in a revision of Colaspidea genus that led to the description of seven new species (Zoia 2014). All these new taxa were formally described by a traditional mor­ phological approach, in some cases molecular data were added to confirm the existence of the new species. Besides the discovery of new taxa, two allochthonous species new to Europe, namely Ophraella communa (Boriani et al. 2013) and Colasposoma dauricum (Montagna et al. in press), were intercepted. O. communa is a leaf beetle of Nearctic origin accidentally introduced in 1996 in Taiwan (Wang and Chiang 1998) and Japan (Takizawa et al. 1999); the species rapidly spread in East Asia and few years ago we in­ tercepted it in the Northern part of Italy (Boriani et al. 2013). C. dauricum is a species originally present in the North and Central-East of Asia, it has never been observed out of its original range until our interception in 2011 in Piedmont (North of Italy). Among the collected samples, the DNA was extracted from 1006 specimens and PCRs targeting a fragment of the cox1 gene performed. PCRs with the selected primer pairs lead to successful amplification in 93.8% of the cases (62 specimens failed the amplification). Among the specimens for which the amplification failed, 43 specimens belong to the subfamily Cryptocephalinae Gyllenhaal, 1813: 18 species of Crypto­ cephalus (40 specimens); interestingly cox1 sequences have never been obtained for Cryptocephalus therondi Franz, 1949, Cryptocephalus cantabricus Franz, 1958 and Cryp­ tocephalus etruscus Sassi, 1995. We can hypothesize the presence of mutations in the Barcoding Chrysomelidae: a resource for taxonomy and biodiversity conservation... 33 annealing region of the used primers. Results and discussion Sequences obtained from Clytra laeviuscula Rat­ zeburg, 1837, Clytra quadripunctata Linnaeus, 1758, Cryptocephalus cristula Dufour, 1843, Cryptocephalus octoguttatus Linnaeus, 1767, Lachnaia tristigma Lacordaire, 1848 and Oomorphus concolor Sturm, 1807 did not possess an open reading frame and were thus considered as nuclear pseudogenes. Twenty-seven sequences were discarded be­ cause of contamination from exogenous DNA. A total of 910 cox1 sequences (267 species corresponding to ~13% of those inhabiting the Mediterranean region) were obtained, the size of the sequences was > 400 bp in ~99% of the cases. q p We observed that only two species, namely Cryptocephalus violaceus Laicharting, 1781 and Cryptocephalus duplicatus Suffrian, 1845, sharing the same haplotype can not be discriminated through DNA barcoding. In this and in similar cases a barcod­ ing failure can be confirmed only ensuring the correct identification of the samples by expert taxonomists. Therefore 99.3% of the species (265) for which we obtained cox1 sequences possessed unique haplotypes, allowing their molecular identification. The mean intraspecific nucleotide distance value is of 2%, while the mean interspecific and intrageneric distances result of, respectively, 25.2% and of 19.8%. The obtained intraspecific value are higher than that inferred in a previous study on Coleoptera (Pentinsaari et al. 2014). This results might be the effect of geographical distances among localities of collection of co-specific specimens; a possible alternative explana­ tion is the presence of cryptic species. Among the species showing high intraspecific nucleotide distance noteworthy are the cases of Cryptocephalus rugicollis (2.8% [0%, 5.5%]), Exosoma lusitanicum (6.7% [0.2%, 9.2%]) and Cryptocephalus quadripuncta­ tus that shows a mean intraspecific distance (3% [0%, 4.9%]). To test the formulated hypotheses further analyses, including the use of other mitochondrial and nuclear markers as well as a wider sample of specimens, are required. Among the nine subfamilies for which cox1 sequences were obtained (Table 1), Cryptocephalinae and Galerucinae Latreille, 1802 were better represented. In the first subfamily are listed 111 species (83 species of Cryptocephalini Gyllenhaal, 1813 and 28 of Clytrini Lacordaire, 1848, 426 specimens in total) while the second counts 88 species (24 species of Galerucini Latreille, 1802 and 64 of Alticini Spinola, 1844, 274 specimens in total). Results and discussion The unbalanced sampling towards Cryptocephalini, which in some way might affect the obtained results, could be explained by the fact that most of the C-bar speci­ mens have been collected by Sassi and Montagna, which mainly work on this clade and are likely to have developed collecting strategies that increase their sampling (Figure 1).hi The metadata related to the specimens (i.e., specimen identification, collection identifier, collecting date, state, province, exact site of collection, latitude, longi­ tude, elevation and collector/s) from which cox1 gene sequences were obtained, are available in a web site dedicated to the project (http://www.c-bar.org). Regarding the specimens collected within Italian administrative boundaries the metadata as­ sociated with the specimens are also available in the Biodiversity Database and GIS platform of the Italian National Network of Biodiversity. These faunistic data are useful because increase the awareness of species presence and distribution in the sampled area. Giulia Magoga et al. / ZooKeys 597: 27–38 (2016) 34 Table 1. List of the barcoded subfamilies and genera with the number of species and specimens belonging to each taxon. to each taxon. Results and discussion Subfamily Genus Ns a bNspec Nb Zeugophorinae Böving and Craighead, 1931 Zeugophora Kunze, 1818 1 1 1 Orsodacninae Thomson, 1859 Orsodacne Latreille, 1802 3 7 2.3 Donacinae Kirby, 1837 Donacia Fabricius, 1775 2 6 3 Criocerinae Latreille, 1804 Crioceris Muller, 1764 3 18 3 Lilioceris Reitter, 1912 1 Lema Fabricius, 1798 1 Oulema Gozis, 1886 1 Cassidinae Gyllenhal, 1813 Cassida Linnaeus, 1758 14 61 3.4 Hypocassida Weise, 1893 2 Hispa Linnaeus, 1767 1 Dicladispa Gestro, 1897 1 Chrysomelinae Latreille, 1802 Chrysolina Motschulsky, 1860 13 117 3.4 Chrysomela Linnaeus, 1758 3 Entomoscelis Chevrolat, 1836 1 Gastrophysa Chevrolat, 1836 1 Gonioctena Motschulsky, 1860 3 Oreina Chevrolat, 1836 6 Plagiosterna Motschulsky, 1860 1 Phratora Chevrolat, 1836 1 Plagiodera Chevrolat, 1836 1 Prasocuris Latreille, 1802 1 Timarcha Latreille, 1829 3 Galerucinae Latreille, 1802 Agelastica Chevrolat, 1836 1 274 3.1 Arima Chapuis, 1875 1 Calomicrus Stephens, 1831 3 Exosoma Jacoby, 1903 2 Diabrotica Chevrolat, 1836 1 Galeruca Geoffroy, 1762 5 Galerucella Crotch, 1873 3 Lochmaea Weise, 1883 2 Luperus Geoffroy, 1762 6 Nymphius Weise, 1900 2 Sermylassa Reitter, 1913 1 Altica Muller, 1764 4 Aphthona Chevrolat, 1842 6 Argopus Fischer von Waldheim, 1824 1 Arrhenocoela Foudras, 1860 1 Chaetocnema Stephens, 1831 2 Crepidodera Chevrolat, 1836 5 Derocrepis Weise, 1886 2 Dibolia Latreille, 1829 2 Epitrix Foudras, 1860 1 Hermaeophaga Foudras, 1860 1 Hippuriphila Foudras, 1860 1 Galerucinae Latreille, 1802 Barcoding Chrysomelidae: a resource for taxonomy and biodiversity conservation... 35 Subfamily Genus Ns a bNspec Nb Longitarsus Berthold, 1827 9 Lythraria Bedel, 1897 1 Neocrepidodera Heikertinger, 1911 6 Phyllotreta Chevrolat, 1836 4 Podagrica Chevrolat, 1836 1 Psylliodes Berthold, 1827 12 Sphaeroderma Stephens, 1831 2 Cryptocephalinae Gyllenhal, 1813 Cryptocephalus Geoffroy, 1762 73 426 3.8 Pachybarchis Chevrolat, 1836 8 Stylosomus Suffrian, 1848 2 Clytra Laicharting, 1781 4 Coptocephala Chevrolat, 1836 3 Labidostomis Chevrolat, 1836 10 Lachnaia Chevrolat, 1836 3 Macrolenes Chevrolat, 1836 1 Smaragdina Chevrolat, 1836 7 Tituboea Lacordaire, 1848 1 Eumolpinae Hope, 1840 Chrysochus Chevrolat, 1836 1 5 1.7 Colaspidea Laporte de Castelnau, 1833 1 Macrocoma Chapuis, 1874 1 aNs indicates the number of barcoded species; bNspec and N indicates respectively the total number and the average number of barcoded specimens belonging to each subfamily aNs indicates the number of barcoded species; bNspec and N indicates respectively the total number and the average number of barcoded specimens belonging to each subfamily Conclusion In this paper, we report that C-Bar project, besides having produced useful data for molecular taxonomy (cox1 sequences were obtained for about 13% of the species in­ habiting the investigated area), has obtained important results also from the viewpoint of the classical taxonomy leading to the morphological description of same new species of Chrysomelidae. A further important achievement has been the interception of al­ lochthonous species. These results have been obtained only thanks to the cooperation amongst the taxonomists specialized in different leaf beetle clades, which have ensured the correct identification of samples, the people involved in the extensive collecting campaigns and the molecular biologists. g g The promising preliminary results that have been obtained encourage us to contin­ ue with this project since they strongly confirm the urgent need to increase the efforts in faunistic studies to uncover the real biodiversity of leaf beetles inhabiting the Mediter­ ranean region. For these reasons, we are confident that the aim of C-bar project of de­ veloping a repository of cox1 sequences for the majority of the species of Chrysomelidae s. l. inhabiting the Mediterranean region may be achieved in the near future.i In conclusion, as demonstrated by the relevant results obtained during the first years of the project, we believe that DNA barcoding projects, when developed with the participation of taxonomists and molecular biologists, represent an opportunity Giulia Magoga et al. / ZooKeys 597: 27–38 (2016) 36 to bring together two different worlds and may be considered the driving force able to revive interest in what can be regarded as the milestone of biological studies that is a-taxonomy, helping to fill the “taxonomy impediment”. Acknowledgements The Authors would like to thank Dr. Stefano Zoia for the work performed and pre­ cious suggestions. 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https://openalex.org/W2087711953
https://europepmc.org/articles/pmc3621509?pdf=render
English
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Cultural background modulates how we look at other persons’ gaze
International journal of behavioral development
2,013
cc-by
5,184
Introduction can we study the effect of postnatal environment on the develop- ment of face gaze in humans? One of the most promising ways is a cross-cultural comparison, because different cultural norms would systematically modulate how the people in each culture would learn to process and interact with others in face-to-face communication. A brief look at another person’s face would tell you a lot about who they are (e.g., identity, age, gender, ethnicity, health and attractive- ness). Faces are also the ‘‘window to the soul’’, because facial expressions signal their emotional states, and gaze direction would tell you what they see and what they know. Having such signifi- cance in human social communication, it is not surprising that faces attract attention from very early in infancy. Even newborns prefer- entially orient to faces (Farroni et al., 2005; Johnson, Dziurawiec, Ellis, & Morton, 1991), especially those with direct gaze (Farroni, Csibra, Simion, & Johnson, 2002). Eye-tracking studies have demonstrated that infants start to show adult-like face-scanning behaviour, such as preferential fixations on the eyes and mouth (Yarbus, 1967), from as early as 6 weeks after birth (Hunnius & Geuze, 2004). Atypical patterns of face scanning behaviour can be found in neurodevelopmental disorders, such as autism spectrum disorders (ASD), whereby individuals show profound difficulties in social interaction and communication (Senju & Johnson, 2009). Although the mechanisms underlying atypical face-scanning behaviour in ASD is still unclear, it highlights the potential relationship between face-scanning behaviour and the development of social skills. Two independent lines of research contrasted face gaze between Western European/NorthAmericancultureand EasternAsian culture, and found clear differences in the face gaze between the two cultures. First, a series of studies (McCarthy, Lee, Itakura, & Muir, 2006, 2008) reported that Canadian participants maintain longer eye contact with an interviewer than Japanese participants when they answer cogni- tively demanding questions. In these studies, the gaze direction of the participants were analysed from the video recording. These studies clearly show the differential face gaze in realistic face-to-face interac- tion, which isconsistent with the cultural normsthat gaze avoidance is perceivedasinsincereinWestern culture,butthesamebehaviourdoes not have such a negative value in Eastern culture; it can even signal respectinsomecontexts(Argyle, Henderson,Bond,Iizuka,&Contar- ello, 1986). However, the video recording does not have a sufficient spatial resolution to examine which part of the face (eyes, nose or mouth) the participants look at. Special issue International Journal of Behavioral Development 37(2) 131–136 ª The Author(s) 2012 Reprints and permissions: sagepub.co.uk/journalsPermissions.nav DOI: 10.1177/0165025412465360 ijbd.sagepub.com Keywords y cross-cultural study, eye contact, eye-tracking, face scanning, gaze processing Atsushi Senju,1 Ange´lina Vernetti,1 Yukiko Kikuchi,2 Hironori Akechi,2 Toshikazu Hasegawa,3 and Mark H. Johnson1 Atsushi Senju,1 Ange´lina Vernetti,1 Yukiko Kikuchi,2 Hironori Akechi,2 Toshikazu Hasegawa,3 and Mark H. Johnson1 Abstract The current study investigated the role of cultural norms on the development of face-scanning. British and Japanese adults’ eye movements were recorded while they observed avatar faces moving their mouth, and then their eyes toward or away from the participants. British participants fixated more on the mouth, which contrasts with Japanese participants fixating mainly on the eyes. Moreover, eye fixations of British participants were less affected by the gaze shift of the avatar than Japanese participants, who shifted their fixation to the correspond- ing direction of the avatar’s gaze. Results are consistent with the Western cultural norms that value the maintenance of eye contact, and the Eastern cultural norms that require flexible use of eye contact and gaze aversion. Corresponding author: Atsushi Senju, Centre for Brain and Cognitive Development, Birkbeck, University of London, Malet Street, London WC1E 7HX, UK. Email: a.senju@bbk.ac.uk Introduction All the faces were initially presented with (e) forward gaze and closed mouth, which was followed by a mouth movement after 1 second, and a gaze shift after another second (i.e., 2 seconds from the onset of the stimulus). After the gaze shift, the face remained still for another 4 seconds. The orientation of the face was right in half of the stimuli and the left in the other half. movements were either smile (Figure 1a, 1b) or mouth opening (Figure 1c, 1d). Eye movements were either direct gaze (Figure 1a, 1c) or averted gaze (Figure 1b, 1d), which involved rotating both eyes laterally by 25 either towards the centre (direct gaze) or away from it (averted gaze). The amount of rotation for direct gaze was selected based on the rating of 10 naı¨ve observers, who rated the perception of ‘‘directedness’’ of the gaze (Todorovic, 2006). The same amount of rotation in the opposite direction was used for averted gaze. In total, 32 animations were generated (4 faces, 2 mouth movements, 2 gaze directions and 2 face orientations). The faces extended 18.4  13.0 cm on the screen. showed morefixation on the centre of the face(Blais, Jack, Scheepers, Fiset, & Caldara, 2008; Kelly et al., 2011). It was also suggested that reduced fixation on the mouth could partly explain cross-cultural dif- ference in facial expression processing (Jack, Blais, Scheepers, Schyns, & Caldara, 2009). These studies clearly show the subtle dif- ferences in face fixations between participants with different cultural backgrounds,but itisnot clear whether itisspecific tothe contextthey analyse facial information from static images, or a more general pat- tern of fixations in a more realistic context where they face dynamic sequences of facial actions. The current study aimed to bridge these gaps in knowledge by investigating how cultural background (British or Japanese) affects the face gaze when they observe dynamic face stimuli. We also examined whether the gaze direction of face stimuli (looking toward or away from the observer) would affect cross-cultural dif- ferences in face gaze. We predicted that British participants would maintain longer and sustained eye contact and make a triangular fixation (that is, more fixation on the mouth than Japanese partici- pants), whereas Japanese participants would show shorter and flex- ible eye fixations and more central fixations. Introduction Second, another series of studies recorded eye movements of Western European (British) and Eastern Asian (mainly Chinese) participants as they processed static images of faces, and found that Western European participants showed trian- gular fixation on both eyes and mouth, but Eastern Asian participants An important question about the development of face gaze is the role of postnatal environment. Several major theories of social skills development emphasize the role of input from their parents (or caregiver) as well as those from other members of the society, which are essential for the infant brain to learn the social world and become an ‘‘expert’’ (Gauthier & Nelson, 2001; Pascalis et al., 2005). For example, Sugita (2008) reared infant monkeys with no exposure to faces, and found that general preference to faces devel- ops without exposure to faces, but fine discrimination of faces do not develop. The results highlight the role of innate capacity to detect face-like shapes, and the role of postnatal learning in shaping the capacity to recognize individual faces. However, such a control of postnatal environment is impossible in human studies. So, how 1 Birkbeck, University of London, UK 2 Japan Society for the Promotion of Science, Japan 3 The University of Tokyo, Japan Corresponding author: Atsushi Senju, Centre for Brain and Cognitive Development, Birkbeck, University of London, Malet Street, London WC1E 7HX, UK. Email: a.senju@bbk.ac.uk International Journal of Behavioral Development 37(2) 132 Figure 1. Sample of the gaze direction and mouth movement of the stimuli. Note. (a) direct gaze, smile; (b) averted gaze, smile; (c) direct gaze, mouth opening; (d) averted gaze, mouth opening. All the faces were initially presented with (e) forward gaze and closed mouth, which was followed by a mouth movement after 1 second, and a gaze shift after another second (i.e., 2 seconds from the onset of the stimulus). After the gaze shift, the face remained still for another 4 seconds. The orientation of the face was right in half of the stimuli and the left in the other half. Figure 1. Sample of the gaze direction and mouth movement of the stimuli. Note. (a) direct gaze, smile; (b) averted gaze, smile; (c) direct gaze, mouth opening; (d) averted gaze, mouth opening. Introduction We also predicted that the response to direct and averted gaze would be modulated by the cultural background because of the different cultural norms on the use of eye contact, but no further specific predictions have been made due to the exploratory nature of the manipulation. Two Tobii T120 eye-trackers (Tobii, Stockholm, Sweden), equipped with an integrated 17-inch display, were used to present stimuli and record eye-movement in London and in Tokyo. Tobii Studio software was used to control stimulus presentation and to analyse the gaze data. Recordings were conducted in a quiet and soundproofed room within each research institute. Participants were instructed to watch the movies of the faces. The same experimenter (AS) conducted the recording in both the UK and Japan, to maintain strictly similar experimental conditions such as instructions. A 9-point calibration was conducted using Tobii Studio software before the recording. Recording consisted of two blocks, and each of 32 animations was presented twice (once in each block), in a randomized order. An experimenter also sat in the same testing room, out of sight of the participant, and monitored the recording with Tobii studio software. Viewing distance was approximately 60–65 cm from the display. Methods Both groups showed longer fixations on the front eye in the direct gaze condition than in averted gaze condition. However, only Japanese participants showed differ- ential fixations on the back eye, with longer fixation in averted gaze condition (Figure 3). No other contrasts reached significance, including any contrast in the bridge area. Further analyses were conducted on the seven 1-second bins of the data, to explore the time-course of the differential fixations on the four AOIs showing the gaze and cultural background interactions; front eye, back eye, mouth and centre. Front eye. No effects reached significance for the first, second and third bins. In the fourth bin, the point right after the gaze shift of the stimuli, both groups showed longer fixation in direct gaze condition than in averted gaze condition. Interestingly, this effect was exaggerated in Japanese participants, who showed even longer fixation than British participants in response to direct gaze and even shorter fixation than British participants in the averted gaze condi- tion (Figure 4a). The same trend remained in the fifth bin, in which only Japanese participants looked longer in the direct gaze condition than in the averted gaze condition. No effect reached significance from the sixth bin. Figure 2. Examples of the area of interest (AOI); front eye; front eye, back eye, bridge, centre and mouth. The size and the location of the AOI were constant across different stimuli. Back eye. In the first, second and third bins, the only significant effects were the differences between groups, with Japanese partici- pants fixating longer than British participants. From the fourth bin onward, however, Japanese participants fixated longer than British participants only in the averted gaze condition, but not in the direct gaze condition (Figure 4b). analysed with mixed-design ANOVAs to test the effects of cultural background (British or Japanese) and the sex (male or female) of the participants, as well as the ethnicity (Caucasian or Eastern Asian), gender (male or female), gaze direction (direct or averted), mouth movement (smile or mouth opening) and the AOI (front eye, back eye, bridge, centre and mouth) of the stimuli. An initial ANOVA was conducted on the whole 7-second data, which were then followed up by the analyses of seven 1-second bins of the data. Methods Nineteen British adults (ten females and nine males, mean age 27.98 years) and 22 Japanese adults (11 females and 11 males, mean age 27.75 years) participated in the study. (Four participants were not included because of excessive eye tracker data loss, under 70% samples.) British adults were recruited in central London, and Japanese adults were recruited in central Tokyo. All the participants have normal or corrected-to-normal acuity. The gaze data were initially processed with Tobii studio software to calculate the total visit time. Then, we calculated the fixation duration for each stimulus, for the following areas of inter- est (AOIs); front eye, back eye, bridge, centre and mouth. Note that faces are tilted either to the right or to the left, one of the eyes is always closer to the observer (i.e., Front Eye) than the other eye (i.e., Back Eye), the latter which is off to the side (Figure 2). The AOIs were selected based on the findings of relevant literature in cross-cultural face scanning studies (Blais et al., 2008; Jack et al., 2009; Kelly et al., 2011). These fixation duration data were extracted from the Tobii Studio for statistical analyses. Four computer-generated faces (one Caucasian female, one Caucasian male, one Eastern Asian female, one Eastern Asian male) were selected from the library of Poser 7 (Smith Micro Software, Aliso Viejo, CA), and were used to create 7-second ani- mations with the same software. All the animations started with a face presented upright, facing 30 to the left or to the right and gaz- ing forward, followed by a mouth movement (1 second after the start) and an eye movement (2 seconds after the start). Mouth The gaze data for different head orientations and the blocks were averaged together. The visiting duration for each AOI were then divided with the total visiting duration of the whole face, to calculate the relative visiting duration. The relative visiting duration was 133 Senju et al. Figure 2. Examples of the area of interest (AOI); front eye; front eye, back eye, bridge, centre and mouth. The size and the location of the AOI were constant across different stimuli. showed longer fixation on the centre, which was only significant in the averted gaze condition (Figure 3). The two groups showed similarities and differences in the response to different gaze directions. Methods For the significant interactions, post-hoc analyses were conducted on each contrast with Wilcoxon sign rank tests with the Bonferroni correction for multiple testing, to provide robust statistics. Mouth. The group difference in mouth fixation remained from the first to the fourth bin (Figure 4e), which then became only significant in averted gaze condition in the fifth bin and non-significant from the sixth bin. Centre. The group differences were significant in the fourth and fifth bins, only in averted gaze condition (Figure 4d). Discussion contrast, we even found a longer fixation in British participants on the centre in averted gaze condition (Figure 4d), which hap- pened later than the increased mouth fixation (Figure 4e) and is possibly explained by the residual effects of fixations on the mouth. It might suggest that the increased central fixation on the face in the Eastern Asian population is specific to the task which requires perceptual analyses of the faces, such as the recognition and categorization tasks used in the previous studies (Blais et al., 2008; Kelly et al., 2011), and does not happen in passive viewing. Another possibility is that Eastern Asians look more to the eyes when the face is expressive, but fixate centrally when they observe faces with neutral expression. Moreover, Japanese partici- pants did not show shorter eye fixation than British participants, which is consistent with some studies (e.g., Jack et al., 2009) but not with others (McCarthy et al., 2006, 2008). Further studies will be required to test whether the shorter face gaze could be observed in Eastern Asian participants in more naturalistic settings. contrast, we even found a longer fixation in British participants on the centre in averted gaze condition (Figure 4d), which hap- pened later than the increased mouth fixation (Figure 4e) and is possibly explained by the residual effects of fixations on the mouth. It might suggest that the increased central fixation on the face in the Eastern Asian population is specific to the task which requires perceptual analyses of the faces, such as the recognition and categorization tasks used in the previous studies (Blais et al., 2008; Kelly et al., 2011), and does not happen in passive viewing. Another possibility is that Eastern Asians look more to the eyes when the face is expressive, but fixate centrally when they observe faces with neutral expression. Moreover, Japanese partici- pants did not show shorter eye fixation than British participants, which is consistent with some studies (e.g., Jack et al., 2009) but not with others (McCarthy et al., 2006, 2008). Further studies will be required to test whether the shorter face gaze could be observed in Eastern Asian participants in more naturalistic settings. contrast, the fixation on the back eye was not affected by the gaze direction in British participants. Discussion As predicted, the interaction between the cultural background, gaze direction and the AOI was significant, F(4, 148) ¼ 4.684, p < .01, Zp 2 ¼ 0.11, demonstrating that British and Japanese participants fixated differently to the face, depending on whether the face was with direct or averted gaze. The effect was modulated by the sex, F(4, 148) ¼ 4.142, p < .01, Zp 2 ¼ 0.10 (see also the supplementary material), but not with other factors such as the mouth movement, the gender or the ethnicity of the face stimuli. These interactions remained significant when we excluded British participants of non-Caucasian ethnic origin (three females), and when we excluded British participants who had stayed two years in East Asian coun- tries (two males, one in China and the other in Thailand) and East Asian participants who had stayed 6 months in the USA (two males). The current study is the first to investigate how the cultural back- ground of the observer affects the face gaze when they observe dynamic faces looking toward or away from the observer. The results clearly demonstrate the cultural difference between British and Japanese participants in the way they look at different parts of the faces, and how they respond to different gaze direction. First, British participants fixated more on the mouth than did Japanese participants, replicating previous studies using static images of faces (Blais et al., 2008; Jack et al., 2009; Kelly et al., 2011). Second, both groups of participants fixated equally long on the eye closer to the observer, but Japanese fixated longer on the other eye than British participants. It is consistent with the finding that Eastern Asian participants fixate longer on the eyes (Jack et al., 2009) but it is a novel finding because to our knowl- edge, this study is the first to use head-turned faces. On the other hand, we did not replicate the longer fixation on the central parts of the faces in Eastern Asian participants, which is reported in previous studies (Blais et al., 2008; Kelly et al., 2011). By Follow-up analyses revealed that Japanese participants fixated longer on the back eye than did British participants, in both direct gaze and averted gaze conditions. British participants, by contrast, fixated longer on the mouth than Japanese participants, in both direct gaze and averted gaze conditions. Discussion British participants also International Journal of Behavioral Development 37(2) 134 0 0.1 0.2 0.3 0.4 0.5 0.6 Front eye 0 0.1 0.2 0.3 0.4 0.5 0.6 Back eye British / Direct Japanese / Direct British / Averted Japanese / Averted 0 0.1 0.2 0.3 0.4 0.5 0.6 Bridge 0 0.1 0.2 0.3 0.4 0.5 0.6 Mouth 0 0.1 0.2 0.3 0.4 0.5 0.6 Centre * * * * * * * * Figure 3. Relative visiting duration on each AOI during the entire period of stimulus presentation, for each cultural background of the participants and the gaze direction of stimuli. Note. * p < .05 (corrected); error bar: standard error. 0 0.1 0.2 0.3 0.4 0.5 0.6 Front eye 0 0.1 0.2 0.3 0.4 0.5 0.6 Back eye * * * * * 0 0.1 0.2 0.3 0.4 0.5 0.6 Front eye 0 0.1 0.2 0.3 0.4 0.5 0.6 Back eye British / Direct Japanese / Direct British / Averted Japanese / Averted * * * * * Back eye British / Direct Japanese / Direct British / Averted Japanese / Averted Japanese / Averted 0 0.1 0.2 0.3 0.4 0.5 0.6 Bridge 0 0.1 0.2 0.3 0.4 0.5 0.6 Mouth 0 0.1 0.2 0.3 0.4 0.5 0.6 Centre * * * Figure 3. Relative visiting duration on each AOI during the entire period of stimulus presentation, for each cultural background of the participants and the gaze direction of stimuli. Note. * p < .05 (corrected); error bar: standard error. 0 0.1 0.2 0.3 0.4 0.5 0.6 Bridge 0 0.1 0.2 0.3 0.4 0.5 0.6 Mouth 0 0.1 0.2 0.3 0.4 0.5 0.6 Centre * * * Figure 3. Relative visiting duration on each AOI during the entire period of stimulus presentation, for each cultural background of the participants and t Figure 3. Relative visiting duration on each AOI during the entire period of stimulus presentation, for each cultural background of the participants and the gaze direction of stimuli. N * 05 ( d) b d d Figure 3. Relative visiting duration on each AOI during the entire period of stimulus presentation, for each cultural background of the participants and the gaze direction of stimuli. Note. * p < .05 (corrected); error bar: standard error. Discussion The results showed that Japanese participants shifted their own fixation to the corresponding direc- tion of the observed gaze shift (i.e., to the front, medial eye in response to direct gaze and to the back, lateral eye in response to averted gaze), as if they ‘‘followed’’ the direction of the face gaze. British participants did not show such a change of fixation follow- ing observed gaze shift. It might suggest that the eye fixation of British participants reflects the cultural expectation to maintain eye contact, but the eye fixation of Japanese participants reflects the cultural norm to conform to others’ behaviour. Note that it is not a general difference in the sensitivity to facial motion, because mouth movements did not exaggerate or diminish the cultural differences in mouth fixation. The effect was more prominent in males than in females (see the Supplementary material), suggesting that the effect of cultural norm on face and gaze processing manifests more strongly in males. Further studies will be required to see how the differences in gender-related cultural norm interact with face-scanning behaviour. Interestingly, these two groups responded differently to the gaze shift of the stimuli. Second-by-second analyses revealed that both groups of participants fixated longer on the front eye immediately (that is, around 1 second) after they saw the gaze shift toward them, but such a change was more exaggerated and lasted longer with Japanese participants than British participants. Moreover, the initial cultural difference in the fixation on the back eye was overridden by the effect of gaze shift, in which Japanese participants fixated lon- ger on the back eye only when they saw an averted gaze. By The current study clearly demonstrates the cultural differences in face gaze in adults, but it cannot tell us how it develops. For example, Kelly et al. (2011) demonstrated that 70% of British-born Chinese adults show a face fixation pattern similar to Eastern culture, whereas 30% of them show a Western pattern of face fixation. Discussion This study suggests that cultural diversity in face fixation is more strongly affected by early familial environment, but could also be affected by societal environment (e g peers) in some individuals stimuli) which contrasts with previous infant research (Liu et al 0 0.1 0.2 0.3 0.4 0.5 0.6 1 2 3 4 5 6 7 1 2 3 4 5 6 7 1 2 3 4 5 6 7 1 2 3 4 5 6 7 1 2 3 4 5 6 7 0 0.1 0.2 0.3 0.4 0.5 0.6 * * * * * * * * * * * * * * 0 0.1 0.2 0.3 0.4 0.5 0.6 (b) (a) (d) (c) 0 0.1 0.2 0.3 0.4 0.5 0.6 0 0.1 0.2 0.3 0.4 0.5 0.6 * * * * * * * * * (e) British / Direct Japanese / Direct British / Averted Japanese / Averted Figure 4. Relative visiting duration on each AOI for each 1-second bin of stimulus presentation, for each cultural background of the participants and the gaze direction of stimuli. Note. (a) front eye, (b) back eye, (c) bridge, (d) centre and (e) mouth. The mouth movement occurred during the second bin, and the eye movement occurred during the third bin. * p < .05 (corrected); error bar: standard error. Senju et al. 135 135 Senju et al. 0 0.1 0.2 0.3 0.4 0.5 0.6 1 2 3 4 5 6 7 1 2 3 4 5 6 7 1 2 3 4 5 6 7 1 2 3 4 5 6 7 0 0.1 0.2 0.3 0.4 0.5 0.6 * * * * * * * * * * * * * * 0 0.1 0.2 0.3 0.4 0.5 0.6 (b) (a) (d) (c) 0 0.1 0.2 0.3 0.4 0.5 0.6 0 4 0.5 0.6 (e) Senju et al. 135 0 0.1 0.2 0.3 0.4 0.5 0.6 1 2 3 4 5 6 7 1 2 3 4 5 6 7 1 2 3 4 5 6 7 1 2 3 4 5 6 7 0 0.1 0.2 0.3 0.4 0.5 0.6 * * * * * * * * * * * * * * 0 0.1 0.2 0.3 0.4 0.5 0.6 (b) (a) (d) (c) 0 0.1 0.2 0.3 0.4 0.5 0.6 Figure 4. Discussion Relative visiting duration on each AOI for each 1-second bin of stimulus presentation, for each cultural background of the participants and the gaze direction of stimuli. Note. (a) front eye, (b) back eye, (c) bridge, (d) centre and (e) mouth. The mouth movement occurred during the second bin, and the eye movement occurred during the third bin. * p < .05 (corrected); error bar: standard error. Figure 4. Relative visiting duration on each AOI for each 1-second bin of stimulus presentation, for each cultural background of the participants and the gaze direction of stimuli. Note. (a) front eye, (b) back eye, (c) bridge, (d) centre and (e) mouth. The mouth movement occurred during the second bin, and the eye movement occurred during the third bin. * p < .05 (corrected); error bar: standard error. stimuli), which contrasts with previous infant research (Liu et al., 2011; Wheeler et al., 2011). Future developmental studies will be essential to assess the role of face familiarity on face scanning throughout the course of development. We also need to examine how the gender difference found in the current study (that is, larger cross-cultural differences in male than in female participants) develops, by testing younger populations. affected by societal environment (e.g., peers) in some individuals. Further studies will be necessary to study the time-course of the emergence of cultural diversity in face gaze early in the develop- ment. Moreover, as in previous adult studies (Blais et al., 2008; Jack et al., 2009; Kelly et al., 2011), we did not replicate the other race effect on face scanning (that is, the significant interaction between the ethnicity of the participants and the ethnicity of the International Journal of Behavioral Development 37(2) 136 Hunnius, S., & Geuze, R. H. (2004). Developmental changes in visual scanning of dynamic faces and abstract stimuli in infants: A longitudinal study. Infancy, 6, 231–255. To summarize, the current study revealed that the cultural background of the participant affects how they look at another person’s eyes and mouth, and how they modulate eye fixation in response to the gaze direction of others. These differences are consistent with the culturally-relevant strategy of perceptual analyses, as well as the cultural norms on the use of eye contact in face-to-face communication. Discussion These results highlight the new frontier of the research about how cultural norms can affect behavioural, cognitive and neural development, which would pro- vide a great opportunity to study the effect of postnatal environment on human behavioural and cognitive development. Jack, R. E., Blais, C., Scheepers, C., Schyns, P. G., & Caldara, R. (2009). Cultural confusions show that facial expressions are not uni- versal. Current Biology, 19, 1543–1548. Johnson, M. H., Dziurawiec, S., Ellis, H., & Morton, J. (1991). New- borns’ preferential tracking of face-like stimuli and its subsequent decline. Cognition, 40, 1–19. Kelly, D. J., Jack, R.E., Miellet, S., De Luca, E., Foreman, K., & Caldara, R. (2011). Social experience does not abolish cultural diversity in eye movements. Frontiers in Psychology, 2: 95. doi: 10.3389/fpsyg.2011.00095 Acknowledgement Liu, S., Quinn, P. C., Wheeler, A., Xiao, N., Ge, L., & Lee, K. (2011). Similarity and difference in the processing of same- and other-race faces as revealed by eye tracking in 4- to 9-month-olds. Journal of Experimental Child Psychology, 108, 180–189. We thank Megan Schulossmacher for the help in collecting the data and Gizelle Anzures for her comments on an earlier version of the draft. McCarthy, A., Lee, K., Itakura, S., & Muir, D. W. (2006). Cultural display rules drive eye gaze during thinking. Journal of Cross-Cultural Psychology, 37, 717–722. Funding This work has been supported by the UK Medical Research Council [grant number G1100252; G0701484]; the UK Economic and Social Research Council [grant number RES-063-27-0207]; the Great Britain Sasakawa Foundation [grant number #3495]; and the Japan Society for the Promotion of Science [grant number B16330192]. McCarthy, A., Lee, K., Itakura, S., & Muir, D. W. (2008). Gaze display when thinking depends on culture and context. Journal of Cross-Cultural Psychology, 39, 716–729. Pascalis, O., Scott, L. S., Kelly, D. J., Shannon, R. W., Nicholson, E., Coleman, M., & Nelson, C. A. (2005). Plasticity of face processing in infancy. Proceedings of National Academy of Science of the United States of America, 102, 5297–5300. References Argyle, M., Henderson, M., Bond, M., Iizuka, Y., & Contarello, A. (1986). Cross-cultural variations in relationship rules. International Journal of Psychology, 21, 287–315. Senju, A., & Johnson, M. H. (2009). Atypical eye contact in autism: Models, mechanisms and development. Neuroscience & Biobeha- vioral Reviews, 33, 1204–1214. Blais, C., Jack, R. E., Scheepers, C., Fiset, D., & Caldara, R. (2008). Culture shapes how we look at faces. PLoS ONE, 3, e3022. Sugita, Y. (2008). Face perception in monkeys reared with no exposure to faces. Proceedings of the National Academy of Sciences of the United States of America, 105, 394–398. Farroni, T., Csibra, G., Simion, F., & Johnson, M. H. (2002). Eye contact detection in humans from birth. Proceedings of the National Acad- emy of Science of the United States of America, 99, 9602–9605. Todorovic, D. (2006). Geometrical basis of perception of gaze direction. Vision Research, 46, 3549–3562. Farroni, T., Johnson, M. H., Menon, E., Zulian, L., Faraguna, D., & Csibra, G. (2005). Newborns’ preference for face-relevant stimuli: Effects of contrast polarity. Proceedings of National Academy of Science of the United States of America, 102, 17245–17250. Wheeler, A., Anzures, G., Quinn, P. C., Pascalis, O., Omrin, D. S., & Lee, K. (2011). Caucasian infants scan own- and other-race faces differently. PLoS ONE, 6(4), e18621. Yarbus, A. (1967). Eye movement and vision. New York, NY: Plenum. Gauthier, I., & Nelson, C. A. (2001). The development of face exper- tise. Current Opinion in Neurobiology, 11, 219–224.
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Perception of Nigerian Dùndún Talking Drum Performances as Speech-Like vs. Music-Like: The Role of Familiarity and Acoustic Cues
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ORIGINAL RESEARCH published: 20 May 2021 doi: 10.3389/fpsyg.2021.652673 ORIGINAL RESEARCH published: 20 May 2021 doi: 10.3389/fpsyg.2021.652673 ORIGINAL RESEARCH published: 20 May 2021 doi: 10.3389/fpsyg.2021.652673 Perception of Nigerian Dùndún Talking Drum Performances as Speech-Like vs. Music-Like: The Role of Familiarity and Acoustic Cues Cecilia Durojaye1,2*†, Lauren Fink1,3†, Tina Roeske1, Melanie Wald-Fuhrmann1,3 and Pauline Larrouy Maestri3 4 Cecilia Durojaye1,2*†, Lauren Fink1,3†, Tina Roeske1, Melanie Wald-Fuhrmann1,3 and Pauline Larrouy-Maestri3,4 1 Department of Music, Max Planck Institute for Empirical Aesthetics, Frankfurt am Main, Germany, 2 Department of Psychology, Arizona State University, Tempe, AZ, United States, 3 Max Planck-NYU, Center for Language, Music, and Emotion, Frankfurt am Main, Germany, 4 Neuroscience Department, Max Planck Institute for Empirical Aesthetics, Frankfurt am Main, Germany Keywords: speech surrogacy, Yorùbá, categorization, amplitude modulation spectrum, pitch, intensity, timbre, timing Edited by: Laura McPherson, Dartmouth College, United States It seems trivial to identify sound sequences as music or speech, particularly when the sequences come from different sound sources, such as an orchestra and a human voice. Can we also easily distinguish these categories when the sequence comes from the same sound source? On the basis of which acoustic features? We investigated these questions by examining listeners’ classification of sound sequences performed by an instrument intertwining both speech and music: the dùndún talking drum. The dùndún is commonly used in south-west Nigeria as a musical instrument but is also perfectly fit for linguistic usage in what has been described as speech surrogates in Africa. One hundred seven participants from diverse geographical locations (15 different mother tongues represented) took part in an online experiment. Fifty-one participants reported being familiar with the dùndún talking drum, 55% of those being speakers of Yorùbá. During the experiment, participants listened to 30 dùndún samples of about 7s long, performed either as music or Yorùbá speech surrogate (n = 15 each) by a professional musician, and were asked to classify each sample as music or speech- like. The classification task revealed the ability of the listeners to identify the samples as intended by the performer, particularly when they were familiar with the dùndún, though even unfamiliar participants performed above chance. A logistic regression predicting participants’ classification of the samples from several acoustic features confirmed the perceptual relevance of intensity, pitch, timbre, and timing measures and their interaction with listener familiarity. In all, this study provides empirical evidence supporting the discriminating role of acoustic features and the modulatory role of familiarity in teasing apart speech and music. Reviewed by: Elizabeth Margulis, Princeton University, United States Aaron Carter-Enyi, Morehouse College, United States *Correspondence: Cecilia Durojaye cdurojay@asu.edu *Correspondence: Cecilia Durojaye cdurojay@asu.edu *Correspondence: Cecilia Durojaye cdurojay@asu.edu †These authors have contributed equally to this work †These authors have contributed equally to this work Specialty section: This article was submitted to Language Sciences, a section of the journal Frontiers in Psychology Received: 12 January 2021 Accepted: 21 April 2021 Published: 20 May 2021 Citation: Durojaye C, Fink L, Roeske T, Wald-Fuhrmann M and Larrouy-Maestri P (2021) Perception of Nigerian Dùndún Talking Drum Performances as Speech-Like vs. Music-Like: The Role of Familiarity and Acoustic Cues. Front. Psychol. 12:652673. doi: 10.3389/fpsyg.2021.652673 INTRODUCTION et al., 2013) are altered as a function of listener familiarity with a stimulus type. Regarding pitch, Weidema et al. (2016) showed that the same contours are perceived differently depending on the context in which they are embedded, with better discrimination in music than speech context. These studies highlight that the role of specific acoustic features in shaping perceptual categorization may, in part, be driven by previous exposure. When we turn on the radio it seems trivial to determine whether what we are hearing is music or someone speaking. Sound sequences can generally be described in terms of pitch, timbre, and timing (e.g., Kraus et al., 2009); acoustical differences have been shown between sequences associated with music and language domains. For instance, speech typically comprises many gliding tones and more variation in pitch trajectory than (Western) music (Patel, 2008), with a temporal modulation spectrum peaking around 5 Hz, approximating the syllable rate (Ding et al., 2017). On the other hand, music is characterized by discrete pitches sustained for longer durations (Zatorre and Baum, 2012), and a temporal modulation spectrum peaking around 2 Hz, approximating the average beat rate (Ding et al., 2017). However, note that such studies often examine material that has different sound sources, such as the human voice vs. musical instruments, which might enhance the distinctive characteristics of the sequences associated with each of the two categories. Acoustically, sound sequences from music and language domains can be defined in several ways, with summary statistics (i.e., mean), as reported earlier, but also in terms of changes over time. In the case of pitch, for example, speech and music are rarely monotonous. In speech, the intonation or pitch direction (e.g., utterances interpreted as statements or questions, Bolinger, 1986; Ladd, 2008), the pitch accent (e.g., Ladd et al., 1999), or the prosodic patterns (e.g., Bänziger and Scherer, 2005; Kraljic and Brennan, 2005; Dilley and McAuley, 2008), play a considerable role in carrying paralinguistic information, such as the emotional state (Banse and Scherer, 1996) or the intention (Hellbernd and Sammler, 2016) of the speaker. Additionally, pitch contrasts and thus changes of pitch over time additionally carry lexical information in tonal languages (Yip, 2002; Carter- Ényì and Carter-Ényì, 2016). In music, changes of pitch over time also provide crucial information that allow listeners to recognize, evaluate, and enjoy a musical performance. INTRODUCTION There exist different musical (cultural) systems that define pitch movements, with specific scales and rules (Krumhansl, 1979; Lerdahl and Jackendoff, 1983; Cross, 2001; Ringer, 2002; Thompson, 2013), but some features such as the presence of small intervals or descending melodies are present in different cultures (e.g., Huron, 2001) and have been described as statistical universals for music (Savage et al., 2015). Besides the relevance of pitch changes over time (in addition to mean pitch), a large range of literature in the music and language domains supports the perceptual relevance of changes in other dimensions such as timbre, intensity, or duration. It is thus important to explore acoustic features that take into account dynamics (rather than means) not only with regard to pitch, but also more broadly in the dimensions of timing, timbre, and intensity. When coming from the same source (e.g., the vocal instrument), music and language categories can show a certain overlap. For instance, certain types of speech are considered more musical than others (e.g., child-directed speech, rhymes, poetry), while certain types of vocal music are considered speech- like (e.g., rap). To better understand the ambiguity of vocal stimuli, Merrill and Larrouy-Maestri (2017) presented several versions of Arnold Schoenberg’s Pierrot lunaire—a piece notable for its use of sprechstimme or “speech-song” (Stadlen, 1981)—to vocal experts and found a large variety in the description of the material, from very spoken-like to very sung-like. Interestingly, the same exact material can be interpreted as either song or speech. A phenomenon called the speech-to-song illusion has been reported by Deutsch et al. (2008, 2011): A spoken sentence can be perceived as sung after several repetitions. In other words, the same acoustic contents can be perceived as belonging to two distinct categories, which makes it difficult to identify clear boundaries between speech and music, even in a culture in which the idea of contrasting categories is widely accepted (see Brown, 2000 for a gradual view of music-language). Further, it has been shown that categorization, in the context of the speech-to-song illusion task, changes when the material is difficult to pronounce (Margulis et al., 2015) or to understand (Jaisin et al., 2016), which supports the role of listeners’ familiarity or prior knowledge in stimulus categorization (Vanden Bosch der Nederlanden et al., 2015) and suggests a potential downside of using vocal stimuli, which are highly familiar to listeners. Citation: Citation: Durojaye C, Fink L, Roeske T, Wald-Fuhrmann M and Larrouy-Maestri P (2021) Perception of Nigerian Dùndún Talking Drum Performances as Speech-Like vs. Music-Like: The Role of Familiarity and Acoustic Cues. Front. Psychol. 12:652673. doi: 10.3389/fpsyg.2021.652673 Citation: Durojaye C, Fink L, Roeske T, Wald-Fuhrmann M and Larrouy-Maestri P (2021) Perception of Nigerian Dùndún Talking Drum Performances as Speech-Like vs. Music-Like: The Role of Familiarity and Acoustic Cues. Front. Psychol. 12:652673. doi: 10.3389/fpsyg.2021.652673 May 2021 | Volume 12 | Article 652673 1 Frontiers in Psychology | www.frontiersin.org Talking Drum Perception Durojaye et al. Frontiers in Psychology | www.frontiersin.org INTRODUCTION In this study, we further examine speech-music specificities by employing an instrument intertwining language and music: the dùndún talking drum. The dùndún is commonly used in south- west Nigeria as a musical instrument. The dùndún of the Yorùbá is played by people of all ages, though mostly men, and in a variety of sacred and secular cultural contexts (Durojaye, 2020). It is used to play musical rhythms without semantic information but also to communicate announcements, warnings, prayer, jokes, proverbs, or poetry (Sotunsa, 2009). While a dùndún ensemble consists of drums of varying sizes and functions, we focus here on the ìyá ìlù dùndún—the lead drum in the ensemble—usually performing the role of “talker” during performance, imitating Yorùbá, which is a tonal language, in what has been described as speech surrogacy (Durojaye et al., in review; McPherson, 2018). Villepastour, in her analysis of bàtá drums—a very close relative of the dùndún—argues for the interdependence of speech tone and music and highlights the significance of relative pitch and rhythm in the surrogacy system of the dùndún (Villepastour, 2010, 2014). In the present study, we focus on the perceptual categorization of non-vocal material into speech-like vs. music-like, as well as the role of familiarity in shaping these categories. Familiarity/culture effects on the perception of speech and music are well-described (Palmer and Krumhansl, 1987; Morrison and Demorest, 2009; Perrachione et al., 2011; Bregman and Creel, 2014; Sharma et al., 2020). With respect to timbre, familiar sound sources are recognized more quickly than unfamiliar ones (Siedenburg and McAdams, 2016). In the time domain, both rhythm (Hannon et al., 2012) and meter perception (Kalender May 2021 | Volume 12 | Article 652673 2 Durojaye et al. Talking Drum Perception FIGURE 1 | Transcription of a Yorùbá proverb played on the dùndún (stimulus 6S in our corpus). The dots placed on the three horizontal lines represent a schematic depiction of the pitch changes (high, medium, or low) as the phrase written underneath would be spoken in the Yorùbá language. INTRODUCTION The phrase translates to “Public or private, there is no place the God cannot see, public or private.” Though the phrase would typically be spoken without the repetition at the end, when played on the dùndún the proverb is elaborated by repetition of the first phrase (gray shaded areas), as repetition is often a means to provide context and remove ambiguity in meaning (Stern, 1957; Arewa and Adekola, 1980). FIGURE 1 | Transcription of a Yorùbá proverb played on the dùndún (stimulus 6S in our corpus). The dots placed on the three horizontal lines represent a schematic depiction of the pitch changes (high, medium, or low) as the phrase written underneath would be spoken in the Yorùbá language. The phrase translates to “Public or private, there is no place the God cannot see, public or private.” Though the phrase would typically be spoken without the repetition at the end, when played on the dùndún the proverb is elaborated by repetition of the first phrase (gray shaded areas), as repetition is often a means to provide context and remove ambiguity in meaning (Stern, 1957; Arewa and Adekola, 1980). MATERIALS AND METHODS The Yorùbá language uses three relative tone levels: Low (grave accent), Middle (usually left unmarked), and High (acute accent). The tone levels are vital to distinguish the meaning of words (Carter-Ényì and Carter-Ényì, 2016). Like the language, the drum also consistently employs three relative tone levels. The dùndún is a waisted (hourglass shaped), double- headed membrane drum, with gut or leather cords securing the skins around the wooden frame of the drum. The cords are manipulated with one hand, while the other strikes the top membrane with a curved stick. Pressure on the cords changes the pitch of the drum, allowing for a full octave range and effects like glissandi (Blades, 1992; Euba, 1990). For the drum to produce the lowest pitch, minimal pressure is applied on the cords; the more the pressure, the higher the frequency. Thus, the drum can manipulate tone levels and contours, as in Yorùbá language. This imitation is confirmed by recent acoustical analyses of mono or disyllabic words performed on the drum which demonstrated that the three Yorùbá tones (Low, Middle, and High) are produced on a global level with three measurably different fundamental frequencies (Akinbo, 2019). The technique of representing syllables can take many forms such as (1) using one drum stroke for each syllable (as for a single tone level and vowel elisions); (2) many strokes for one syllable; (3) one drum stroke for two or more syllables; (4) one drum stroke for a syllable with many speech tone levels as would be the case for some glides, or assimilations (see also, Euba, 1990; Villepastour, 2010, for bàtá drums). A transcription of dùndún “talking” is provided as an example in Figure 1. The experimental procedure was in accordance with guidelines ethically approved by the Ethics Council of the Max Planck Society. Participants provided informed consent before proceeding with the study. Participants One hundred and seven participants (36 self-reported as females, 71 as males, aged from 18 to 75 years old, M = 39.22, SD = 15.06) were recruited via the research participant database of the Max Planck Institute for Empirical Aesthetics and via personal contacts. From various geographical locations (15 different mother tongues were represented), fifty-one participants reported being familiar with the dùndún talking drum (i.e., they knew about the dùndún prior to the survey). Of these 51 participants, 28 (55%) were speakers of Yorùbá. Participants declared to have normal hearing ability and reported various musical training levels. Participants received no financial compensation. Frontiers in Psychology | www.frontiersin.org Material Thirty-six dùndún samples were created from performances by one professional dùndún musician from Ibadan, South- West Nigeria. The performer (male) is a native Yorùbá and fluent English speaker with more than 25 years of experience playing the drum. Performances were recorded at a local music studio with a SHURE SM57 dynamic microphone directed at the face of the drum, at a 3-inch distance, sampling at 44.1 kHz. Note that clicks and environmental noise can be heard in some recordings. In the experiment outlined below, we seek to identify acoustic features associated with the perception of speech-like vs. music- like dùndún performances and the potential role of familiarity in such classification. To do so, we first compared speech-like vs. music-like dùndún performances with regard to different acoustic features related to pitch, intensity, timbre, and timing. Second, we presented the same samples to familiar and unfamiliar listeners and examined their ability to classify the performances as intended by the performer, as well as their confidence in the classification. Finally, a statistical model was created to quantify the role of listener familiarity and acoustic features of dùndún performance, in predicting listeners’ perception of dùndún as speech-like vs. music-like. Half of the performances were music-like material consisting of Yorùbá àlùjó rhythms (literally dance drumming); the other half were speech-like material, composed of Yorùbá proverbs and oríkì (poetry). The performer was first asked to use the drum to say different phrases (“talk”), after which he was asked to “drum” (the equivalent of music). All instructions were given in the Yorùbá language. Note that in Yorùbá dùndún performances, when drummers say they “talk” with the drum, they refer to the performance of oríkì, proverbs, or the signal mode of the drum. Similarly, when they talk May 2021 | Volume 12 | Article 652673 Frontiers in Psychology | www.frontiersin.org 3 Durojaye et al. Talking Drum Perception had failed, we manually outlined note onsets with custom- written MATLAB code. about “drumming” or “playing music” (for those who use the English term), they are making a reference to àlùjó. These categories were confirmed by the performer after the task. Also note that dance rhythms, proverbs, and poetry are used for any occasion (e.g., weddings, burials, religious events). In the current performances, the “talking” contents covered various themes, such as a praise to a deity, prayers, vilification, and admonition. Procedure The task was implemented as an online experiment in Unipark Enterprise Feedback Suite (QuestBack GmbH, Cologne, Germany). Prior to the classification task, a brief presentation of the dùndún was given (origin, uses, description, picture) without sound examples. The aim was to provide a basic knowledge for those who reported being unfamiliar with the drum or the potential use of this instrument as speech surrogates. To determine participants’ familiarity with the dùndún, we asked if they knew about the dùndún prior to the survey. Participants were instructed to listen to each excerpt and to indicate whether it was best described as “speech-like” or “music-like.” For each excerpt, the forced choice identification was followed by a confidence rating on a 4 point-scale (1 = not confident, 4 = very confident). The order of stimuli and response pattern (speech- like button as the first or the second option) was randomized for each participant. (p y g p ) Besides these time-related measures, we selected various features typically used to describe auditory signals: pitch, intensity, and timbre measures. At the note level and between consecutive notes, for each stimulus, we calculated: pitch height, intensity, and Wiener entropy (timbre, the maximum value is a pure sine tone). Scaling was performed within recording, on the millisecond-wise acoustic features between the 0.5 and 99.5th percentiles (instead of between minimum and maximum) to exclude outliers. Amplitude envelope and Wiener entropy were extracted from the audio in 10 ms time windows and steps of 1 ms using the MATLAB package Sound Analysis for MATLAB (by Sigal Saar). The pitch function from MATLAB Audio Toolbox (The MathWorks Inc., 2020) was used for pitch extraction. For pitch, intensity, and timbre measures (as well as IOI and ratio), we also calculated the probability densities for each group of stimuli (music-like and speech-like). Additionally, we computed the average of each feature, across each stimulus, resulting in the following final measures: mean pitch, mean intensity, and mean timbre; as well as average of absolute differences between consecutive tones (leading to mean intensity change, mean timbre change, and mean pitch change measures). Note that scaled values (0–1) were used to compute the measures capturing changes between consecutive notes. Acoustic Measures of Interest First, we computed the amplitude modulation spectra of the recordings, following the procedure of Ding et al. (2017), with MATLAB code kindly provided by Nai Ding. In brief, we extracted the sound envelope in narrow frequency bands (corresponding to frequency bands used by the human cochlea), then, following a re-scaling procedure, we calculated the root mean square of the Discrete Fourier Transform of each frequency band and binned over frequencies. High frequencies in the amplitude modulation spectrum correspond to fast modulations of intensity, low frequencies to slow modulations (for details, see Ding et al., 2017). We then calculated the peak frequency in the spectrum (i.e., the frequency exhibiting the greatest amplitude modulation), for each recording. To further analyze differences in timing, we also computed the inter-onset interval (IOI, in ms) between notes, the two-interval ratio: interval1/(interval1+interval2), and the pulse clarity. The first measure corresponds to the timing at the note level and the second to the change in timing between consecutive time intervals. A short interval preceding a long interval would result in a ratio < 0.5, a short interval following a longer one has a ratio > 0.5, and an isochronous rhythm of two similar intervals has a ratio of 0.5. Pulse clarity, a measure that estimates the temporal regularity of events in the signal (Lartillot et al., 2008), was calculated using all recommended default parameters of the mirpulseclarity function from the music information retrieval toolbox v.1.7.2 (i.e., using a frame length of 5 s, a hop factor of 10%, and the maximum value of the autocorrelation curve to define clarity). Pulse clarity ranges between 0 (no clear pulse) and 1 (perfectly regular pulse). To confirm that the performances clearly represented the category of speech or music, all recordings were presented to three independent professional dùndún drummers located in Nigeria and South Africa. The judges were asked to categorize the performances according to whether they represented speech or music. Like the performer, they used the terms àlùjó, oríkì, owe (proverbs), in their responses. The 30 samples on which the judges unanimously agreed to represent speech (n = 15) or music (n = 15) were selected. The duration of the samples ranged from 5 to 10 s (M = 7.37 s, SD = 1.1 s). All recordings can be accessed at: https://edmond.mpdl.mpg.de/imeji/collection/ovmWl7 rLtIiGSv1v. Acoustic Analyses The analysis of acoustic features was carried out in MATLAB 2018b (The MathWorks, Inc., Natick, Massachusetts, United States). The analysis of acoustic features was carried out in MATLAB 2018b (The MathWorks, Inc., Natick, Massachusetts, United States). Acoustic Properties of Dùndún Performances We first investigated whether the amplitude modulation spectrum (AMS) systematically differs between speech-like and music-like dùndún stimuli, as the AMS has previously been shown to distinguish between speech (around 4–6 Hz) and different kinds of Western music (around 2 Hz). As illustrated in Figure 2A and confirmed via t-test, however, the AMS of the two types of stimuli did not significantly differ [t(27.93) = 1.60, p = 0.120, d = 0.59]; both peaked around 5 Hz, which corresponds to the previously established amplitude modulation rate typical of speech (Ding et al., 2017; Figure 2A, bottom panel). In terms of pulse clarity, we find a significant difference between the two stimulus categories, with a greater pulse clarity (i.e., greater temporal regularity) in the music-like stimuli, t(26.14) = 4.03, p < 0.001, d = 1.47 (Figure 2B). accuracy = TP + TN TP + TN + FP + FN However, accuracy has been shown to be biased if the classes are unbalanced (e.g., if there would be more instances of perceived speech than music). Therefore, as widely used and recommended, we computed Matthews Correlation Coefficient (MCC): However, accuracy has been shown to be biased if the classes are unbalanced (e.g., if there would be more instances of perceived speech than music). Therefore, as widely used and recommended, we computed Matthews Correlation Coefficient (MCC): MCC = TP × TN −FP × FN √(TP + FP) × (TP + FN) × (TN + FP) × (TN + FN) p g In addition to the AMS measure, we examined four types of features (see section “Materials and Methods” for description of all measures) that could capture differences between music- and speech-like stimuli: intensity, pitch, timbre, and timing. Figure 3 illustrates each measure at the note level (mean for each stimulus and their distribution). As reported in Table 1, we observed significant differences for intensity and timing, with higher intensity level (Figure 3A) and longer IOI (Figure 3D) in the speech-like recordings. Figure 4 illustrates each measure at the consecutive note level (mean of differences between consecutive notes for each stimulus and their distribution). Besides being louder, consecutive notes also varied less in intensity in the speech-like stimuli (Figure 4A), in line with the distribution depicted in Figure 3A, with a narrower range for the speech-like stimuli. Also, we observed that near-isochrony is very common in both speech- and music-like excerpts (Figure 4D). Statistical Analyses Statistical analyses were conducted in R Core Team (2013). To compare acoustic measures between speech- and music- like dùndún recordings, we performed independent, two-tailed t-tests. For measures relative to changes between consecutive notes, absolute (rather than signed) differences were used. At an alpha of 0.05, the threshold p-value after correcting for multiple comparisons (10 t-tests) was p = 0.005. Cohen’s d is used to report effect sizes and was calculated in R using the cohen.d function in the effsize package (Torchiano, 2020). To predict participants’ perception of the stimuli as music- or speech-like, a generalized linear mixed effects logistic regression model was fit via maximum likelihood using the Laplace approximation method, with bound optimization by quadratic approximation, implemented using the glmer function from the lme4 package (Bates et al., 2015). Acoustic variables were centered and scaled before being entered into the model. Multicollinearity was checked using variance inflation factors (VIFs). Some predictors had a VIF > 5 and, therefore, required removal from the model. Specifically, inter-onset-interval (VIF = 5.90) and Behavioral Analyses amplitude difference between adjacent notes (VIF = 8.27) were removed. Correlations between means of all acoustic features are provided in Supplementary Figure 1. Note that inter-onset interval has a high correlation with intensity [r(28) = 0.63, p < 0.001] and intensity difference [r(28) = −0.82, p < 0.001], and that intensity and intensity difference have a high correlation with each other [r(28) = −0.71, p < 0.001]. Participants’ Classification of Stimuli Participants’ responses on the task can be summarized using a 2 × 2 contingency table, or confusion matrix M: M = hTP FN FP TN i where we arbitrarily define music as positive and speech as negative, such that true positives (TP) represent music intended by the performer and classified by the listener, while true negatives (TN) represent speech intended by the performer and similarly classified by listener. TPs (music) and TNs (speech) represent correct classifications. False positives (FP) designate instances of intended speech perceived as music, while false negatives (FN) indicate cases of intended music perceived as speech. Perfect classification is thus defined as 0 FPs and FNs: h n+ 0 0 n− i . Accuracy on the task is defined as the number of correct classifications, divided by the total number of observations: where we arbitrarily define music as positive and speech as negative, such that true positives (TP) represent music intended by the performer and classified by the listener, while true negatives (TN) represent speech intended by the performer and similarly classified by listener. TPs (music) and TNs (speech) represent correct classifications. False positives (FP) designate instances of intended speech perceived as music, while false negatives (FN) indicate cases of intended music perceived as speech. Perfect classification is thus defined as 0 FPs and FNs: h n+ 0 0 n− i . Accuracy on the task is defined as the number of correct classifications, divided by the total number of observations: Acoustic Properties of Dùndún Performances However, in music-like ones, intervals tend to speed up (the second interval in an isochronous pair being a little shorter, moving the near- isochronous peak slightly right) while slowing down in speech- like ones (near-isochronous peak moved slightly left). Some very high and very low ratios (due to a short interval next to a long one) become apparent in the wider spread of speech-like data, in the top right scatter plot, and in the peaks of the probability density plot, marked by arrowheads. MCC is robust to unbalanced datasets, and has been shown to be a more reliable measure than accuracy (Chicco and Jurman, 2020). It is a measure of classification performance across all classes that takes into account the size of each class. MCC ranges between -1 and 1, with 0 indicating chance performance, 1 perfect performance, and -1 perfect misclassification. As is common practice with MCC, in the case of 0 in the denominator, we set the denominator to 1. Frontiers in Psychology | www.frontiersin.org Segmentation Segmentation of single notes was performed semi-automatically on each recording’s amplitude envelope, using an adaptive threshold. First, amplitude envelopes were slightly smoothed with a Hodrick-Prescott (HP) filter (coefficient = 50). Then, the adaptive segmentation threshold was created by applying a stronger HP filter to the amplitude envelope (coefficient = #107). The difference between the slightly smoothed amplitude envelope and the adaptive filter provided robust segmentation in most recordings. We visually inspected the segmented waveforms and sonograms while listening to the audio to validate segmentation. In cases where the automated segmentation May 2021 | Volume 12 | Article 652673 Frontiers in Psychology | www.frontiersin.org 4 Talking Drum Perception Durojaye et al. Listeners’ Classification of Dùndún Performances As can be seen in Figure 5A, participants clearly separated the stimuli into two distinct speech vs. music categories that largely aligned with the intention of the performer. We May 2021 | Volume 12 | Article 652673 5 Talking Drum Perception Durojaye et al. FIGURE 2 | (A) Amplitude modulation spectra of the dùndún performances (top panel). Thin lines are for AMS of individual recordings, thick lines represent the mean for 15 music (blue) and 15 speech-like (red) stimuli. Bottom panel shows AMS for speech and music corpora from Ding et al. (2017) for comparison. Dashed arrow illustrates that dùndún recordings peak at higher modulation rates than most Western music, namely rates that are typical for speech (black line) but also occur in rock music (teal line). The lower panel in A is reprinted from Neuroscience and Biobehavioral Reviews, 81, Nai Ding, Aniruddh D. Patel, Lin Chen, Henry Butler, Cheng Luo, David Poeppel, Temporal modulations in speech and music, 7, Copyright Elsevier Ltd. (2017), with permission from Elsevier. (B) Violin plots representing the pulse clarity of the music (blue) and speech-like (red) stimuli. The black horizontal line indicates the median of both distributions. Black dots represent pulse clarity for individual stimuli. FIGURE 2 | (A) Amplitude modulation spectra of the dùndún performances (top panel). Thin lines are for AMS of individual recordings, thick lines represent the mean for 15 music (blue) and 15 speech-like (red) stimuli. Bottom panel shows AMS for speech and music corpora from Ding et al. (2017) for comparison. Dashed arrow illustrates that dùndún recordings peak at higher modulation rates than most Western music, namely rates that are typical for speech (black line) but also occur in rock music (teal line). The lower panel in A is reprinted from Neuroscience and Biobehavioral Reviews, 81, Nai Ding, Aniruddh D. Patel, Lin Chen, Henry Butler, Cheng Luo, David Poeppel, Temporal modulations in speech and music, 7, Copyright Elsevier Ltd. (2017), with permission from Elsevier. (B) Violin plots representing the pulse clarity of the music (blue) and speech-like (red) stimuli. The black horizontal line indicates the median of both distributions. Black dots represent pulse clarity for individual stimuli. speech). Within the speech category, 13S was most clearly perceived as speech, while 5S was most often confused for music. Readers can access all stimuli online (link in section “Materials and Methods”). Listeners’ Classification of Dùndún Performances observed that only four participants categorized every sample as music (solid blue rows near the bottom of the plot), whereas the large majority showed few confusions. Twelve participants exhibited perfect classification (top rows). In the figure, within the speech and music categories, stimuli (columns) are sorted by the number of errors made per stimulus (i.e., the left-most column, stimulus 13M, was least often confused for speech, while 3M was most often confused for A confusion matrix for perceived vs. intended music and speech-like performances is plotted in Figure 5B. Overall, the average accuracy of participants on the task was 66%. The average rate people perceived speech when the performance was intended to be music was 12%, while the average rate at which people perceived music when it was intended to be speech was 29%. Collectively, these latter two rates indicate that participants were more likely to perceive speech as music than music as speech. The illustration of confidence ratings (underlying histograms in Figure 5B, with ratings from 1 to 4) showed similar patterns, with moderately high confidence even in the case of false classifications. Note, however, that the listeners who were unfamiliar (gray) with the dùndún seem to be least confident when they perceive the stimulus to be speech (right column in confusion matrix). Confidence means for unfamiliar (gray) and familiar (gold) participants are displayed in the lower left and right corners of each quadrant, respectively. TABLE 1 | Output of the independent t-tests carried out for the four types of acoustic features (Intensity, Pitch, Timbre, and Timing) between music- and speech-like stimuli. Intensity Pitch Timbre Timing Mean for note level t(18.18) = −5.37 t(20.61) = 2.06 t(18.84) = −1.38 t(16.18) = −8.53 p < 0.001 p = 0.052 p = 0.185 p < 0.001 d = −1.96 d = 0.75 d = −0.50 d = −3.12 Mean change for consecutive notes t(26.32) = 8.95 t(17.53) = −0.75 t(27.60) = 4.09 t(15.74) = 2.17 p < 0.001 p = 0.465 p < 0.001 p = 0.046 d = 3.27 d = −0.27) d = 1.49 d = 0.79 For the four types of comparisons, measures consisted of the mean of all notes (Figure 2) and the mean of the absolute difference between all consecutive notes (Figure 3). Note that the threshold p-value (for an alpha of 0.05) after correcting for multiple comparisons was p = 0.005. Listeners’ Classification of Dùndún Performances Thicker frames highlight the measures significantly differing after correction. TABLE 1 | Output of the independent t-tests carried out for the four types of acoustic features (Intensity, Pitch, Timbre, and Timing) between music- and speech-like stimuli. Given the imbalance in perceiving speech vs. music, and the statistical properties outlined in the methods, our main metric of interest for participants’ classifications of the stimuli was the Matthews Correlation Coefficient (MCC). An MCC of 1 indicates perfect classification, 0 chance, and -1 perfect misclassification. Participants’ average MCC was 0.61 (±0.33). For the four types of comparisons, measures consisted of the mean of all notes (Figure 2) and the mean of the absolute difference between all consecutive notes (Figure 3). Note that the threshold p-value (for an alpha of 0.05) after correcting for multiple comparisons was p = 0.005. Thicker frames highlight the measures significantly differing after correction. For the four types of comparisons, measures consisted of the mean of all notes (Figure 2) and the mean of the absolute difference between all consecutive notes (Figure 3). Note that the threshold p-value (for an alpha of 0.05) after correcting for multiple comparisons was p = 0.005. Thicker frames highlight the measures significantly differing after correction. May 2021 | Volume 12 | Article 652673 Frontiers in Psychology | www.frontiersin.org 6 Talking Drum Perception Durojaye et al. FIGURE 3 | Acoustic measures computed at the note level for music-like (blue) and speech-like (red) dùndún performances. (A) Mean intensity, (B) Mean pitch, and (C) Mean timbre (Wiener entropy) (D) Mean inter onset intervals (IOI). Left panels show mean note values for 15 speech- and music-like pieces each in gray, with mean and SEM per piece in color. Right panels show the respective probability densities. Participants who were familiar1 with the dùndún exhibited a significantly higher MCC, compared to those who were unfamiliar with the dùndún [Welch’s independent two-tailed, t-test: t(93 08) = 6.12, p < 0.001, Cohen’s d = 1.16, 1MCC = 0.33]; FIGURE 3 | Acoustic measures computed at the note level for music-like (blue) and speech-like (red) dùndún performances. (A) Mean intensity, (B) Mean pitch, and FIGURE 3 | Acoustic measures computed at the note level for music-like (blue) and speech-like (red) dùndún performances. (A) Mean intensity, (B) Mean pitch, and (C) Mean timbre (Wiener entropy) (D) Mean inter onset intervals (IOI). Left panels show mean note values for 15 speech- and music-like pieces each in gray, with mean and SEM per piece in color. Right panels show the respective probability densities. Participants who were familiar1 with the dùndún exhibited a significantly higher MCC, compared to those who were unfamiliar with the dùndún [Welch’s independent two-tailed, t-test: t(93.08) = 6.12, p < 0.001, Cohen’s d = 1.16, 1MCC = 0.33]; see Figure 5C; N.B. each participants’ familiarity (gold = familiar; gray = unfamiliar) is plotted in the left color bar of Figure 5A). Nonetheless, familiarity is not required to perform the task, as unfamiliar participants still exhibited an average MCC 1We ran an additional t-test comparing the MCC of the subset of familiar participants who did not speak Yorùbá to those unfamiliar and still found a large, significant effect of familiarity, t(58.74) = 3.18, p = 0.002, d = 1.16, 1MCC = 0.211. May 2021 | Volume 12 | Article 652673 Frontiers in Psychology | www.frontiersin.org 7 Talking Drum Perception Durojaye et al. FIGURE 4 | Acoustic measures computed at the level of consecutive notes for music-like (blue) and speech-like (red) dùndún performances. All measures are scaled from 0 to 1. (A) Mean intensity change: The top panel shows, for each pair of consecutive notes, the intensity of the pair’s first note on the x-axis, and of the second note on the y-axis. The diagonal line indicates where note pairs with two similar note intensities would fall. The bottom panel shows the probability density of intensity differences. (B,C) are the Mean pitch change and Mean timbre change, illustrated following the same logic as (A). (D) Similar illustration for change in IOIs, as measured by ratio = interval1 / (interval1+interval2). In the probability density plot (bottom), green boxes and dashed lines mark interval ratios in terms of “interval1: interval2,” an alternative way to describe interval ratios, commonly used in music theory. FIGURE 5 | Participants’ classification of music vs. speech-like dùndún performances. (A) Each participant’s (vertical axis) judgment of each stimulus (horizontal axis) as music (blue) or speech (red). Participant performance is sorted descending from least to most errors. Columns are sorted from left to right, within each category (speech and music), from least to most errors per stimulus. The color bar to the left of the plot indicates whether each participant was familiar (golden) or unfamiliar (gray) with the dùndún and whether they spoke Yorùbá (black) or not (white). (B) Confusion matrix for perceived vs. intended stimulus classes, with histograms of participants’ confidence ratings (1–4) for each response type (i.e., quadrant), grouped by familiarity (gold = familiar; gray = unfamiliar). Confidence rating densities were computed within each response type (quadrant) for each familiarity group separately. Means for unfamiliar (gray) and familiar (gold) groups are displayed in the lower left and right corners of each quadrant, respectively. (C) Violin plots and underlying data points indicating the Matthews Correlation Coefficient (MCC) for each participant, separated according to those who were familiar with the dùndún (golden) vs. unfamiliar (gray). The bottom and top horizontal black lines in each distribution represent the 25th and 75th percentiles, the middle line represents the median. An MCC of 1 indicates perfect classification; 0 represents chance, and -1 perfect misclassification. of 0.46, well above chance (0), t(55) = 10.10, p < 0.001, d = 1.35. logistic regression model. The binary dependent variable was participants’ perception (speech = 0, music = 1). On the stimulus level, fixed effects included all measures reported in Figures 2– 4, except intensity difference and inter-onset-interval, which had to be excluded due to high correlation with intensity and each other (see section “Materials and Methods”). Since we observed an effect of familiarity on the classification performance index, with better classification for listeners who were familiar with the dùndún, we included familiarity as a fixed effect and in Frontiers in Psychology | www frontiersin org 8 May 2021 | Volume 12 | Article 652673 FIGURE 4 | Acoustic measures computed at the level of consecutive notes for music-like (blue) and speech-like (red) dùndún performances. All measures are scaled from 0 to 1. (A) Mean intensity change: The top panel shows, for each pair of consecutive notes, the intensity of the pair’s first note on the x-axis, and of the second note on the y-axis. The diagonal line indicates where note pairs with two similar note intensities would fall. The bottom panel shows the probability density of intensity differences. (B,C) are the Mean pitch change and Mean timbre change, illustrated following the same logic as (A). (D) Similar illustration for change in IOIs, as measured by ratio = interval1 / (interval1+interval2). In the probability density plot (bottom), green boxes and dashed lines mark interval ratios in terms of “interval1: interval2,” an alternative way to describe interval ratios, commonly used in music theory. May 2021 | Volume 12 | Article 652673 8 Durojaye et al. Talking Drum Perception FIGURE 5 | Participants’ classification of music vs. speech-like dùndún performances. (A) Each participant’s (vertical axis) judgment of each stimulus (horizontal axis) as music (blue) or speech (red) Participant performance is sorted descending from least to most errors Columns are sorted from left to right within each Predictors of Listeners’ Perception of Speech vs. Music In an effort to understand which acoustic features were most relevant in participants’ perception of the dùndún excerpts as music vs. speech-like, we built a linear mixed effects May 2021 | Volume 12 | Article 652673 Frontiers in Psychology | www.frontiersin.org 9 Durojaye et al. Talking Drum Perception FIGURE 6 | Odds ratios, with confidence interval (CI), for each fixed effect in a logistic regression model predicting participants’ perception of stimuli as music-like (1) or speech-like (0). The vertical line at 1 indicates no effect (i.e., any fixed effect predictor whose odds ratio CI overlaps 1 does not significantly predict participants’ perception). Fixed effects with an odds ratio <1 (red) indicate that a high value on that feature leads to the perception of speech. Odds ratios >1 (blue) indicate that a high value on that feature leads to the perception of music. The significance of each fixed effect is indicated with stars (***p < 0.001, **p < 0.01, and *p < 0.05). Number of observations: 3,210. Familiarity was a binary predictor (0 = no; 1 = yes); confidence ranged from 0 to 3; all other variables were continuous. FIGURE 6 | Odds ratios, with confidence interval (CI), for each fixed effect in a logistic regression model predicting participants’ perception of stimuli as music-like (1) or speech-like (0). The vertical line at 1 indicates no effect (i.e., any fixed effect predictor whose odds ratio CI overlaps 1 does not significantly predict participants’ perception). Fixed effects with an odds ratio <1 (red) indicate that a high value on that feature leads to the perception of speech. Odds ratios >1 (blue) indicate that a high value on that feature leads to the perception of music. The significance of each fixed effect is indicated with stars (***p < 0.001, **p < 0.01, and *p < 0.05). Number of observations: 3,210. Familiarity was a binary predictor (0 = no; 1 = yes); confidence ranged from 0 to 3; all other variables were continuous. confident were more likely to classify the stimulus as music. These confidence/familiarity results are in line with the general trends presented in Figure 5B. interaction with all acoustic measures. Confidence ratings were also entered as fixed effects. Random intercepts were included for participants and stimuli. Figure 6 shows the odds ratios and confidence intervals for each fixed effect in the model. Predictors of Listeners’ Perception of Speech vs. Music Fixed effects with an odds ratio <1 (red) indicate that a high value on that feature leads to the perception of speech. Odds ratios >1 (blue) indicate that a high value on that feature leads to the perception of music. Overall, the model had a prediction accuracy of 85% and an MCC of 0.70. The model explained a significant proportion of variance in the data, with a marginal R2 of 0.46 (amount of variance explained by fixed effects alone) and a conditional R2 of 0.65 (amount of variance explained by fixed and random effects). Frontiers in Psychology | www.frontiersin.org DISCUSSION Note also that, while mean IOI was significantly different between the two types of stimuli, it unfortunately could not be included into our statistical model (like the mean intensity change measure), as it had high correlation with other features (Supplementary Figure 1). Thus, it could well be the case that participants are using IOI (as well as intensity changes) in their classification. This issue could be addressed by systematically manipulating IOI, as well as mean intensity and intensity change, to tease apart their perceptual relevance. Participants could accurately classify the dùndún performances in the category intended by the performer, with an unsurprising bias toward the music-like category, given that drums are more commonly associated with music than speech. Listeners who were familiar with the instrument seem to have a clearer representation of what is grouped into speech or music categories, as visible by their better classification performance. Such results could be driven by the fact that slightly over half of the familiar participants also reported speaking Yorùbá, though the familiarity effect remained even when Yorùbá-speaking participants were removed from the analysis. In any case, if familiarity (broadly defined) or Yorùbá fluency sharpens the categories, it is not required to perform the task, as shown by the above chance level MCC and relatively high confidence for participants who were not familiar with the instrument. Such results suggest the relevance of commonly recognized acoustic cues that shape each category but become fine-tuned through repeated exposure. In the current study, we asked broadly about listeners’ familiarity and thus are not able to disentangle what exactly underlies the familiarity effect. Teasing apart perceptual vs. cultural vs. linguistic familiarity might all be of interest in future research. Additionally, manipulation of familiarity, via priming or additional explicit information about the dùndún, might allow one to quantify the amount and type of previous exposure that affects the classification. Further, exploration of listeners’ perception of dùndún performances with less constrained answer types might reveal other categories that could include both music and speech-like performances or less strong boundaries between these two culturally shaped categories (Brown, 2000). Regarding the dynamic aspect of timing, the present study focused mainly on consecutive notes or intervals, though we did include a measure of pulse clarity. DISCUSSION In this study, we used an instrument capable of speech surrogacy to explore the boundaries between speech and music. While several instruments such as trumpets (Kaminski, 2008), flutes (Moore and Meyer, 2014), xylophones (Strand, 2009; Zemp and Soro, 2010; McPherson, 2018), and whistling (Stern, 1957) can produce speech surrogates, we focussed here on the dùndún talking drum to examine listeners’ perception of music and speech and, more specifically, the role of acoustic features in distinguishing these two categories. To do so, we recorded expert dùndún performances, which have the advantage of being able to create both musical and speech-like stimuli without requiring the human voice (highly familiar to all listeners). Acoustic measures relative to pitch, timbre, intensity, and timing were used to describe the stimuli and we measured listeners’ ability to classify each performance into two pre-defined categories intended by the performer: speech- or music-like. Greater pulse clarity predicted perception of music. At the note level, greater mean intensity predicted speech. In terms of changes between notes, greater changes in pitch predicted speech, while greater changes in timbre predicted music. However, mean intensity, pitch and pulse clarity all interacted with participants’ familiarity (same direction of the overall effects but enhanced magnitude). Additionally, confidence predicted the categorization “music-like,” and interacted with familiarity, such that those familiar and confident were more likely to classify a given stimulus as speech, while those not familiar and May 2021 | Volume 12 | Article 652673 10 Talking Drum Perception Durojaye et al. Durojaye et al. et al., 2020). In the current study, peaks stand around 5 Hz for both music and speech-like dùndún. Though some musical styles have been shown to also peak around 5 Hz (e.g., rock), this rate is consistent with the modulation rate of speech (Ding et al., 2017). However, while Ding et al. (2017) analyzed a variety of Indo-European languages (American and British English, French, German, Swedish, Dutch, Danish, Norwegian; exception: Chinese), they did not include any Niger-Congo languages, such as Yorùbá, and only included Western types of music, which limits the generalizability of their findings. Here we extend Ding et al.’s AMS analysis to non-Western stimuli (dùndún) and show that its peak closely resembles that of speech. Future work might extend the AMS analyses to spoken Yorùbá and compare with that of the dùndún to better understand the findings about the temporal aspect reported here. DISCUSSION Our pulse clarity metric was related to maxima in the autocorrelation function of our stimuli (i.e., periodic repetitions), but our measure set could also be extended to perceived beat and/or meter (e.g., Tomic and Janata, 2008; London et al., 2017) or the detection of repeated patterns (e.g., via recurrence quantification analysis Fukino et al., 2016 or multi-fractal analysis Roeske et al., 2018). The analysis/evaluation of longer stimuli would allow for application of a more extensive set of timing measures to investigate their role in speech vs. music distinction. Also, though our stimulus set is novel in that it consists of speech-like and music-like performances on the dùndún from the same performer, it is also limited in scope. Future studies might consider developing larger corpora with more examples of speech-like and music-like material from multiple performers. In addition, future research might also more closely consider the relationship between measures like IOI, perceived beat and meter, and AMS. Though IOI, AMS, and perceived pulse / meter all have previously reported preferred temporal ranges, which broadly seem to align with each other (e.g., Fraisse, 1963; Parncutt, 1994; Farbood et al., 2013; Gotham, 2015; Ding et al., 2017), it is likely that IOI, AMS, and perceived pulse / meter do not form a trivial and/or consistent relationship to one another across all timescales. y p g The model proposed to examine the predictors of participants’ classification revealed that participants relied on several features. Pulse clarity, mean note intensity, and mean timbre change between consecutive notes were significant predictors. Additionally, familiarity seemed to amplify the effects of pulse clarity and intensity in predicting music vs. speech, respectively. These perceptual results are in line with those we find to distinguish acoustically between the two different stimulus categories. However, we also observed that features which did not significantly differ between the speech and music recordings nonetheless contributed significantly to the perceptual model. Specifically, mean pitch did not differ acoustically between the two types of stimuli performed by the musician, though it interacted with familiarity in predicting the perception of music. Similarly, mean pitch change between consecutive notes did not significantly differ acoustically but was associated with the perception of the stimuli as speech. Frontiers in Psychology | www.frontiersin.org REFERENCES Durojaye, C. (2020). 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Altogether, our findings confirm the relevance of acoustic features relative to intensity, pitch, timbre, and timing in distinguishing speech and music, as well as the role of culture and/or exposure in defining such categories. different conceptual domains, which paves the way to a more in-depth understanding of speech/music differences/similarities. CD, MW-F, and PL-M designed the study. CD collected the data. TR conducted acoustic analyses. LF conducted perceptual analyses and mixed modeling. LF and PL-M drafted the manuscript. All authors edited and approved the manuscript. ACKNOWLEDGMENTS The datasets presented in this study can be found in the links below: https://edmond.mpdl.mpg.de/imeji/collection/ ovmWl7rLtIiGSv1v, https://github.com/lkfink/Dundun. We thank David Poeppel for helpful discussion and feedback on analyses. We thank the performer for providing the stimuli and the dùndún experts who assisted in classifying the stimuli for final use. SUPPLEMENTARY MATERIAL The studies involving human participants were reviewed and approved by Ethics Council of the Max Planck Society. The participants provided their written informed consent to participate in this study. The Supplementary Material for this article can be found online at: https://www.frontiersin.org/articles/10.3389/fpsyg. 2021.652673/full#supplementary-material The Supplementary Material for this article can be found online at: https://www.frontiersin.org/articles/10.3389/fpsyg. 2021.652673/full#supplementary-material DISCUSSION In the future, the nature of the music and speech categories (that are slightly modulated by the familiarity of the listeners) could be clarified by means of psychophysical experiments that parametrically manipulate the relevant acoustic features reported here. 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Sci. 112, 8987–8992. doi: 10.1073/pnas.1414495112 May 2021 | Volume 12 | Article 652673 Frontiers in Psychology | www.frontiersin.org 13
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Knockout of Babesia bovis rad51 ortholog and its complementation by expression from the BbACc3 artificial chromosome platform
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. CC-BY 4.0 International license a certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made available under The copyright holder for this preprint (which was not this version posted April 11, 2019. ; https://doi.org/10.1101/606590 doi: bioRxiv preprint . CC-BY 4.0 International license a certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made available under The copyright holder for this preprint (which was not this version posted April 11, 2019. ; https://doi.org/10.1101/606590 doi: bioRxiv preprint . CC-BY 4.0 International license a certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made available under The copyright holder for this preprint (which was not this version posted April 11, 2019. ; https://doi.org/10.1101/606590 doi: bioRxiv preprint 1 Knockout of Babesia bovis rad51 ortholog and its 2 complementation by expression from the BbACc3 artificial 3 chromosome platform 4 5 Short title: Knockout of the B. bovis rad51 gene and its phenotype 6 7 Erin A. Mack1¶#, Yu-Ping Xiao1¶, and David R. Allred1,2,3* 8 9 1Department of Infectious Diseases and Immunology, College of Veterinary Medicine, 10 University of Florida, Gainesville, Florida, United States of America 11 2Genetics Institute, University of Florida, Gainesville, Florida, United States of America 12 3Emerging Pathogens Institute, University of Florida, Gainesville, Florida, United States of 13 America 14 15 *Corresponding author 16 E-mail: allredd@ufl.edu 17 18 ¶MEA and YP-X contributed equally to this project. 19 20 #Current address: Department of Biological Sciences, University of Idaho, Moscow, Idaho, 21 United States of America 22 1 Knockout of Babesia bovis rad51 ortholog and its 22 . CC-BY 4.0 International license a certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made available under The copyright holder for this preprint (which was not this version posted April 11, 2019. ; https://doi.org/10.1101/606590 doi: bioRxiv preprint . CC-BY 4.0 International license a certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made available under The copyright holder for this preprint (which was not this version posted April 11, 2019. ; https://doi.org/10.1101/606590 doi: bioRxiv preprint 23 Abstract 24 Babesia bovis establishes persistent infections of long duration in cattle, despite the development 25 of effective anti-disease immunity. One mechanism used by the parasite to achieve persistence is 26 rapid antigenic variation of the VESA1 cytoadhesion ligand through segmental gene conversion 27 (SGC), a phenomenon thought to be a form of homologous recombination (HR). To begin 28 investigation of the enzymatic basis for SGC we initially identified and knocked out the Bbrad51 29 gene encoding the B. bovis Rad51 ortholog. BbRad51 was found to be non-essential for in vitro 30 growth of asexual-stage parasites. However, its loss resulted in hypersensitivity to 31 methylmethane sulfonate (MMS) and an apparent defect in HR. This defect rendered attempts to 32 complement the knockout phenotype by reinsertion of the Bbrad51 gene into the genome 33 unsuccessful. To circumvent this difficulty, we constructed an artificial chromosome, BbACc3, 34 into which the complete Bbrad51 locus was inserted, for expression of BbRad51 under 35 regulation by autologous elements. Maintenance of BbACc3 makes use of centromeric 36 sequences from chromosome 3 and telomeric ends from chromosome 1 of the B. bovis C9.1 line. 37 A selection cassette employing human dihydrofolate reductase enables recovery of transformants 38 by selection with pyrimethamine. We demonstrate that the BbACc3 platform is stably 39 maintained once established, assembles nucleosomes to form native chromatin, and expands in 40 telomere length over time. Significantly, the MMS-sensitivity phenotype observed in the absence 41 of Bbrad51 was successfully complemented at essentially normal levels. We provide cautionary 42 evidence, however, that in HR-competent parasites BbACc3 can recombine with native 43 chromosomes, potentially resulting in crossover. We propose that, under certain circumstances 44 this platform can provide a useful alternative for the genetic manipulation of this group of 45 parasites, particularly when regulated gene expression under the control of autologous elements 46 may be important. 49 Keywords 50 artificial chromosome; Babesia bovis; chromatin; gene complementation; homologous 51 recombination; Rad51 52 . CC-BY 4.0 International license a certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made available under The copyright holder for this preprint (which was not this version posted April 11, 2019. ; https://doi.org/10.1101/606590 doi: bioRxiv preprint . CC-BY 4.0 International license a certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made available under The copyright holder for this preprint (which was not this version posted April 11, 2019. ; https://doi.org/10.1101/606590 doi: bioRxiv preprint 53 Introduction 54 Babesiosis is a tick-borne disease caused by apicomplexan parasites of the genus Babesia. 55 Humans are not the natural host for any babesial parasite but may be an incidental host, 56 acquiring zoonotic infections with a variety of different species. Babesia microti is the most 57 common species of Babesia to infect humans, although in western Europe infections commonly 58 occur with Babesia divergens. In the U.S. infections have been observed with Babesia duncani 59 and B. divergens-like organisms, as well as the unspeciated WA1 and MO1 isolates (reviewed in 60 [1]). Many individuals may carry asymptomatic infections [2], including as a result of inadequate 61 drug treatment of acute parasitemia [3, 4], posing a serious risk to the blood supply [5]. In cattle 62 babesiosis may be caused by at least five different species, with Babesia bovis generally 63 considered the most virulent. B. bovis shares many parallels with the human malarial parasite, 64 Plasmodium falciparum, including immune evasion via cytoadhesion and antigenic variation, 65 and the capacity for development of a lethal cerebral disease [6]. 66 67 Bovine babesiosis caused by B. bovis is quite severe. Cattle that survive the acute disease 68 develop a strong anti-disease immunity, but remain persistently infected for periods of at least 69 several years. This parasite makes use of at least two mechanisms to effect persistence: (i) 70 cytoadhesion of infected red blood cells (iRBCs) to capillary and post-capillary venous 71 endothelium, presumably in order to avoid splenic clearance; and (ii) rapid antigenic variation of 72 the cytoadhesion ligand, VESA1, to avoid antibody-mediated forced re-entry into the circulation 73 [7]. Cytoadhesion is therefore a behavior that is immunologically sensitive, and which the 74 parasite correspondingly has evolved means to protect. The development of the ability to 75 abrogate antigenic variation would allow for ready elimination of the parasite; this capability is a 76 desirable goal for parasite control through disruption of an essential aspect of its biology. 77 78 There is a great need to understand the molecular bases of virulence mechanisms in parasites. 79 One informative way to facilitate such studies is through the ability to genetically manipulate the 80 organisms. To date, the tools developed for the genetic manipulation of B. bovis are limited and 81 remain most appropriate for use in characterizing individual targets, although there is a need for 82 high-throughput methodologies [8]. 53 Introduction The expression of exogenous genes has been demonstrated, 83 using babesial promoter sequences to drive transcription of the target sequences. This has been 54 Babesiosis is a tick-borne disease caused by apicomplexan parasites of the genus Babesia. 55 Humans are not the natural host for any babesial parasite but may be an incidental host, 56 acquiring zoonotic infections with a variety of different species. Babesia microti is the most 57 common species of Babesia to infect humans, although in western Europe infections commonly 58 occur with Babesia divergens. In the U.S. infections have been observed with Babesia duncani 59 and B. divergens-like organisms, as well as the unspeciated WA1 and MO1 isolates (reviewed in 60 [1]). Many individuals may carry asymptomatic infections [2], including as a result of inadequate 61 drug treatment of acute parasitemia [3, 4], posing a serious risk to the blood supply [5]. In cattle 62 babesiosis may be caused by at least five different species, with Babesia bovis generally 63 considered the most virulent. B. bovis shares many parallels with the human malarial parasite, 64 Plasmodium falciparum, including immune evasion via cytoadhesion and antigenic variation, 65 and the capacity for development of a lethal cerebral disease [6]. 66 . CC-BY 4.0 International license a ertified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made available under The copyright holder for this preprint (which was not this version posted April 11, 2019. ; https://doi.org/10.1101/606590 doi: bioRxiv preprint . CC-BY 4.0 International license a certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made available under The copyright holder for this preprint (which was not this version posted April 11, 2019. ; https://doi.org/10.1101/606590 doi: bioRxiv preprint 84 achieved transiently through transfection with circular plasmids [9-13]. The longevity of this 85 class of vector has been improved by the inclusion of a centromeric segment, improving the 86 segregation of the circular molecules [12]. Long-term, stable expression has been achieved by 87 single-crossover insertional mutagenesis of chromosomal loci [12, 14]. This approach 88 demonstrates the capacity for creating targeted gene disruptions and the potential for the use of 89 promoter trapping strategies. Significantly, double-crossover knockout of the thioredoxin 90 peroxidase (tpx) gene has been achieved, providing complete gene knockouts [12]. 53 Introduction 91 Complementation of tpx was achieved by re-integration of the gene ectopically into the EF1 92 locus, resulting in high level expression. However, ectopic expression of exogenous genes in this 93 manner may suffer from improper expression due to local chromatin modifications, including 94 silencing or significant overexpression, compromising interpretation of genetic contributions to 95 phenotype. Despite the available tools there remains a need for convenient platforms that are 96 stable, efficiently and accurately segregated during mitosis, allow the transcription of large 97 sequences, and which may be properly regulated relative to metabolic need. 98 99 Antigenic variation in B. bovis is known to heavily utilize segmental gene conversion (SGC) to 100 modify the VESA1 ligand (BAK), and may participate in in situ transcriptional switching (isTS) 101 as well. SGC is thought to be a form of homologous recombination (HR), much the same as gene 102 conversion, a process that is dependent upon Rad51 proteins. To initiate investigation of the 103 enzymatic underpinnings of antigenic variation by SGC in B. bovis, we describe here the 104 knockout of the B. bovis rad51 gene by double crossover replacement [15]. Given the potential 105 for adverse effects if expressed at inappropriate levels, we did not want to overexpress Bbrad51 106 during complementation. Moreover, because of the knockout of the Bbrad51 gene, homologous 107 recombination was lost and complementation of the knockout by reintroduction of the gene into 108 the genome was not possible. We have addressed this issue through artificial chromosome 109 technology, an approach that allows the complementation of disrupted genes by reintroduction of 110 the entire locus, providing gene control through its autologous regulatory elements. An artificial 111 chromosome platform has already been developed for Plasmodium berghei, enabling 112 transformation efficient enough for shotgun screening to recover genes associated with drug 113 resistance [16, 17]. We have created an analogous platform for use with B. bovis through the 114 assembly of an artificial chromosome, BbACc3. BbACc3 is comprised of B. bovis centromeric 84 achieved transiently through transfection with circular plasmids [9-13]. The longevity of this 85 class of vector has been improved by the inclusion of a centromeric segment, improving the 86 segregation of the circular molecules [12]. Long-term, stable expression has been achieved by 87 single-crossover insertional mutagenesis of chromosomal loci [12, 14]. 53 Introduction This approach 88 demonstrates the capacity for creating targeted gene disruptions and the potential for the use of 89 promoter trapping strategies. Significantly, double-crossover knockout of the thioredoxin 90 peroxidase (tpx) gene has been achieved, providing complete gene knockouts [12]. 91 Complementation of tpx was achieved by re-integration of the gene ectopically into the EF1 92 locus, resulting in high level expression. However, ectopic expression of exogenous genes in this 93 manner may suffer from improper expression due to local chromatin modifications, including 94 silencing or significant overexpression, compromising interpretation of genetic contributions to 95 phenotype. Despite the available tools there remains a need for convenient platforms that are 96 stable, efficiently and accurately segregated during mitosis, allow the transcription of large 97 sequences, and which may be properly regulated relative to metabolic need. 98 99 Antigenic variation in B. bovis is known to heavily utilize segmental gene conversion (SGC) to 100 modify the VESA1 ligand (BAK), and may participate in in situ transcriptional switching (isTS) 101 as well. SGC is thought to be a form of homologous recombination (HR), much the same as gene 102 conversion, a process that is dependent upon Rad51 proteins. To initiate investigation of the 103 enzymatic underpinnings of antigenic variation by SGC in B. bovis, we describe here the 104 knockout of the B. bovis rad51 gene by double crossover replacement [15]. Given the potential 105 for adverse effects if expressed at inappropriate levels, we did not want to overexpress Bbrad51 106 during complementation. Moreover, because of the knockout of the Bbrad51 gene, homologous 107 recombination was lost and complementation of the knockout by reintroduction of the gene into 108 the genome was not possible. We have addressed this issue through artificial chromosome 109 technology, an approach that allows the complementation of disrupted genes by reintroduction of 110 the entire locus, providing gene control through its autologous regulatory elements. An artificial 111 chromosome platform has already been developed for Plasmodium berghei, enabling 112 transformation efficient enough for shotgun screening to recover genes associated with drug 113 resistance [16, 17]. We have created an analogous platform for use with B. bovis through the 114 assembly of an artificial chromosome, BbACc3. BbACc3 is comprised of B. bovis centromeric . CC-BY 4.0 International license a certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. 53 Introduction It is made available under The copyright holder for this preprint (which was not this version posted April 11, 2019. ; https://doi.org/10.1101/606590 doi: bioRxiv preprint . CC-BY 4.0 International license a ertified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made available under The copyright holder for this preprint (which was not this version posted April 11, 2019. ; https://doi.org/10.1101/606590 doi: bioRxiv preprint 115 and telomeric ends in a modified pBluescript-KS(+) backbone. This construct employs the 116 human dihydrofolate reductase (hDHFR) gene as selectable marker, conferring resistance to 117 pyrimethamine. BbACc3 was constructed and is manipulated as the circular plasmid, pBACc3, 118 which is linearized for transfection. Linearization with PmeI removes a spacer sequence and 119 exposes legitimate telomeric ends. Here, we demonstrate that BbACc3 transforms B. bovis to 120 pyrimethamine resistance, is maintained as a distinct linear chromosome that assembles into 121 chromatin, and undergoes expansion of its telomeres. Significantly, the Bbrad51 gene expressed 122 from BbACc3 under regulation by its autologous elements provided normal levels of 123 complementation of the Bbrad51 knockout upon challenge with methylmethane sulfonate 124 (MMS). Although further optimization is desirable, this platform may facilitate long-term 125 complementation of essential genes for studies connecting genotype and phenotype, particularly 126 where improper regulation would be problematic. 127 130 The orthologous Bbrad51 gene was identified. The B. bovis rad51 gene 131 (Bbrad51) was identified by first searching the genus Babesia non-redundant proteome (S1 132 Table). The search was initiated with the Saccharomyces cerevisiae Rad51 sequence 133 (CAA45563) as bait, using BLAST, with default parameters [18]. In this initial search, proteins 134 XP_001609877 (E= 2e-103) and XP_001609660 (E= 4e-15) were identified as candidates. To 135 cast a wider net and ensure capture of all candidates, CAA45563 was again used to initiate an 136 iterative search of the genus Babesia non-redundant protein database, using Psi-BLAST [19, 20] 137 with default parameters [19]. After three iterations the top B. bovis candidate, XP_001609877, 138 had a significance score of E = 3e-156, and three additional proteins of the Rad51/DMC1/RadA 139 superfamily were also identified. Each of these proteins then was used individually in reciprocal 140 iterative searches of the NCBI non-redundant protein database, excluding the genus Babesia, 141 using Delta-BLAST and composition-based statistics to provide their presumptive identities [19, 142 20]. These proteins included the second-best fit, XP_001609660 (E = 3e-86; XRCC3-like), 143 XP_001610815 (E= 8e-16; Rad51 homolog 2-like), and XP_001609995 (E= 0.077; XRCC2- 144 like). Genes encoding putative orthologs of all four proteins are found among various other . CC-BY 4.0 International license a ertified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made available under The copyright holder for this preprint (which was not this version posted April 11, 2019. ; https://doi.org/10.1101/606590 doi: bioRxiv preprint . CC-BY 4.0 International license a certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made available under The copyright holder for this preprint (which was not this version posted April 11, 2019. ; https://doi.org/10.1101/606590 doi: bioRxiv preprint 145 piroplasms (S1 Table; adapted from PiroplasmaDB). For XP_001609877 this search yielded over 146 200 significant hits of E= 0.0, all of which were confirmed or putative eukaryotic Rad51 147 molecules sharing 47-49% identity. Alignment of the four candidates with a number of 148 confirmed or predicted Rad51 proteins demonstrated excellent alignment of all functional 149 domains and motifs (S1 Fig.). An alignment tree was created by the Neighbor-joining method, 150 using Jukes-Cantor protein distance measures and 100 bootstraps to assess robustness. The tree 151 shows that, among the apicomplexan parasites Rad51 proteins cluster in apparent clades (Fig. 130 The orthologous Bbrad51 gene was identified. The B. bovis rad51 gene 152 1A, colored blocks), suggesting shared selection pressures within individual groups. P. knowlesi 153 was the only exception, failing to cluster with other Plasmodium spp. XP_001609877 clusters 154 with other eukaryotic Rad51 proteins, forming an apparent clade with other piroplasmids, yet is 155 clearly quite different. By contrast, the other three B. bovis RecA/RadA/Rad51-related proteins 156 cluster more closely with the Escherichia coli RecA protein included as outlier. An analysis of 157 XP_001609877 for probable structural domains by the Conserved Domains Database yielded 158 specific hits of PTZ00035 ("Rad51 protein, provisional"; residues 6-343; E= 0e+00) and 159 cd01123 ("Rad51_DMC1_radA, P-loop NTPase superfamily"; residues 105-339; E= 3.33e-111), 160 as well as a lower-confidence hit in the N-terminus for pfam14520, a small "helix-hairpin-helix" 161 domain (residues 39-83; E= 2.02e-03). The pfam14520 "helix-hairpin-helix" domain, which is 162 common to Rad51 proteins, was lacking from the other three candidates. Further support for the 163 putative assignment of this protein as a legitimate Rad51 ortholog was obtained by ab initio 164 prediction of its three-dimensional structure, using Robetta software (http://robetta.bakerlab.org/) 165 [21, 22]. The predicted structure models were superimposed onto the crystal structure of the S. 166 cerevisiae Rad51 H352Y mutant protein (PDB accession 3LDA) [23], using the Chimera 167 Matchmaker algorithm [24]. The predicted three-dimensional structure of XP_001609877 168 (model 4) was found to coordinate spatially extremely well with the 3LDA crystal structure, 169 including Walker A and B motifs, residue Q298 regulating access of ATP to the active site [23], 170 and the interaction domain sequences pivotal to filament formation (Fig. 1B). Based on these 171 results we refer to XP_001609877 as BbRad51, and BBOV_II003540 as the Bbrad51 gene 172 encoding this protein. The Bbrad51 gene was annotated with two predicted introns [25], a 173 structural model that was confirmed by RT-PCR amplification, cloning, and sequencing of the 174 full-length transcript. Flanking sequences were recovered through 5'- and 3'-RACE reactions (see 175 Methods). The results exactly matched the published B. bovis T2Bo isolate genome sequence, 145 piroplasms (S1 Table; adapted from PiroplasmaDB). For XP_001609877 this search yielded over 146 200 significant hits of E= 0.0, all of which were confirmed or putative eukaryotic Rad51 147 molecules sharing 47-49% identity. Alignment of the four candidates with a number of 148 confirmed or predicted Rad51 proteins demonstrated excellent alignment of all functional 149 domains and motifs (S1 Fig.). 130 The orthologous Bbrad51 gene was identified. The B. bovis rad51 gene ; https://doi.org/10.1101/606590 doi: bioRxiv preprint . CC-BY 4.0 International license a certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made available under The copyright holder for this preprint (which was not this version posted April 11, 2019. ; https://doi.org/10.1101/606590 doi: bioRxiv preprint 176 including predicted start and stop codons, and intron boundaries (Fig. 2A; [15, 25]). Active 177 transcription of Bbrad51 by asexual stages has been reported previously [26], and was confirmed 178 here by RT-PCR (S2 Fig., panel A). 180 Fig. 1. Relationship of BbRad51 to other Rad51 proteins. A. Alignment tree of the four B. 181 bovis RecA/RadA/Rad51-related proteins (accession numbers in red) with established or 182 predicted Rad51 proteins of other species (Neighbor-joining; Kimura protein distance 183 measure; 100 bootstrap replicates; numbers at nodes are bootstrap values). Direct 184 experimental evidence supports the catalysis of canonical RecA/RadA/Rad51 activities by 185 those proteins whose accession number is indicated in blue. BbRad51 (black arrow) clusters 186 with, but is comparatively dissimilar to, putative Rad51 proteins of other Piroplasmida (pink 187 panel). In contrast, the three remaining B. bovis proteins cluster more closely with the 188 prokaryotic (E. coli) RecA protein included as outlier to the alignment, and with several algal 189 Rad51 proteins. All four B. bovis proteins cluster away from other apicomplexan parasites 190 (Haemosporida, green panel; Coccidia, yellow panel) or higher eukaryotes. Interestingly, 191 Cryptosporidium spp. cluster separately from other Coccidia (blue panel). B. The predicted 192 three-dimensional structure of BbRad51 (blue ribbon), superimposed upon the crystal 193 structure of the S. cerevisiae Rad51 H352Y mutant (3LDA [23], tan ribbon), shows excellent 194 conformational coherence. Indicated on BbRad51 are the Walker A (red) and Walker B 195 (green) motifs, subunit interaction domains (orange), and the position of residue Q298 that 196 acts as “gatekeeper” controlling access of ATP to the active site (purple). Corresponding 197 ScRad51 residues are shown in yellow. ScRad51 has a disordered N-terminal extension that 198 fails to yield usable crystal data [71], resulting in a lack of sequence to align with that region 199 of BbRad51. 201 Fig. 2. Bbrad51 locus and knock-out strategy. A. The wt Bbrad51 locus structure was 202 confirmed to be comprised of three exons/ two introns. 130 The orthologous Bbrad51 gene was identified. The B. bovis rad51 gene An alignment tree was created by the Neighbor-joining method, 150 using Jukes-Cantor protein distance measures and 100 bootstraps to assess robustness. The tree 151 shows that, among the apicomplexan parasites Rad51 proteins cluster in apparent clades (Fig. 152 1A, colored blocks), suggesting shared selection pressures within individual groups. P. knowlesi 153 was the only exception, failing to cluster with other Plasmodium spp. XP_001609877 clusters 154 with other eukaryotic Rad51 proteins, forming an apparent clade with other piroplasmids, yet is 155 clearly quite different. By contrast, the other three B. bovis RecA/RadA/Rad51-related proteins 156 cluster more closely with the Escherichia coli RecA protein included as outlier. An analysis of 157 XP_001609877 for probable structural domains by the Conserved Domains Database yielded 158 specific hits of PTZ00035 ("Rad51 protein, provisional"; residues 6-343; E= 0e+00) and 159 cd01123 ("Rad51_DMC1_radA, P-loop NTPase superfamily"; residues 105-339; E= 3.33e-111), 160 as well as a lower-confidence hit in the N-terminus for pfam14520, a small "helix-hairpin-helix" 161 domain (residues 39-83; E= 2.02e-03). The pfam14520 "helix-hairpin-helix" domain, which is 162 common to Rad51 proteins, was lacking from the other three candidates. Further support for the 163 putative assignment of this protein as a legitimate Rad51 ortholog was obtained by ab initio 164 prediction of its three-dimensional structure, using Robetta software (http://robetta.bakerlab.org/) 165 [21, 22]. The predicted structure models were superimposed onto the crystal structure of the S. 166 cerevisiae Rad51 H352Y mutant protein (PDB accession 3LDA) [23], using the Chimera 167 Matchmaker algorithm [24]. The predicted three-dimensional structure of XP_001609877 168 (model 4) was found to coordinate spatially extremely well with the 3LDA crystal structure, 169 including Walker A and B motifs, residue Q298 regulating access of ATP to the active site [23], 170 and the interaction domain sequences pivotal to filament formation (Fig. 1B). Based on these 171 results we refer to XP_001609877 as BbRad51, and BBOV_II003540 as the Bbrad51 gene 172 encoding this protein. The Bbrad51 gene was annotated with two predicted introns [25], a 173 structural model that was confirmed by RT-PCR amplification, cloning, and sequencing of the 174 full-length transcript Flanking sequences were recovered through 5'- and 3'-RACE reactions (see . CC-BY 4.0 International license a certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made available under The copyright holder for this preprint (which was not this version posted April 11, 2019. 130 The orthologous Bbrad51 gene was identified. The B. bovis rad51 gene For brevity, CE11rad51ko1H5 is 222 hereafter referred to as “ko1H5”. The structure of the modified Bbrad51 locus and loss or gain of 223 Bbrad51 or gfp-bsd transcription, respectively, was confirmed by diagnostic PCR, RT-PCR, and 224 Southern blotting, confirming the replacement of Bbrad51 with the gfp-bsd selection cassette 225 from pRad51KO (Fig. 2B; S2 and S3 Figures). PCR-mediated cloning and sequencing of the 226 modified locus confirmed that error-free double-crossover homologous recombination (HR) had 227 occurred. Two additional independent knockout clonal lines, CE11rad51ko2 and 228 CE11rad51ko3 (for brevity, referred to as “ko2” and “ko3”, respectively), were established 229 approximately one year later. These two lines were confirmed by diagnostic PCR and sequencing 230 of the insertion sites (not shown). Because of the lag in obtaining the latter two lines, tests of 231 phenotype initially were performed with ko1H5 and subsequently confirmed with the latter two. 232 As the results were generated in different experiments they are presented separately. 233 234 Bbrad51 knockout had no effects on growth or morphology Knockout 207 reactions and Southern blotting (S2 Table), and the sites to which each anneals, are indicated. 208 B. Diagnostic PCR was used to initially confirm Bbrad51 knock-out in each line, using the 209 primer pairs indicated. Locus structure was fully confirmed for the ko1H5 mutant line (lanes 210 ko1H5), using PCR, Southern blotting and sequencing. Bbrad51 knock-out was supported by 211 diagnostic PCR for CE11rad51ko2 and CE11rad51ko3 (lanes Rad51ko2 and Rad51ko3, 212 respectively). 214 Bbrad51-null B. bovis lines were established. Bbrad51 is a single copy gene in 215 the haploid B. bovis genome [25]. Therefore, a single-locus double-crossover strategy was used 216 to replace the gene with a selection cassette expressing GFP-Blasticidin-s deaminase fusion 217 protein (gfp-bsd; a gift from C.E. Suarez [10, 14]). The transfection plasmid, pRad51KO (Fig. 218 2A), was constructed and used to transform B. bovis CE11 line parasites to blasticidin-s 219 resistance. The transformed culture, and the clonal line CE11rad51ko1, and subclones 220 CE11rad51ko1H5, CE11rad51ko1C3, and CE11rad51ko1H6 derived from it, were screened by 221 PCR (primers used in this study are provided in S2 Table). For brevity, CE11rad51ko1H5 is 222 hereafter referred to as “ko1H5”. 130 The orthologous Bbrad51 gene was identified. The B. bovis rad51 gene The structure of the modified Bbrad51 locus and loss or gain of 223 Bbrad51 or gfp-bsd transcription, respectively, was confirmed by diagnostic PCR, RT-PCR, and 224 Southern blotting, confirming the replacement of Bbrad51 with the gfp-bsd selection cassette 225 from pRad51KO (Fig. 2B; S2 and S3 Figures). PCR-mediated cloning and sequencing of the 226 modified locus confirmed that error-free double-crossover homologous recombination (HR) had 227 occurred. Two additional independent knockout clonal lines, CE11rad51ko2 and 228 CE11rad51ko3 (for brevity, referred to as “ko2” and “ko3”, respectively), were established 229 approximately one year later. These two lines were confirmed by diagnostic PCR and sequencing 230 of the insertion sites (not shown). Because of the lag in obtaining the latter two lines, tests of 231 phenotype initially were performed with ko1H5 and subsequently confirmed with the latter two. 232 As the results were generated in different experiments they are presented separately. 130 The orthologous Bbrad51 gene was identified. The B. bovis rad51 gene The gene was replaced by double 203 homologous crossover, with a selectable gfp-bsd cassette (purple arrow) driven by the B. 204 bovis EF1-B hemi-promoter [10, 14], using flanking sequences from the locus (crossover 205 regions shown as green arrows). The structures of the Bbrad51 wt locus (top), pRAD51KO 206 plasmid (center), and the disrupted Bbrad51 locus (bottom) are shown. Primers used in PCR . CC-BY 4.0 International license a certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made available under The copyright holder for this preprint (which was not this version posted April 11, 2019. ; https://doi.org/10.1101/606590 doi: bioRxiv preprint . CC-BY 4.0 International license a ertified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made available under The copyright holder for this preprint (which was not this version posted April 11, 2019. ; https://doi.org/10.1101/606590 doi: bioRxiv preprint . CC-BY 4.0 International license a certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made available under The copyright holder for this preprint (which was not this version posted April 11, 2019. ; https://doi.org/10.1101/606590 doi: bioRxiv preprint 207 reactions and Southern blotting (S2 Table), and the sites to which each anneals, are indicated. 208 B. Diagnostic PCR was used to initially confirm Bbrad51 knock-out in each line, using the 209 primer pairs indicated. Locus structure was fully confirmed for the ko1H5 mutant line (lanes 210 ko1H5), using PCR, Southern blotting and sequencing. Bbrad51 knock-out was supported by 211 diagnostic PCR for CE11rad51ko2 and CE11rad51ko3 (lanes Rad51ko2 and Rad51ko3, 212 respectively). 213 214 Bbrad51-null B. bovis lines were established. Bbrad51 is a single copy gene in 215 the haploid B. bovis genome [25]. Therefore, a single-locus double-crossover strategy was used 216 to replace the gene with a selection cassette expressing GFP-Blasticidin-s deaminase fusion 217 protein (gfp-bsd; a gift from C.E. Suarez [10, 14]). The transfection plasmid, pRad51KO (Fig. 218 2A), was constructed and used to transform B. bovis CE11 line parasites to blasticidin-s 219 resistance. The transformed culture, and the clonal line CE11rad51ko1, and subclones 220 CE11rad51ko1H5, CE11rad51ko1C3, and CE11rad51ko1H6 derived from it, were screened by 221 PCR (primers used in this study are provided in S2 Table). 234 Bbrad51 knockout had no effects on growth or morphology. Knockout Mean differences in growth rate of as little as 2% per generation would result in 246 larger differences than were observed. Thus, within the limits of sensitivity of our assay no 247 differences in growth rates were observed between wild type and knockout lines. 248 249 Fig. 3. Growth of wild type and Bbrad51 knockout parasites. Shown is the comparative 250 growth over a 48h period (mean ± 1 s.d.) of A. B. bovis CE11 wt and the Bbrad51 knockout 251 clonal line, ko1H5, and of B. CE11 wt and the CE11rad51ko2 and CE11rad51ko3 lines. The 252 48h period of the assay represents 5-6 cell cycles. The data in A. and B. were generated in 253 independent experiments separated by a year, and so are presented separately. CE11, white 254 bars; ko1H5, hatched bars; CE11rad51ko2, gray bars; CE11rad51ko3, dark gray bars. 255 256 Assessment of MMS-sensitivity. One common means of assessing contributions of a 257 protein to DNA repair is to determine sensitivity to DNA damage deliberately induced by 258 chemical insult. In this study, we employed exposure to methylmethane sulfonate (MMS), a 259 chemical which alkylates guanines to 7-methylguanine and adenines to 3-methyladenine [28]. To 260 apply that approach in this situation, sensitivity of B. bovis CE11 to MMS first was titrated over 261 a range from 0 to 2000 M, employing a strategy of exposure to MMS for 90 minutes, with a 262 buffer washout of the MMS, followed by growth for 72 hours (S4 Fig.). Initially, we used a 263 microscopic approach to follow parasite growth, to avoid any concerns about MMS effects on 264 erythrocytes or residual DNA fluorescence from dead parasites. The results ranged from little or 265 no effect at ≤250 M to complete killing by exposure to 2000 M MMS. B. bovis CE11 and 266 ko1H5 lines then were compared routinely over a range from 125- 1000 M. B. bovis ko1H5 was 249 Fig. 3. Growth of wild type and Bbrad51 knockout parasites. Shown is the comparative 250 growth over a 48h period (mean ± 1 s.d.) of A. B. bovis CE11 wt and the Bbrad51 knockout 251 clonal line, ko1H5, and of B. CE11 wt and the CE11rad51ko2 and CE11rad51ko3 lines. The 252 48h period of the assay represents 5-6 cell cycles. The data in A. and B. 234 Bbrad51 knockout had no effects on growth or morphology. Knockout 235 of Bbrad51 had no observable effects on parasite morphology at the level of light microscopy 236 (not shown). Despite the expected significance of Rad51 function to DNA repair and genome . CC-BY 4.0 International license a certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made available under The copyright holder for this preprint (which was not this version posted April 11, 2019. ; https://doi.org/10.1101/606590 doi: bioRxiv preprint . CC-BY 4.0 International license a ertified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made available under The copyright holder for this preprint (which was not this version posted April 11, 2019. ; https://doi.org/10.1101/606590 doi: bioRxiv preprint 237 stability, no significant differences were observed in the growth rates of wild type and Bbrad51 238 knockout lines (Fig. 3). The growth data shown was obtained with a SybrGreen-based assay of 239 cumulative DNA content to avoid the subjectivity associated with microscopic assays, although 240 microscopic assessments of the percentage of parasitized erythrocytes on Giemsa-stained smears 241 of cultured parasites confirmed these results (not shown). Growth was followed for 48 hours 242 only, to avoid any artifactual differences caused by a need for media changes or cell 243 manipulations. However, as these parasites are not developmentally synchronous in their growth, 244 and have an 8-10 hour asexual cell cycle [27], this period represents approximately 5-6 complete 245 cell cycles. Mean differences in growth rate of as little as 2% per generation would result in 246 larger differences than were observed. Thus, within the limits of sensitivity of our assay no 247 differences in growth rates were observed between wild type and knockout lines. 248 239 cumulative DNA content to avoid the subjectivity associated with microscopic assays, although 240 microscopic assessments of the percentage of parasitized erythrocytes on Giemsa-stained smears 241 of cultured parasites confirmed these results (not shown). Growth was followed for 48 hours 242 only, to avoid any artifactual differences caused by a need for media changes or cell 243 manipulations. However, as these parasites are not developmentally synchronous in their growth, 244 and have an 8-10 hour asexual cell cycle [27], this period represents approximately 5-6 complete 245 cell cycles. 234 Bbrad51 knockout had no effects on growth or morphology. Knockout were generated in 253 independent experiments separated by a year, and so are presented separately. CE11, white 254 bars; ko1H5, hatched bars; CE11rad51ko2, gray bars; CE11rad51ko3, dark gray bars. 256 Assessment of MMS-sensitivity. One common means of assessing contributions of a 257 protein to DNA repair is to determine sensitivity to DNA damage deliberately induced by 258 chemical insult. In this study, we employed exposure to methylmethane sulfonate (MMS), a 259 chemical which alkylates guanines to 7-methylguanine and adenines to 3-methyladenine [28]. To 260 apply that approach in this situation, sensitivity of B. bovis CE11 to MMS first was titrated over 261 a range from 0 to 2000 M, employing a strategy of exposure to MMS for 90 minutes, with a 262 buffer washout of the MMS, followed by growth for 72 hours (S4 Fig.). Initially, we used a 263 microscopic approach to follow parasite growth, to avoid any concerns about MMS effects on 264 erythrocytes or residual DNA fluorescence from dead parasites. The results ranged from little or 265 no effect at ≤250 M to complete killing by exposure to 2000 M MMS. B. bovis CE11 and 266 ko1H5 lines then were compared routinely over a range from 125- 1000 M. B. bovis ko1H5 was . CC-BY 4.0 International license a ertified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made available under The copyright holder for this preprint (which was not this version posted April 11, 2019. ; https://doi.org/10.1101/606590 doi: bioRxiv preprint . CC-BY 4.0 International license a certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made available under The copyright holder for this preprint (which was not this version posted April 11, 2019. ; https://doi.org/10.1101/606590 doi: bioRxiv preprint 267 significantly more susceptible to DNA damage at 250 or 500 M MMS than CE11 wild-type 268 parasites (Fig. 4). In the CE11 line there was little or no effect seen at 250 M. B. bovis CE11 269 parasites exposed to 500 M showed a severe reduction in growth rate that quickly increased, 270 approaching that of 0 M controls by 48 hours. In contrast, ko1H5 parasites were significantly 271 reduced in growth at 250 M, and 500 M-treated parasites failed to regain normal growth even 272 by 72 hours post-treatment. 234 Bbrad51 knockout had no effects on growth or morphology. Knockout These results indicate that BbRad51 plays a role in recovery from 273 DNA damage due to nucleotide alkylation [29-32]. 274 275 Fig. 4. Effects of environmental insult on growth of Bbrad51 knockouts. B. bovis CE11 276 wild-type (CE11), ko1H5 (ko1), and CE11 parasites with pBbrad51wt_comp integrated at the 277 Bbrad51 locus (CE11_comp) were tested for relative sensitivity of growth to a 90-minute 278 exposure to varying concentrations of MMS, in a 72h terminal experiment (upper left panel). 279 Growth characteristics over time are shown for CE11 (upper right), ko1H5 (lower left), and 280 CE11_comp (lower right) following MMS exposure at the various concentrations. These 281 results suggest that the Bbrad51 locus is neither affected by nor refractory to manipulation by 282 recombination. 283 284 Re-integration of Bbrad51 gene. To confirm the contribution of BbRad51 to this 285 phenotype, we attempted to complement B. bovis ko1H5 parasites by replacement of the gfp-bsd 286 selection cassette integrated at the Bbrad51 locus with the vector, pBbrad51wt_comp. 287 pBbrad51wt_comp was designed to integrate by double crossover HR (S5 Fig.), replacing the 288 knockout construct with the intact Bbrad51 wt gene, along with a selection cassette expressing 289 the hDHFR gene [33]. CE11 wild-type parasites also were transformed with this vector to 290 control for any effects on parasite viability or sensitivity to MMS caused by manipulation of the 291 Bbrad51 locus. Importantly, successful complementation by double crossover HR would have 292 resulted in identical Bbrad51 locus structures in both CE11 and ko1H5 parasites. CE11 parasites 293 incorporated this vector on the first attempt. A similar vector, differing only by the addition of 294 enhanced green fluorescent protein (EGFP) sequences to the 5’ end of the Bbrad51 open reading 295 frame, also successfully integrated into the CE11 parasite genome without error in two of two 296 attempts, although each crossed over 3’ to the EGFP sequences and failed to incorporate the 284 Re-integration of Bbrad51 gene. To confirm the contribution of BbRad51 to this 285 phenotype, we attempted to complement B. bovis ko1H5 parasites by replacement of the gfp-bsd 286 selection cassette integrated at the Bbrad51 locus with the vector, pBbrad51wt_comp. 287 pBbrad51wt_comp was designed to integrate by double crossover HR (S5 Fig.), replacing the 288 knockout construct with the intact Bbrad51 wt gene, along with a selection cassette expressing 289 the hDHFR gene [33]. 234 Bbrad51 knockout had no effects on growth or morphology. Knockout CE11 wild-type parasites also were transformed with this vector to 290 control for any effects on parasite viability or sensitivity to MMS caused by manipulation of the 291 Bbrad51 locus. Importantly, successful complementation by double crossover HR would have 292 resulted in identical Bbrad51 locus structures in both CE11 and ko1H5 parasites. CE11 parasites 293 incorporated this vector on the first attempt. A similar vector, differing only by the addition of 294 enhanced green fluorescent protein (EGFP) sequences to the 5’ end of the Bbrad51 open reading 295 frame, also successfully integrated into the CE11 parasite genome without error in two of two 296 attempts, although each crossed over 3’ to the EGFP sequences and failed to incorporate the . CC-BY 4.0 International license a ertified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made available under The copyright holder for this preprint (which was not this version posted April 11, 2019. ; https://doi.org/10.1101/606590 doi: bioRxiv preprint . CC-BY 4.0 International license a certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made available under The copyright holder for this preprint (which was not this version posted April 11, 2019. ; https://doi.org/10.1101/606590 doi: bioRxiv preprint 297 fluorescent tag. However, HR readily occurred at this locus. In contrast, ko1H5 parasites could not 298 be complemented in this manner in 11 transformation attempts, 7 performed with linearized 299 vector (favoring double crossover homologous recombination) and 4 with circular vector 300 (favoring single-site integration of the entire plasmid). No integration of the transfection plasmid 301 occurred at the Bbrad51 locus through the intended double-crossover HR, or by single-site 302 integration at the Bbrad51 locus or ectopically at any other site. Our inability to achieve 303 integration via double-crossover HR in ko1H5 suggests that HR is significantly impaired in the 304 absence of BbRad51, consistent with BbRad51 playing a role in HR-based DNA repair. When 305 tested for MMS sensitivity, CE11/pBbrad51wt_comp parasites were indistinguishable from 306 CE11 wild-type (Fig. 4), indicating that modification of the Bbrad51 locus in this way had not 307 affected the ability of the parasite to repair DNA damage caused by MMS. 234 Bbrad51 knockout had no effects on growth or morphology. Knockout While we recognize 308 that CE11/pBbrad51wt_comp does not represent the complementation of the defect on a ko1H5 309 background that we had attempted to achieve, we suggest that this represents the probable 310 outcome of successful complementation of the knockout at the Bbrad51 locus. 311 312 Identification of putative B. bovis centromeres. Because we were unable to 313 complement the knockout phenotype through re-integration of the Bbrad51 gene into the 314 genome, we chose instead to attempt its stable expression from an episomal location. Prior work 315 with an artificial chromosome platform in P. berghei [16] suggested this could be a productive 316 approach for expression of exogenous genes in an HR-compromised parasite line. To create one 317 for B. bovis we first had to recover native B. bovis centromeric and telomeric sequences. Putative 318 B. bovis centromeres were identified by focusing on key features common to centromeres in 319 other organisms, including very high percent A+T content, internal repetitive sequences, and a 320 size large enough to mediate attachment of a kinetochore [34, 35]. In this study we searched the 321 genome for A+T-rich regions that are ≥ 2.5 S.D. below the mean G+C content, using a 322 computational window of 1000 bp and length cutoff of ≥ 2 Kbp. Each region so identified was 323 further analyzed by dotplot analysis for internal repetitive structure. Four regions fitting these 324 criteria were identified in the B. bovis C9.1 line nuclear genome [36], one per region 325 corresponding to the four B. bovis T2Bo chromosomes [25]. The internal repeat structures of the 326 B. bovis T2Bo chromosome 2 centromere and of the C9.1 line chromosome 3 centromere were 327 confirmed by dotplot analyses (S6 Fig.). This was done by plotting each sequence against itself, . CC-BY 4.0 International license a certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made available under The copyright holder for this preprint (which was not this version posted April 11, 2019. ; https://doi.org/10.1101/606590 doi: bioRxiv preprint . CC-BY 4.0 International license a certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made available under The copyright holder for this preprint (which was not this version posted April 11, 2019. ; https://doi.org/10.1101/606590 doi: bioRxiv preprint 328 using a 21 nucleotide sliding window (not shown). Each candidate matched well with the 329 predicted centromeric regions of the B. bovis T2Bo genome [25], and used by Kawazu and 330 coworkers in constructing the circular centromere-containing plasmid vector, pDHFR-gfp- 331 Bbcent2 [12]. We elected to use the putative centromere from the B. 312 Identification of putative B. bovis centromeres. Because we were unable to bovis C9.1 line chromosome 332 3 due to its shorter length of 2.3 Kbp, as compared with 3.5- 5 Kbp of the other three, and to 333 ensure full compatibility with derivatives of the B. bovis Mexico isolate, such as the C9.1 and 334 CE11 lines. 335 336 Stability of BbACc3. Parasites transformed with BbACc3 (Fig. 5) could be selected with 337 10 M pyrimethamine, the approximate IC90 for this drug, after approximately three weeks. 338 Parasite growth was somewhat slower than non-transformed parasites, with parasites typically 339 reaching a maximum PPE of approximately 3%. To assess stability of BbACc3, established 340 parasites that had been maintained under pyrimethamine selection for 49 days were split into 341 two. One culture was maintained in the presence of 2 M pyrimethamine, whereas the second 342 was grown in the absence of drug for 99 days. Each then was grown for 72h in the presence of a 343 range of pyrimethamine concentrations to measure effective IC50 values, along with B. bovis 344 CE11 and the integrated plasmid line, CE11/pBbrad51wt_comp. As seen in Fig. 6, B. bovis 345 CE11 had an IC50 value for pyrimethamine of approximately 2.5 M, whereas the line carrying 346 the integrated pBbrad51wt_comp plasmid was highly drug-resistant with an IC50 of 34 M. B. 347 bovis CE11/BbACc3 that had been maintained under drug selection exhibited an IC50 of 348 approximately 24 M, indicative of high resistance to drug and expression of hDHFR. In 349 contrast, CE11/BbACc3 that had been grown in the absence of drug pressure was 350 indistinguishable from wild type CE11 parasites (IC50 = 2.7 M). These results demonstrate that 351 significant expression of the exogenous gene, hDHFR, occurs from BbACc3, resulting in a ten- 352 fold increase in IC50. The drop to wild type levels of drug resistance in parasites grown in the 353 absence of continued selection suggests that either silencing of the hDHFR marker or loss of 354 BbACc3 occurred. We did not pursue identification of the cause, but it is clear that when 355 maintained under drug pressure BbACc3 was maintained and that the hDHFR marker remained 356 actively expressed. 336 Stability of BbACc3. Parasites transformed with BbACc3 (Fig. 5) could be selected with 337 10 M pyrimethamine, the approximate IC90 for this drug, after approximately three weeks. 312 Identification of putative B. bovis centromeres. Because we were unable to 338 Parasite growth was somewhat slower than non-transformed parasites, with parasites typically 339 reaching a maximum PPE of approximately 3%. To assess stability of BbACc3, established 340 parasites that had been maintained under pyrimethamine selection for 49 days were split into 341 two. One culture was maintained in the presence of 2 M pyrimethamine, whereas the second 342 was grown in the absence of drug for 99 days. Each then was grown for 72h in the presence of a 343 range of pyrimethamine concentrations to measure effective IC50 values, along with B. bovis 344 CE11 and the integrated plasmid line, CE11/pBbrad51wt_comp. As seen in Fig. 6, B. bovis 345 CE11 had an IC50 value for pyrimethamine of approximately 2.5 M, whereas the line carrying 346 the integrated pBbrad51wt_comp plasmid was highly drug-resistant with an IC50 of 34 M. B. 347 bovis CE11/BbACc3 that had been maintained under drug selection exhibited an IC50 of 348 approximately 24 M, indicative of high resistance to drug and expression of hDHFR. In 349 contrast, CE11/BbACc3 that had been grown in the absence of drug pressure was 350 indistinguishable from wild type CE11 parasites (IC50 = 2.7 M). These results demonstrate that 351 significant expression of the exogenous gene, hDHFR, occurs from BbACc3, resulting in a ten- 352 fold increase in IC50. The drop to wild type levels of drug resistance in parasites grown in the 353 absence of continued selection suggests that either silencing of the hDHFR marker or loss of 354 BbACc3 occurred. We did not pursue identification of the cause, but it is clear that when 355 maintained under drug pressure BbACc3 was maintained and that the hDHFR marker remained 356 actively expressed. 357 . CC-BY 4.0 International license a certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made available under The copyright holder for this preprint (which was not this version posted April 11, 2019. ; https://doi.org/10.1101/606590 doi: bioRxiv preprint . CC-BY 4.0 International license a certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made available under The copyright holder for this preprint (which was not this version posted April 11, 2019. ; https://doi.org/10.1101/606590 doi: bioRxiv preprint 358 Fig. 5. Components and structure of pBbACc3 and pBbACc3_Bbrad51wt. A. 312 Identification of putative B. bovis centromeres. Because we were unable to CC-BY 4.0 International license a certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made available under The copyright holder for this preprint (which was not this version posted April 11, 2019. ; https://doi.org/10.1101/606590 doi: bioRxiv preprint 389 partially-digested genomic DNAs from B. bovis CE11 wild-type (CE11), 390 ko1H5/BbACc3_Bbrad51wt (ko1H5/Br51), and ko1H5 Bbrad51 knockouts (ko1H5). Lanes 391 are: (1) PstI-cut purified DNA, (lane 2) chromatin partially-digested with 0 U, (lane 3) 0.0075 392 U, (lane 4) 0.015 U, (lane 5) 0.03 U, or (lane 6) 0.06 U of MNase prior to isolation of DNA. 393 Asterisks demonstrate correspondence among the nucleosomal patterns generated from the 394 samples, which form a ladder with 156-159 bp spacing between bands, as previously reported 395 [37]. (center panel) Southern blot of the same gel as in the left panel, probed with -lactamase 396 sequences to detect the pBluescript backbone of BbACc3_Bbrad51wt. (right panel) The same 397 blot stripped and re-probed with Bbrad51 coding sequences to detect endogenous Bbrad51 398 (CE11), both BbACc3_Bbrad51wt-provided and non-deleted 3’ sequences (ko1H5/Br51), and 399 remaining non-deleted sequences from the 3’ end of Bbrad51 (ko1H5). B. Southern blot 400 probing of plasmids and genomic DNAs from B. bovis lines with Bbrad51 coding sequences 401 (left panel). The same blot was stripped and re-probed with -lactamase sequences (right 402 panel). Overall telomeric lengthening of BbACc3_Bbrad51wt by 2-3 Kbp can be seen by 403 comparing lanes 4 and 7 (both probes, arrowheads). Telomeric lengthening of 1-1.5 Kbp at 404 each end of BbACc3_Bbrad51wt can be observed with either probe (compare lanes 11 and 405 13; arrowheads). Extreme lengthening of the “left” half of BbACc3 (compare lanes 3 and 6, 406 or 10 and 12, asterisks) when using the -lactamase probe, perhaps due to crossover with a 407 native chromosome. Lanes are: (1) B. bovis CE11 wt gDNA; (2) B. 312 Identification of putative B. bovis centromeres. Because we were unable to The 359 structure of pBbACc3 in circular plasmid form, prior to removal of stuffer DNA. B. 360 Linearized form of BbACc3_Bbrad51wt following removal of stuffer DNA by cleavage with 361 PmeI restriction endonuclease. Rap1 3’UTR, B. bovis rhoptry-associated protein-1 gene 3’ 362 untranslated region; bla, -lactamase coding sequences; CDS, coding sequences; Bb Ch3, B. 363 bovis C9.1 line chromosome 3; hDHFR, human dihydrofolate reductase coding sequences. 364 365 Fig. 6. Pyrimethamine-sensitivity in the presence of BbACc3. Sensitivity profiles are 366 shown for B. bovis CE11, and CE11 transformed with BbACc3 or pBbrad51wt_comp. The 367 presence of either the artificial chromosome, BbACc3, or the integrated construct, 368 pBbrad51wt_comp, results in significant resistance to pyrimethamine. The IC50 value for each 369 line was approximately 2.5 M for CE11, 34 M for CE11/pBbrad51wt_comp, 24 M for 370 CE11/BbACc3 (maintained under constant pyrimethamine pressure; BbACc3 (+pyr)), and 2.7 371 M for CE11/BbACc3 three weeks after relief of drug pressure (BbACc3 (no pyr)). 372 373 Adapatation of BbAcC3 derivatives over time. For BbAcC3 and modified 374 derivatives to be used successfully for many purposes would require that this construct behave 375 like a real chromosome, including assembly into chromatin and expansion of telomeric ends for 376 stability. To test for such adaptation over time, we performed partial micrococcal nuclease 377 (MNase) digestion of isolated chromatin. Digestion products were probed by staining with 378 SybrGreen to detect total chromatin, and by Southern blotting to detect BbAcC3_Bbrad51- 379 derived sequences. BbAcC3_Bbrad51wt sequences provided a ladder of digestion products with 380 a nucleosomal periodicity of approximately 157-158 bp, matching that of total native chromatin 381 (Fig. 7A) and previously reported values [37], indicative of assembly on nucleosomes. Southern 382 blot probing of isolated plasmid or genomic DNAs, either uncut or cut with informative 383 restriction endonucleases, revealed the expansion of both ends of established BbAcC3 and 384 BbAcC3_Bbrad51 chromosomes, indicative of telomere lengthening (Fig. 7B). 385 386 Fig. 7. Assembly of BbACc3_Bbrad51wt into chromatin. Isolated parasite nuclei were 387 subjected to digestion with titrated concentrations of MNase, then Southern-blotted and 388 probed with different sequences. A. (left panel) SybrGreen-stained agarose gel of MNase 386 Fig. 7. Assembly of BbACc3_Bbrad51wt into chromatin. Isolated parasite nuclei were 387 subjected to digestion with titrated concentrations of MNase, then Southern-blotted and 388 probed with different sequences. A. (left panel) SybrGreen-stained agarose gel of MNase . 312 Identification of putative B. bovis centromeres. Because we were unable to bovis ko1H5 gDNA; (3) 408 ko1H5/ BbACc3 gDNA (uncut); (4) ko1H5/ BbACc3_Bbrad51 gDNA (uncut); (5) CE11/ 409 pBbrad51wt_comp gDNA (uncut); (6) pBbACc3 plasmid (+ PmeI); (7) 410 pBbACc3_Bbrad51wt plasmid (+ PmeI); (8) CE11 gDNA (+ BamHI); (9) ko1H5 gDNA (+ 411 BamHI); (10) ko1H5/ BbACc3 gDNA (+ BamHI); (11) ko1H5/ BbACc3_Bbrad51wt gDNA (+ 412 BamHI); (12) pBbACc3 plasmid (+ BamHI, PmeI); (13) pBbACc3_Bbrad51wt plasmid (+ 413 BamHI, PmeI); (14) CE11 gDNA (+ EcoRI); (15) CE11/ pBbrad51wt comp gDNA (+ 389 partially-digested genomic DNAs from B. bovis CE11 wild-type (CE11), 390 ko1H5/BbACc3_Bbrad51wt (ko1H5/Br51), and ko1H5 Bbrad51 knockouts (ko1H5). Lanes 391 are: (1) PstI-cut purified DNA, (lane 2) chromatin partially-digested with 0 U, (lane 3) 0.0075 392 U, (lane 4) 0.015 U, (lane 5) 0.03 U, or (lane 6) 0.06 U of MNase prior to isolation of DNA. 393 Asterisks demonstrate correspondence among the nucleosomal patterns generated from the 394 samples, which form a ladder with 156-159 bp spacing between bands, as previously reported 395 [37]. (center panel) Southern blot of the same gel as in the left panel, probed with -lactamase 396 sequences to detect the pBluescript backbone of BbACc3_Bbrad51wt. (right panel) The same 397 blot stripped and re-probed with Bbrad51 coding sequences to detect endogenous Bbrad51 398 (CE11), both BbACc3_Bbrad51wt-provided and non-deleted 3’ sequences (ko1H5/Br51), and 399 remaining non-deleted sequences from the 3’ end of Bbrad51 (ko1H5). B. Southern blot 400 probing of plasmids and genomic DNAs from B. bovis lines with Bbrad51 coding sequences 401 (left panel). The same blot was stripped and re-probed with -lactamase sequences (right 402 panel). Overall telomeric lengthening of BbACc3_Bbrad51wt by 2-3 Kbp can be seen by 403 comparing lanes 4 and 7 (both probes, arrowheads). Telomeric lengthening of 1-1.5 Kbp at 404 each end of BbACc3_Bbrad51wt can be observed with either probe (compare lanes 11 and 405 13; arrowheads). Extreme lengthening of the “left” half of BbACc3 (compare lanes 3 and 6, 406 or 10 and 12, asterisks) when using the -lactamase probe, perhaps due to crossover with a 407 native chromosome. Lanes are: (1) B. bovis CE11 wt gDNA; (2) B. bovis ko1H5 gDNA; (3) 408 ko1H5/ BbACc3 gDNA (uncut); (4) ko1H5/ BbACc3_Bbrad51 gDNA (uncut); (5) CE11/ 409 pBbrad51wt_comp gDNA (uncut); (6) pBbACc3 plasmid (+ PmeI); (7) 417 Complementation of Bbrad51 knockout phenotype. Because we were It is thus important that we understand well the biology of these 446 parasites, and prepare to defend against their expansion. Key to understanding the biology of 420 CE11 wild type and ko1H5 knockout parasites were transformed by transfection with the artificial 421 chromosome constructs, BbACc3 (as negative control) and BbACc3_Bbrad51wt. 420 CE11 wild type and ko1H5 knockout parasites were transformed by transfection with the artificial 421 chromosome constructs, BbACc3 (as negative control) and BbACc3_Bbrad51wt. 420 CE11 wild type and ko1H5 knockout parasites were transformed by transfection with the artificial 421 chromosome constructs, BbACc3 (as negative control) and BbACc3_Bbrad51wt. 422 pBbACc3_Bbrad51wt is built upon pBbACc3 but also contains the entire uninterrupted Bbrad51 423 locus, including promoter and 3’-termination sequences to facilitate proper regulation (Fig. 5B). 424 ko1H5/ BbAC_Bbrad51wt parasites displayed reduced sensitivity to MMS compared with ko1H5 425 or ko1H5/ BbACc3 parasites, that was not significantly different from wild-type. While not quite 426 reaching wild type levels, this result indicates successful complementation by providing the 427 Bbrad51 locus on the BbACc3 platform. The contribution of ko1H5/ BbBACc3_Bbrad51wt is 428 especially clear when compared with ko1H5/ BbACc3 parasites, which appear to be somewhat 429 less robust overall than ko1H5 (Fig. 8). 431 Fig. 8. Complementation of the MMS-sensitivity phenotype by Bbrad51 expressed from 432 the BbACc3 platform. B. bovis parasites of the CE11 (wild type), ko1H5 (Bbrad51 knockout), 433 ko1H5/BbACc3_Bbrad51wt, and ko1Ht/BbACc3 lines were exposed to varying concentrations 434 of MMS for 90 minutes, followed by drug washout. Parasites then were placed back into 435 culture for 72h, and growth assessed by the SybrGreen method. Growth characteristics were 436 plotted as the growth of each line relative to itself, employing 0 M as maximal growth and 437 1000 M MMS as no growth. Asterisks indicate a difference from the CE11 sample at a 438 significance of p  0.05. The presence of BbACc3_Bbrad51wt complemented ko1H5 439 sensitivity to 250 M MMS to a level that was not significantly different from wild-type 440 parasites. The presence of BbACc3 sequences, however, appears to impact somewhat the 441 overall robustness of parasites. 443 Discussion. With increasing human population density and environmental encroachment, 444 as well as climate change, many zoonotic diseases are emerging and/or expanding in range, 445 including babesiosis [38-40]. It is thus important that we understand well the biology of these 446 parasites, and prepare to defend against their expansion. 417 Complementation of Bbrad51 knockout phenotype. Because we were 418 unable to complement the loss of Bbrad51 by knock-in of the gene via double crossover HR, we 419 attempted complementation with the entire Bbrad51 locus provided via the BbACc3 platform. . CC-BY 4.0 International license a certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made available under The copyright holder for this preprint (which was not this version posted April 11, 2019. ; https://doi.org/10.1101/606590 doi: bioRxiv preprint . CC-BY 4.0 International license a ertified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made available under The copyright holder for this preprint (which was not this version posted April 11, 2019. ; https://doi.org/10.1101/606590 doi: bioRxiv preprint 420 CE11 wild type and ko1H5 knockout parasites were transformed by transfection with the artificial 421 chromosome constructs, BbACc3 (as negative control) and BbACc3_Bbrad51wt. 422 pBbACc3_Bbrad51wt is built upon pBbACc3 but also contains the entire uninterrupted Bbrad51 423 locus, including promoter and 3’-termination sequences to facilitate proper regulation (Fig. 5B). 424 ko1H5/ BbAC_Bbrad51wt parasites displayed reduced sensitivity to MMS compared with ko1H5 425 or ko1H5/ BbACc3 parasites, that was not significantly different from wild-type. While not quite 426 reaching wild type levels, this result indicates successful complementation by providing the 427 Bbrad51 locus on the BbACc3 platform. The contribution of ko1H5/ BbBACc3_Bbrad51wt is 428 especially clear when compared with ko1H5/ BbACc3 parasites, which appear to be somewhat 429 less robust overall than ko1H5 (Fig. 8). 430 431 Fig. 8. Complementation of the MMS-sensitivity phenotype by Bbrad51 expressed from 432 the BbACc3 platform. B. bovis parasites of the CE11 (wild type), ko1H5 (Bbrad51 knockout), 433 ko1H5/BbACc3_Bbrad51wt, and ko1Ht/BbACc3 lines were exposed to varying concentrations 434 of MMS for 90 minutes, followed by drug washout. Parasites then were placed back into 435 culture for 72h, and growth assessed by the SybrGreen method. Growth characteristics were 436 plotted as the growth of each line relative to itself, employing 0 M as maximal growth and 437 1000 M MMS as no growth. Asterisks indicate a difference from the CE11 sample at a 438 significance of p  0.05. The presence of BbACc3_Bbrad51wt complemented ko1H5 439 sensitivity to 250 M MMS to a level that was not significantly different from wild-type 440 parasites. 417 Complementation of Bbrad51 knockout phenotype. Because we were The presence of BbACc3 sequences, however, appears to impact somewhat the 441 overall robustness of parasites. 442 443 Discussion. With increasing human population density and environmental encroachment, 444 as well as climate change, many zoonotic diseases are emerging and/or expanding in range, 445 including babesiosis [38-40]. It is thus important that we understand well the biology of these 446 parasites, and prepare to defend against their expansion. Key to understanding the biology of 447 parasites at the molecular level is the ability to manipulate them genetically, something which is 420 CE11 wild type and ko1H5 knockout parasites were transformed by transfection with the artificial 421 chromosome constructs, BbACc3 (as negative control) and BbACc3_Bbrad51wt. 422 pBbACc3_Bbrad51wt is built upon pBbACc3 but also contains the entire uninterrupted Bbrad51 423 locus, including promoter and 3’-termination sequences to facilitate proper regulation (Fig. 5B). 424 ko1H5/ BbAC_Bbrad51wt parasites displayed reduced sensitivity to MMS compared with ko1H5 425 or ko1H5/ BbACc3 parasites, that was not significantly different from wild-type. While not quite 426 reaching wild type levels, this result indicates successful complementation by providing the 427 Bbrad51 locus on the BbACc3 platform. The contribution of ko1H5/ BbBACc3_Bbrad51wt is 428 especially clear when compared with ko1H5/ BbACc3 parasites, which appear to be somewhat 429 less robust overall than ko1H5 (Fig. 8). 430 431 Fig. 8. Complementation of the MMS-sensitivity phenotype by Bbrad51 expressed from 432 the BbACc3 platform. B. bovis parasites of the CE11 (wild type), ko1H5 (Bbrad51 knockout), 433 ko1H5/BbACc3_Bbrad51wt, and ko1Ht/BbACc3 lines were exposed to varying concentrations 434 of MMS for 90 minutes, followed by drug washout. Parasites then were placed back into 435 culture for 72h, and growth assessed by the SybrGreen method. Growth characteristics were 436 plotted as the growth of each line relative to itself, employing 0 M as maximal growth and 437 1000 M MMS as no growth. Asterisks indicate a difference from the CE11 sample at a 438 significance of p  0.05. The presence of BbACc3_Bbrad51wt complemented ko1H5 439 sensitivity to 250 M MMS to a level that was not significantly different from wild-type 440 parasites. The presence of BbACc3 sequences, however, appears to impact somewhat the 441 overall robustness of parasites. 442 443 Discussion. With increasing human population density and environmental encroachment, 444 as well as climate change, many zoonotic diseases are emerging and/or expanding in range, 445 including babesiosis [38-40]. 417 Complementation of Bbrad51 knockout phenotype. Because we were This was initially attempted 467 with pBbrad51wt_comp, which was designed to replace sequences associated with pBbrad51ko 468 and reinsert the Bbrad51 gene back into the genome at its native locus, where it would be under 469 control of its own promoter and terminator elements. However, in 11 attempts, eight performed 470 with linearized vector and three with circular plasmid, no successful transformations were 471 achieved, whereas three out of three attempts were successful in wild-type B. bovis CE11 472 parasites, using linearized vector. We interpret this difference as support for the pivotal 473 importance of Rad51 proteins to HR, including in B. bovis. However, while supportive, these 474 results also left the Bbrad51 gene without complementation to provide formal proof of its 475 involvement. Therefore, we sought to achieve complementation by another means. 476 477 In recent years, progress has been made in the ability to genetically modify B. bovis, with the 478 development of methods for transient and stable transfection with episomally-maintained 479 plasmids [9, 11, 14, 44-46], and by targeted integration via single- [12, 47] or double-crossover 450 parasite which, if compromised, could lead to its control during infection [41]. At least two 451 mechanisms are used to evade an ongoing immune response: cytoadhesion in the deep 452 vasculature and rapid antigenic variation of the cytoadhesion ligand [6]. Previously, we have 453 shown that antigenic variation relies in large part upon segmental gene conversion (SGC) for 454 modification of the expressed member of the ves gene family [42]. With SGC assumed to be a 455 form of homologous recombination, a process thought to be dependent upon Rad51 proteins, we 456 knocked out the Bbrad51 gene to begin an assessment of its contribution to this phenomenon. 457 Here, we demonstrate that a lack of BbRad51 led to hypersensitivity to MMS, an alkylating 458 agent that methylates adenosine bases [43]. The methyl adducts can result in stalled replication 459 forks and/or single-stranded DNA breaks, both of which can advance to double-stranded DNA 460 breaks, and lead to MMS-hypersensitivity in organisms lacking Rad51 [29-31]. 461 462 We confirmed in two ways that the MMS-hypersensitive phenotype observed in the Bbrad51 463 knockout lines was due to the loss of BbRad51 and not some artifactual secondary effect caused 464 by modification of the genome. 417 Complementation of Bbrad51 knockout phenotype. Because we were Key to understanding the biology of 447 parasites at the molecular level is the ability to manipulate them genetically, something which is 448 not yet well developed for babesial parasites. We have been especially interested in the 449 mechanisms of immune evasion employed by B. bovis as a potential “Achilles heel” of this . CC-BY 4.0 International license a certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made available under The copyright holder for this preprint (which was not this version posted April 11, 2019. ; https://doi.org/10.1101/606590 doi: bioRxiv preprint . CC-BY 4.0 International license a ertified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made available under The copyright holder for this preprint (which was not this version posted April 11, 2019. ; https://doi.org/10.1101/606590 doi: bioRxiv preprint 450 parasite which, if compromised, could lead to its control during infection [41]. At least two 451 mechanisms are used to evade an ongoing immune response: cytoadhesion in the deep 452 vasculature and rapid antigenic variation of the cytoadhesion ligand [6]. Previously, we have 453 shown that antigenic variation relies in large part upon segmental gene conversion (SGC) for 454 modification of the expressed member of the ves gene family [42]. With SGC assumed to be a 455 form of homologous recombination, a process thought to be dependent upon Rad51 proteins, we 456 knocked out the Bbrad51 gene to begin an assessment of its contribution to this phenomenon. 457 Here, we demonstrate that a lack of BbRad51 led to hypersensitivity to MMS, an alkylating 458 agent that methylates adenosine bases [43]. The methyl adducts can result in stalled replication 459 forks and/or single-stranded DNA breaks, both of which can advance to double-stranded DNA 460 breaks, and lead to MMS-hypersensitivity in organisms lacking Rad51 [29-31]. 461 462 We confirmed in two ways that the MMS-hypersensitive phenotype observed in the Bbrad51 463 knockout lines was due to the loss of BbRad51 and not some artifactual secondary effect caused 464 by modification of the genome. First, we integrated pBbrad51wt_comp into the Bbrad51 locus 465 and demonstrated that the phenotype was identical to wt upon exposure to MMS. Secondly, we 466 complemented the phenotype in parasites lacking the Bbrad51 gene. 417 Complementation of Bbrad51 knockout phenotype. Because we were First, we integrated pBbrad51wt_comp into the Bbrad51 locus 465 and demonstrated that the phenotype was identical to wt upon exposure to MMS. Secondly, we 466 complemented the phenotype in parasites lacking the Bbrad51 gene. This was initially attempted 467 with pBbrad51wt_comp, which was designed to replace sequences associated with pBbrad51ko 468 and reinsert the Bbrad51 gene back into the genome at its native locus, where it would be under 469 control of its own promoter and terminator elements. However, in 11 attempts, eight performed 470 with linearized vector and three with circular plasmid, no successful transformations were 471 achieved, whereas three out of three attempts were successful in wild-type B. bovis CE11 472 parasites, using linearized vector. We interpret this difference as support for the pivotal 473 importance of Rad51 proteins to HR, including in B. bovis. However, while supportive, these 474 results also left the Bbrad51 gene without complementation to provide formal proof of its 475 involvement. Therefore, we sought to achieve complementation by another means. 476 477 In recent years, progress has been made in the ability to genetically modify B. bovis, with the 478 development of methods for transient and stable transfection with episomally-maintained 479 plasmids [9, 11, 14, 44-46], and by targeted integration via single- [12, 47] or double-crossover 480 recombination [15, 45]. Although each approach is very useful, each also has its own limitations. . CC-BY 4.0 International license a certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made available under The copyright holder for this preprint (which was not this version posted April 11, 2019. ; https://doi.org/10.1101/606590 doi: bioRxiv preprint . CC-BY 4.0 International license a ertified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made available under The copyright holder for this preprint (which was not this version posted April 11, 2019. ; https://doi.org/10.1101/606590 doi: bioRxiv preprint . CC-BY 4.0 International license a certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made available under The copyright holder for this preprint (which was not this version posted April 11, 2019. 417 Complementation of Bbrad51 knockout phenotype. Because we were ; https://doi.org/10.1101/606590 doi: bioRxiv preprint 481 Transient transfection provides a short-term phenotype only, does not affect the entire parasite 482 population, and may not accurately reflect gene regulation. Segregation of episomes during 483 mitosis is inaccurate, resulting in a heterogeneous parasite population and plasmid loss from the 484 population over time. Such heterogeneity could compromise the ability to interpret 485 complementation of a phenotype that is not dramatic, such as the MMS-sensitivity phenotype of 486 Bbrad51 knockout parasites. This problem was partially solved by the inclusion of centromeric 487 sequences into the plasmid which dramatically improved segregation and resulted in sufficient 488 stability for a plasmid to be maintained in the absence of drug selection for two months [12]. 489 However, a circular construct retains the potential to integrate by single-crossover through 490 sequence elements within the plasmid, potentially compromising ongoing studies. We sought to 491 address this issue through the construction of a linear mini-chromosome, BbACc3, containing 492 centromeric and subtelomeric sequences, and telomeric repeats. BbACc3 is manipulated as a 493 plasmid (pBACc3) in E. coli for modification and production. It is then linearized for 494 transfection into B. bovis, where it is maintained as a linear mini-chromosome with legitimate 495 telomeric ends. Once established, BbACc3_Bbrad51 was found to assemble with nucleosomes 496 into chromatin (Fig. 7A), and to undergo expansion of the telomeric repeat regions (Fig. 7B). 497 These behaviors suggest that, once established, the BbACc3 platform behaves in a manner 498 consistent with a native chromosome. Here, BbACc3_Bbrad51, carrying the Bbrad51 gene and 499 its native flanking 5’ and 3’ regulatory sequences, was demonstrated to complement the lack of 500 BbRad51 in knockout parasites with regard to MMS-sensitivity. Moreover, complementation 501 occurred at levels not significantly different from wild-type, strongly supporting the notion that 502 BbRad51 contributes to DNA repair and consistent with a possible role in segmental gene 503 conversion. The ability to express sequences in a regulated fashion opens the possibility of using 504 BbACc3 as a platform to complement knockout of essential genes, or to perform studies of 505 promoter structure and functional interactions. Moreover, constructs carrying exogenous genes 506 could be used to engender transmission-blocking immunity by including vector proteins, to 507 immunize against other co-transmitted parasites by inclusion of appropriate vaccine genes, or 508 other similar applications. 417 Complementation of Bbrad51 knockout phenotype. Because we were 509 510 In the course of these studies we found it useful to construct and employ an artificial 481 Transient transfection provides a short-term phenotype only, does not affect the entire parasite 482 population, and may not accurately reflect gene regulation. Segregation of episomes during 483 mitosis is inaccurate, resulting in a heterogeneous parasite population and plasmid loss from the 484 population over time. Such heterogeneity could compromise the ability to interpret 485 complementation of a phenotype that is not dramatic, such as the MMS-sensitivity phenotype of 486 Bbrad51 knockout parasites. This problem was partially solved by the inclusion of centromeric 487 sequences into the plasmid which dramatically improved segregation and resulted in sufficient 488 stability for a plasmid to be maintained in the absence of drug selection for two months [12]. 489 However, a circular construct retains the potential to integrate by single-crossover through 490 sequence elements within the plasmid, potentially compromising ongoing studies. We sought to 491 address this issue through the construction of a linear mini-chromosome, BbACc3, containing 492 centromeric and subtelomeric sequences, and telomeric repeats. BbACc3 is manipulated as a 493 plasmid (pBACc3) in E. coli for modification and production. It is then linearized for 494 transfection into B. bovis, where it is maintained as a linear mini-chromosome with legitimate 495 telomeric ends. Once established, BbACc3_Bbrad51 was found to assemble with nucleosomes 496 into chromatin (Fig. 7A), and to undergo expansion of the telomeric repeat regions (Fig. 7B). 497 These behaviors suggest that, once established, the BbACc3 platform behaves in a manner 498 consistent with a native chromosome. Here, BbACc3_Bbrad51, carrying the Bbrad51 gene and 499 its native flanking 5’ and 3’ regulatory sequences, was demonstrated to complement the lack of 500 BbRad51 in knockout parasites with regard to MMS-sensitivity. Moreover, complementation 501 occurred at levels not significantly different from wild-type, strongly supporting the notion that 502 BbRad51 contributes to DNA repair and consistent with a possible role in segmental gene 503 conversion. The ability to express sequences in a regulated fashion opens the possibility of using 504 BbACc3 as a platform to complement knockout of essential genes, or to perform studies of 505 promoter structure and functional interactions. Moreover, constructs carrying exogenous genes 506 could be used to engender transmission-blocking immunity by including vector proteins, to 507 immunize against other co-transmitted parasites by inclusion of appropriate vaccine genes, or 508 other similar applications. 417 Complementation of Bbrad51 knockout phenotype. Because we were 509 510 In the course of these studies we found it useful to construct and employ an artificial 481 Transient transfection provides a short-term phenotype only, does not affect the entire parasite 482 population, and may not accurately reflect gene regulation. Segregation of episomes during 483 mitosis is inaccurate, resulting in a heterogeneous parasite population and plasmid loss from the 484 population over time. Such heterogeneity could compromise the ability to interpret 510 In the course of these studies we found it useful to construct and employ an artificial 511 chromosome, BbACc3, as a platform for the reintroduction of the Bbrad51 gene into B. bovis. . CC-BY 4.0 International license a certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made available under The copyright holder for this preprint (which was not this version posted April 11, 2019. ; https://doi.org/10.1101/606590 doi: bioRxiv preprint . CC-BY 4.0 International license a certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made available under The copyright holder for this preprint (which was not this version posted April 11, 2019. ; https://doi.org/10.1101/606590 doi: bioRxiv preprint 512 This allowed us to circumvent the need for homologous recombination to reintegrate Bbrad51 513 sequences into the genome in order to acquire a uniform population. Significantly, 514 BbACc3_Bbrad51wt enabled the complementation of the Bbrad51 knockout phenotype at near- 515 normal levels in response to MMS challenge. Once established, the BbACc3 platform behaved 516 like a native chromosome, forming chromatin and lengthening its telomeres. Despite these 517 positive attributes, a cautionary note must also be provided. Southern blot results suggest that 518 within CE11 wild-type parasites, the major parasite population to grow up during selection 519 appears to have undergone a crossover event between BbACc3 and one of the native 520 chromosomes (Fig. 7B). This appears to have occurred through subtelomeric or telomeric repeat 521 sequences at the “left” end near the -lactamase coding sequences, resulting in significant 522 expansion of that end of BbACc3, and an overall much longer artificial chromosome than in 523 ko1H5/BbACc3_Bbrad51wt, even after expansion of its telomeric repeats. An event of this nature 524 would always be a potential hazard in any HR-competent parasite line. 542 Parasites and culture conditions. B. bovis parasites of the CE11 clonal line were 542 Parasites and culture conditions. B. bovis parasites of the CE11 clonal line were 543 used in experiments [7]. Parasites were grown under microaerophilous stationary phase culture 544 conditions, with an atmosphere of 90:5:5 nitrogen: oxygen: carbon dioxide [49, 50]. Defibrinated 545 bovine blood products were obtained locally from Holstein cows (approved by University of 546 Florida Institutional Animal Care and Use Committee, protocol #201102216) or commercially 547 (Hemostat Laboratories; Dixon, CA). Parasite sensitivity to pyrimethamine was determined as 548 described [51]. Transformed parasites were maintained in medium containing 2.0 M 549 pyrimethamine. 542 Parasites and culture conditions. B. bovis parasites of the CE11 clonal line were 543 used in experiments [7]. Parasites were grown under microaerophilous stationary phase culture 544 conditions, with an atmosphere of 90:5:5 nitrogen: oxygen: carbon dioxide [49, 50]. Defibrinat 545 bovine blood products were obtained locally from Holstein cows (approved by University of 546 Florida Institutional Animal Care and Use Committee, protocol #201102216) or commercially 547 (Hemostat Laboratories; Dixon, CA). Parasite sensitivity to pyrimethamine was determined as 548 described [51]. Transformed parasites were maintained in medium containing 2.0 M 549 pyrimethamine. 550 551 Validation of Bbrad51 gene and BbRad51 identity. Identification of 552 BbRad51 (accession no. XP_001609877) was achieved by reciprocal Psi-BLAST searches of th 553 B. bovis T2Bo genome [25], using S. cerevisiae Rad51 (accession no. CAA45563) to initiate th 554 query. Alignments used sequences publicly available through Genbank, EuPathDB database 555 (http://eupathdb.org/eupathdb/), and/or Wellcome- Sanger Institute pathogens ftp site 556 (ftp://ftp.sanger.ac.uk/pub/pathogens/). Annotation of Bbrad51 gene structure was confirmed a 557 both the genome (GeneID: 5478106) and transcript (XM_001609827.1) levels as described 558 below. Virtual translation and routine manipulations performed with CLC-Bio Main Workbenc 559 (CLC-Bio, Arrhenius, Denmark). Conserved Domains search was performed through the 560 National Center for Biotechnology CDD webserver 561 (http://www.ncbi.nlm.nih.gov/Structure/cdd/cdd.shtml) [52, 53]. Three-dimensional structural 562 modeling was performed through the Robetta webserver (http://robetta.bakerlab.org/) [22, 54]. 563 Structural superimpositions and graphics were created with the Chimera software package of th 564 Resource for Biocomputing, Visualization, and Informatics at the University of California, San 565 Francisco (www.cgl.ucsf.edu/chimera/), using the Matchmaker algorithm [24]. 566 417 Complementation of Bbrad51 knockout phenotype. Because we were A useful improvement of 525 BbACc3 could be made through the inclusion of replication origin sequences that might improve 526 the ease of initial establishment of the chromosome, which in these experiments was no faster 527 than selection of double crossover integration mutants. Thus, while artificial chromosome 528 technology holds considerable promise for certain applications and was invaluable in this study, 529 it will require optimization to meet its full potential. Regardless, BbACc3 provides a new option 530 in the toolkit for genetic manipulation of B. bovis and perhaps other babesial parasites where its 531 centromeric and regulatory sequences may function, and will be made freely available to the 532 scientific community. . CC-BY 4.0 International license a certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made available under The copyright holder for this preprint (which was not this version posted April 11, 2019. ; https://doi.org/10.1101/606590 doi: bioRxiv preprint . CC-BY 4.0 International license a certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made available under The copyright holder for this preprint (which was not this version posted April 11, 2019. ; https://doi.org/10.1101/606590 doi: bioRxiv preprint 551 Validation of Bbrad51 gene and BbRad51 identity. Identification of 552 BbRad51 (accession no. XP_001609877) was achieved by reciprocal Psi-BLAST searches of the 553 B. bovis T2Bo genome [25], using S. cerevisiae Rad51 (accession no. CAA45563) to initiate the 554 query. Alignments used sequences publicly available through Genbank, EuPathDB database 555 (http://eupathdb.org/eupathdb/), and/or Wellcome- Sanger Institute pathogens ftp site 556 (ftp://ftp.sanger.ac.uk/pub/pathogens/). Annotation of Bbrad51 gene structure was confirmed at 557 both the genome (GeneID: 5478106) and transcript (XM_001609827.1) levels as described 558 below. Virtual translation and routine manipulations performed with CLC-Bio Main Workbench 559 (CLC-Bio, Arrhenius, Denmark). Conserved Domains search was performed through the 560 National Center for Biotechnology CDD webserver 561 (http://www.ncbi.nlm.nih.gov/Structure/cdd/cdd.shtml) [52, 53]. Three-dimensional structural 562 modeling was performed through the Robetta webserver (http://robetta.bakerlab.org/) [22, 54]. 563 Structural superimpositions and graphics were created with the Chimera software package of the 564 Resource for Biocomputing, Visualization, and Informatics at the University of California, San 565 Francisco (www.cgl.ucsf.edu/chimera/), using the Matchmaker algorithm [24]. 567 Validation of Bbrad51 gene structure. B. bovis gDNAs were isolated as described Tobacco acid pyrophosphatase was used to remove the 5’-cap structure for adapter 590 addition, and reverse transcription using M-MuLV and random decamers. Nested PCR 591 amplification of 5’-untranslated sequences was performed using primers EAM2 and 5’-RACE 592 outer, and EAM4 and 5’-RACE inner primers. Products were cloned into pCR-TOPO-Blunt and 593 sequenced. The 3’-untranslated sequences were obtained by addition of the 3’-RACE adapter, 594 and nested PCR amplification with primers EAM1 and 3’-RACE outer, and EAM3 and 3’- 595 RACE inner. Products were directly cloned into pCR-TOPO-Blunt and sequenced. 596 572 [58]. When needed, cultures were grown to elevated levels of percent parasitized erythrocytes by 573 “dilution enrichment” [59]. Packed erythrocytes were emulsified with either TRIzol reagent 574 (Invitrogen; Waltham, MA) or RiboZol reagent (Amresco; Solon, OH), extracted twice with 575 chloroform and precipitated from 2-propanol. A 2244 bp segment containing the 1134 bp 576 Bbrad51 gene and its 5´ and 3´ intergenic regions was amplified from B. bovis C9.1 gDNA by 577 polymerase chain reaction (PCR) with Phusion High-Fidelity DNA Polymerase (New England 578 BioLabs; Beverley, MA) using primers EAM6 and EAM18. This segment was cloned into 579 pCR2.1-TOPO-TA (Invitrogen) to create plasmid pBbRad51. Bbrad51 structure was confirmed 580 by PCR amplification of the 1134 bp gene within the 2244 bp amplicon, using flanking primers 581 EAM6 and EAM18. The amplicon was cloned into pCR2.1-TOPO-TA (Invitrogen) and 582 sequenced through the University of Florida Sanger Sequencing Core by primer-walking. To 583 characterize Bbrad51 transcripts, cDNA was made from B. bovis C9.1 line total RNA, using M- 584 MuLV Reverse Transcriptase (New England Biolabs; Beverley, MA) and oligo-d(T) primer. 585 Full-length coding sequences were obtained by PCR amplification of cDNA with primers 586 EAM50 and EAM51. The amplicon was cloned into pCR2.1-TOPO-TA and sequenced. To 587 obtain non-coding transcript sequences, RNA Ligase-mediated rapid amplification of cDNA 588 ends (RLM-RACE) was performed, using the FirstChoice RLM-RACE kit (Ambion; Waltham, 589 MA). Tobacco acid pyrophosphatase was used to remove the 5’-cap structure for adapter 590 addition, and reverse transcription using M-MuLV and random decamers. Nested PCR 591 amplification of 5’-untranslated sequences was performed using primers EAM2 and 5’-RACE 592 outer, and EAM4 and 5’-RACE inner primers. Products were cloned into pCR-TOPO-Blunt and 593 sequenced. 567 Validation of Bbrad51 gene structure. B. bovis gDNAs were isolated as described 567 Validation of Bbrad51 gene structure. B. bovis gDNAs were isolated as described 568 [55, 56], but with prior ammonium chloride lysis of the erythrocytes [57]. Alternatively, QIAmp 569 Mini Spin Columns (Qiagen) were sometimes used, following manufacturer’s instructions. For 570 RNA extractions, cultures were grown in erythrocytes from which host leukocytes were removed 571 by filtration through Whatman CF-11 cellulose (GE Healthcare Life Sciences; Pittsburgh, PA) . CC-BY 4.0 International license a certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made available under The copyright holder for this preprint (which was not this version posted April 11, 2019. ; https://doi.org/10.1101/606590 doi: bioRxiv preprint . CC-BY 4.0 International license a ertified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made available under The copyright holder for this preprint (which was not this version posted April 11, 2019. ; https://doi.org/10.1101/606590 doi: bioRxiv preprint 572 [58]. When needed, cultures were grown to elevated levels of percent parasitized erythrocytes by 573 “dilution enrichment” [59]. Packed erythrocytes were emulsified with either TRIzol reagent 574 (Invitrogen; Waltham, MA) or RiboZol reagent (Amresco; Solon, OH), extracted twice with 575 chloroform and precipitated from 2-propanol. A 2244 bp segment containing the 1134 bp 576 Bbrad51 gene and its 5´ and 3´ intergenic regions was amplified from B. bovis C9.1 gDNA by 577 polymerase chain reaction (PCR) with Phusion High-Fidelity DNA Polymerase (New England 578 BioLabs; Beverley, MA) using primers EAM6 and EAM18. This segment was cloned into 579 pCR2.1-TOPO-TA (Invitrogen) to create plasmid pBbRad51. Bbrad51 structure was confirmed 580 by PCR amplification of the 1134 bp gene within the 2244 bp amplicon, using flanking primers 581 EAM6 and EAM18. The amplicon was cloned into pCR2.1-TOPO-TA (Invitrogen) and 582 sequenced through the University of Florida Sanger Sequencing Core by primer-walking. To 583 characterize Bbrad51 transcripts, cDNA was made from B. bovis C9.1 line total RNA, using M- 584 MuLV Reverse Transcriptase (New England Biolabs; Beverley, MA) and oligo-d(T) primer. 585 Full-length coding sequences were obtained by PCR amplification of cDNA with primers 586 EAM50 and EAM51. The amplicon was cloned into pCR2.1-TOPO-TA and sequenced. To 587 obtain non-coding transcript sequences, RNA Ligase-mediated rapid amplification of cDNA 588 ends (RLM-RACE) was performed, using the FirstChoice RLM-RACE kit (Ambion; Waltham, 589 MA). 567 Validation of Bbrad51 gene structure. B. bovis gDNAs were isolated as described The 3’-untranslated sequences were obtained by addition of the 3’-RACE adapter, 594 and nested PCR amplification with primers EAM1 and 3’-RACE outer, and EAM3 and 3’- 595 RACE inner. Products were directly cloned into pCR-TOPO-Blunt and sequenced. 596 597 Bbrad51 knockout plasmid assembly. The Bbrad51 gene was knocked out by a 598 double crossover homologous recombination strategy [60], with plasmid pRad51KO (Fig. 2A). 599 Plasmid pRad51KO was designed to replace all but the 3’-terminal 388 bp of Bbrad51 coding 600 sequences with a selectable gfp-bsd marker (a gift from C.E. Suarez). This region was retained 601 because of the close proximity (320 bp) of Bbrad51 to the gene downstream encoding a 602 conserved mechanosensitive channel-like protein, XP_001609876. To assemble pRad51KO, the 597 Bbrad51 knockout plasmid assembly. The Bbrad51 gene was knocked out by a 598 double crossover homologous recombination strategy [60], with plasmid pRad51KO (Fig. 2A). 599 Plasmid pRad51KO was designed to replace all but the 3’-terminal 388 bp of Bbrad51 coding 600 sequences with a selectable gfp-bsd marker (a gift from C.E. Suarez). This region was retained 601 because of the close proximity (320 bp) of Bbrad51 to the gene downstream encoding a 602 conserved mechanosensitive channel-like protein, XP_001609876. To assemble pRad51KO, the . CC-BY 4.0 International license a certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made available under The copyright holder for this preprint (which was not this version posted April 11, 2019. ; https://doi.org/10.1101/606590 doi: bioRxiv preprint . CC-BY 4.0 International license a ertified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made available under The copyright holder for this preprint (which was not this version posted April 11, 2019. ; https://doi.org/10.1101/606590 doi: bioRxiv preprint 603 EF1 gene “B” hemi-promoter for driving the bsd selectable marker was PCR-amplified from B. 604 bovis CE11 line gDNA with primers EAM42 and EAM43. Bbrad51 3’-end targeting sequences 605 were amplified from plasmid pBbRAD51, using primers EAM49 and EAM56. The EF1-B 606 segment and Bbrad51 3’ targeting sequences were combined by crossover PCR [61] with 607 EAM43 and EAM56 to form fragment 3’-rad51-EF1-B. The 92 bp B. bovis -tubulin 608 terminator region (T3) was amplified from plasmid pDS-bsd [11], using primers EAM45 and 609 EAM46. 567 Validation of Bbrad51 gene structure. B. bovis gDNAs were isolated as described The gfp-bsd fusion gene from plasmid pGFP-Bsd [14], was combined with the T3 610 fragment by crossover PCR, using primers EAM44 and EAM46 and including the T3 fragment 611 in the reaction. The Bbrad51 upstream targeting sequence was added to the gfp-bsd-T3 fragment 612 by crossover PCR with primers EAM44 and EAM47, and including pBbRad51 in the reaction, to 613 form fragment gfp-bsd-T3-5’-rad51. The two fragments were combined by crossover PCR, using 614 primers EAM56 and EAM47 and inserted into pBluescript II-KS(+). A single missense mutation 615 was identified within the bsd sequence. This was corrected by PCR-amplification of the two 616 halves of the insert with EAM61 and EAM47, and EAM62 and EAM56. The two fragments 617 were fused by crossover PCR, using only EAM47 and EAM56, and reinserted into pBluescript 618 II-KS(+). The corrected plasmid, pRAD51KO, was transformed into Escherichia coli DH5. 619 Proper construction was confirmed by full sequencing, and the corrected plasmid used for 620 subsequent plasmid production. 622 Validation of Bbrad51 knock-out. Parasite populations recovered initially were 623 screened directly with the Phusion Blood Direct PCR Kit (Thermo Scientific), using primers 624 EAM75 and EAM77 within the coding sequences of the immediately flanking genes. Non- 625 mutated parasites generated a band of 2.4 Kbp, whereas a proper knockout yielded a band of 3.4 626 Kbp. RT-PCR with primers EAM50 and EAM51 confirmed the lack of Bbrad51 transcripts. 627 Southern blots were performed as described [42]. DNA fragments, alkaline-transferred to nylon 628 membranes (Amersham Hybond-N+, GE Healthcare), were cross-linked with 50 mJ ultraviolet in 629 a GS Gene Linker UV chamber (BioRad; Hercules, CA). Oligonucleotide probes were end- 630 labeled with -[32P]ATP, using polynucleotide kinase. 631 632 Parasite growth assays. Parasite growth was assayed by counting Giemsa-stained . CC-BY 4.0 International license a certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made available under The copyright holder for this preprint (which was not this version posted April 11, 2019. ; https://doi.org/10.1101/606590 doi: bioRxiv preprint . CC-BY 4.0 International license a ertified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made available under The copyright holder for this preprint (which was not this version posted April 11, 2019. 567 Validation of Bbrad51 gene structure. B. bovis gDNAs were isolated as described ; https://doi.org/10.1101/606590 doi: bioRxiv preprint 633 smears, with samples collected at 0, 24, and 48h growth (approximately 0, 3 and 6 cell cycles 634 [62]). Alternatively, in some experiments a DNA-based SYBR Green I method was performed, 635 essentially as described [63, 64], on parasites grown in bovine erythrocytes depleted of 636 leukocytes [58]. For measuring sensitivity to pyrimethamine, parasites were grown in complete 637 medium containing serial 10-fold dilutions of pyrimethamine for 72 hours, at concentrations 638 from 0 to 100 M. For experiments involving MMS, parasites were exposed at 2% ppe to 639 various concentrations of MMS (diluted in complete medium) for 90 minutes at room 640 temperature, then were washed two times at 6000 x g.min at room temperature with 1x VYM 641 buffer [65] to remove MMS. Parasites then were resuspended in complete medium, and placed 642 under normal culture conditions. In initial experiments, cells were diluted 1/10 into a 10% 643 packed cell volume suspension of untreated uninfected erythrocytes, and were placed back into 644 culture. Smears were made at 0, 24, 48, and 72 hours post-treatment, and Giemsa-stained for 645 microscopic counting of percent parasitized erythrocytes. After validation of effect, subsequent 646 experiments were performed using the SybrGreen method, with starting values of 0.2% 647 parasitized erythrocytes, and terminal collection at 72 hours only. An initial titration experiment 648 with B. bovis CE11 established 125 M to 1000 M as the informative range. 649 647 parasitized erythrocytes, and terminal collection at 72 hours only. An initial titration experiment 648 with B. bovis CE11 established 125 M to 1000 M as the informative range. 650 Assembly of complementation constructs. Two different complementation 650 Assembly of complementation constructs. Two different complementation 651 strategies were used in this project. (i) In the first strategy, plasmid pBbrad51wt_comp (S5 Fig.) 652 was engineered to replace all sequences associated with the knockout plasmid by double 653 crossover homologous recombination. The goal was to replace them with the wild-type Bbrad51 654 locus, concomitant with integration of a human dihydrofolate reductase (hDHFR) expression 655 cassette, to enable selection of transformants with pyrimethamine [51]. pBbrad51wt_comp was 656 made by PCR-amplifying Bbrad51 3’ terminator sequences from B. bovis C9.1 line gDNA with 657 primers DA255 + DA256. The hDHFR selection cassette was moved from pBACc3 by 658 amplification with primers DA254 + DA253. pBluescript-SK(+) was linearized with XhoI + 659 HindIII restriction endonucleases, and the three sequences were assembled simultaneously, using 660 InFusion reagents (Clontech). The Bbrad51 locus, including complete promoter through 661 immediate 3’ sequences, was amplified from B. bovis C9.1 line gDNA with primers DA248 + 662 DA252. The Bbrad51 locus was inserted, using InFusion reagents, into the intermediate 663 construct that had been opened with XbaI + HindIII. (ii) The second strategy was to reintroduce . CC-BY 4.0 International license a ertified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made available under The copyright holder for this preprint (which was not this version posted April 11, 2019. ; https://doi.org/10.1101/606590 doi: bioRxiv preprint . CC-BY 4.0 International license a certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made available under The copyright holder for this preprint (which was not this version posted April 11, 2019. ; https://doi.org/10.1101/606590 doi: bioRxiv preprint 664 the Bbrad51 locus in the context of the BbACc3 artificial chromosome, which did not require 665 reintegration into the genome but allowed expression controlled by autologous regulatory 666 elements. Construction of pBACc3 (circular plasmid form) was begun with destruction of the 667 NotI site (nt 669-676) within the multiple cloning site (mcs) of pBluescript-KS(+), by a fill-in 668 reaction with T4 DNA polymerase and blunt ligation [66]. This product was partially digested 669 with RcaI, and amplified by inverse-PCR with Phusion Hotstart II polymerase (New England 670 Biolabs; Beverley, MA), using primers DA162 and DA163 [67]. 650 Assembly of complementation constructs. Two different complementation PCR-amplification of the hDHFR coding sequences 700 with DA164 + DA165, the B. bovis actin promoter with DA166 + DA167, and the Rap1 gene 3' 701 terminator sequences with DA168 + DA169 was used to recover these segments. A two-step 702 crossover PCR was used to combine hDHFR coding sequence with promoter and terminator 703 [61], simultaneously removing all HindIII, EcoRV, and PstI sites. The full-length cassette first 704 was inserted into the SalI site of pBS2. The complete cassette then was removed with KpnI and 705 PstI, and integrated into the artificial chromosome construct, using Gibson Assembly reagent 706 mix (New England Biolabs; Beverley, MA) [69], yielding pBACc3. The final pBACc3 basic 707 vector construct is 11,030 bp (Fig. 5A). To make BbACc3_Bbrad51wt, the entire Bbrad51 locus 708 was amplified from pBbrad51wt_comp with primers DA248 + DA281, and inserted with 709 NEBuilder reagents (New England Biolabs) into pBACc3 opened with BamHI and PstI. All 710 added sequences and modified regions of the plasmid were fully Sanger-sequenced to confirm 711 proper construction. Constructs were transfected into E. coli, using strains DH5 (Invitrogen), 712 NEB10 (New England Biolabs), or Stellar (Clontech), by electroporation. For transfection, 713 pBACc3_Bbrad51wt was linearized with PmeI to release the stuffer sequence and expose the 714 telomeric ends (Fig. 5B). pBACc3 will be made available upon request. 650 Assembly of complementation constructs. Two different complementation CC-BY 4.0 International license a ertified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made available under The copyright holder for this preprint (which was not this version posted April 11, 2019. ; https://doi.org/10.1101/606590 doi: bioRxiv preprint 695 single candidate was identified from chromosome 3, corresponding to position 2922284- 696 2925108. Centromeric sequences were amplified with primers DA174 and DA175, and inserted 697 into the NotI site by ligation with T4 DNA ligase. The hDHFR selection cassette was moved 698 from the plasmid pDHFR-gfp-Bbcent2 (a gift from Shin-Ichiro Kawazu [12]) into the 699 intermediate construct, in multiple steps. PCR-amplification of the hDHFR coding sequences 700 with DA164 + DA165, the B. bovis actin promoter with DA166 + DA167, and the Rap1 gene 3' 701 terminator sequences with DA168 + DA169 was used to recover these segments. A two-step 702 crossover PCR was used to combine hDHFR coding sequence with promoter and terminator 703 [61], simultaneously removing all HindIII, EcoRV, and PstI sites. The full-length cassette first 704 was inserted into the SalI site of pBS2. The complete cassette then was removed with KpnI and 705 PstI, and integrated into the artificial chromosome construct, using Gibson Assembly reagent 706 mix (New England Biolabs; Beverley, MA) [69], yielding pBACc3. The final pBACc3 basic 707 vector construct is 11,030 bp (Fig. 5A). To make BbACc3_Bbrad51wt, the entire Bbrad51 locus 708 was amplified from pBbrad51wt_comp with primers DA248 + DA281, and inserted with 709 NEBuilder reagents (New England Biolabs) into pBACc3 opened with BamHI and PstI. All 710 added sequences and modified regions of the plasmid were fully Sanger-sequenced to confirm 711 proper construction. Constructs were transfected into E. coli, using strains DH5 (Invitrogen), 712 NEB10 (New England Biolabs), or Stellar (Clontech), by electroporation. For transfection, 713 pBACc3_Bbrad51wt was linearized with PmeI to release the stuffer sequence and expose the 714 telomeric ends (Fig. 5B). pBACc3 will be made available upon request. 715 695 single candidate was identified from chromosome 3, corresponding to position 2922284- 696 2925108. Centromeric sequences were amplified with primers DA174 and DA175, and inserted 697 into the NotI site by ligation with T4 DNA ligase. The hDHFR selection cassette was moved 698 from the plasmid pDHFR-gfp-Bbcent2 (a gift from Shin-Ichiro Kawazu [12]) into the 699 intermediate construct, in multiple steps. 650 Assembly of complementation constructs. Two different complementation Upon circularization of the 671 amplicon nt 2888-2940 were deleted, replacing the RcaI site at position 2881-2886 with a new 672 NotI site, and creating the 2920 bp intermediate plasmid, pBS2. B. bovis C9.1 line telomeric 673 sequences were inserted into the remaining RcaI site at nt 1873-1878 (original numbering), as 674 follows: B. bovis C9.1 line genomic DNA (gDNA) was PCR-amplified with Phusion polymerase 675 (New England Biolabs; Beverley, MA), using primers specific to unique sequences at nt 676 2,592,052-2,592,072 (DA170) and 948-923 (DA171) of the B. bovis T2Bo chromosome 3, 677 coupled with primers DA172 or DA173, respectively. The identification of centromeric 678 sequences is provided in S6 Fig.. The 5’ halves of DA170 and DA171 were complementary to 679 vector sequences flanking the remaining RcaI site. The 3’ halves of primers DA172 and DA173 680 each represented three telomeric repeats. The 3'-most repeat of each contained an extra T, an 681 infrequent imperfection in the telomeric repeats of B. bovis (T2Bo isolate reference genome, 682 accession no. AAXT01000001.1) [25], whereas the 5’ halves overlapped with Anaplasma 683 marginale genomic sequences. A 483 bp stuffer fragment, corresponding to nucleotides 145014- 684 145482 of the A. marginale Florida isolate genome (accession no. NC_012026), then was 685 amplified with primers DA152 and DA153, and a two-step crossover PCR approach was used to 686 assemble all three amplicons into one fragment containing PmeI restriction sites flanking the 687 stuffer [61]. The fused amplicon was inserted into the RcaI site, using InFusion reagents 688 (Clontech Laboratories, Inc.; Mountain View, CA). Putative centromeres were identified in the 689 B. bovis C9.1 line genome (ftp://ftp.sanger.ac.uk/pub/pathogens/Babesia/). Artemis v.16.0.0 690 (http://www.sanger.ac.uk/science/tools/artemis) was used to observe sequence %GC content, 691 identifying stretches > 2.5 standard deviations below the mean in a running window of 1000 nt 692 [68]. Sequences were considered candidates when the AT-rich segment was 2 Kbp or longer and 693 possessed no annotated open reading frames. Candidate sequences were further analyzed by dot- 694 plot to identify those containing significant internal repetitive sequence structure (S6 Fig.). A . CC-BY 4.0 International license a certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made available under The copyright holder for this preprint (which was not this version posted April 11, 2019. ; https://doi.org/10.1101/606590 doi: bioRxiv preprint . 716 Parasite transformation, and maintenance of BbACc3. Parasites were 717 washed into cytomix buffer [70], then transfected with knockout or complementation vectors 718 dissolved in cytomix. Transfection was achieved by electroporation with 10 pmol of DNA, under 719 conditions of 1.25 kV, 25 F, and 200  resistance, as described [11]. Prior to transfection, 720 pRad51ko and pBbrad51wt_comp were linearized with NotI. pBACc3_Bbrad51wt linearized 721 with PmeI, releasing the A. marginale "stuffer" DNA and generating linear BbACc3_Bbrad51wt 722 with telomeric ends. It was found not to be necessary to purify BbACc3 away from cleaved 723 stuffer DNA prior to transfection. In some experiments parasites were transfected with pBACc3 724 as supercoiled plasmid. Following transfection, parasites were placed into culture for 24 h in the 725 absence of drug selection. To effect selection, pyrimethamine then was added and maintained at . CC-BY 4.0 International license a certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made available under The copyright holder for this preprint (which was not this version posted April 11, 2019. ; https://doi.org/10.1101/606590 doi: bioRxiv preprint . CC-BY 4.0 International license a ertified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made available under The copyright holder for this preprint (which was not this version posted April 11, 2019. ; https://doi.org/10.1101/606590 doi: bioRxiv preprint 726 a concentration of 2 M (the approximate IC90 concentration), beginning 24h post-transfection. 727 In some experiments, drug pressure was removed for 21 days (approximately ≥ 60 cell cycles) 728 from already-transformed parasites, then was reapplied at varying concentrations to assess for 729 loss of drug-resistance. 730 731 Observation of chromosome maintenance and chromatin assembly. 732 Maintenance of BbACc3_Bbrad51wt as a linear chromosomal element was confirmed by 733 isolation of genomic DNA [55], with resolution of uncut and restriction-digested DNAs on a 734 0.7% agarose gel. DNAs were alkaline-transferred to nylon membranes as described [42], and 735 hybridized with probes to Bbrad51, -lactamase, centromeric, and hDHFR sequences. Probes 736 were generated by PCR amplification of pBACc3_Bbrad51wt sequences with primers EAM8 + 737 EAM11, DA290 + DA291, DA189 + DA190, or DA164 + DA165, etc., respectively. One g of 738 each amplicon was labeled with digoxigenin, and detected with anti-digoxigenin-HRP antibodies 739 and CSPD substrate, according to supplier’s instructions (Sigma Chemical; St. Louis, MO). 716 Parasite transformation, and maintenance of BbACc3. Parasites were Blots 740 were imaged with a FluorChemR instrument (Protein Simple; San Jose, CA) by detection of 741 luminescence. The stability of telomeric end lengths was tested by assessing the lengths of 742 BamHI restriction fragments of PmeI-linearized BbACc3 constructs used to transfect, and 743 BbACc3 constructs recovered after establishment of transformed parasites in culture. gDNAs 744 were cleaved with BamHI, and probed with the above-mentioned probes. pBACc3 digested with 745 PmeI to release stuffer sequences was used as control. Probes were labeled with 11-dUTP- 746 digoxigenin, following manufacturer’s instructions, and detected as described above. To observe 747 assembly of BbACc3 and BbACc3_Bbrad51wt into chromatin, nuclei were isolated from 748 transformed parasites, the chromatin partially digested with micrococcal nuclease (MNase), and 749 the fragments isolated as described [37]. To observe overall nucleosomal assembly and spacing, 750 digestion products were stained in-gel with SybrGold. Similar gels were also alkaline-blotted to 751 nylon membranes and probed by Southern blotting, as described above. Alkali-labile probes 752 were used to facilitate stripping of blots and re-probing 726 a concentration of 2 M (the approximate IC90 concentration), beginning 24h post-transfection. 727 In some experiments, drug pressure was removed for 21 days (approximately ≥ 60 cell cycles) 728 from already-transformed parasites, then was reapplied at varying concentrations to assess for 729 loss of drug-resistance. 731 Observation of chromosome maintenance and chromatin assembly. 732 Maintenance of BbACc3_Bbrad51wt as a linear chromosomal element was confirmed by 733 isolation of genomic DNA [55], with resolution of uncut and restriction-digested DNAs on a 734 0.7% agarose gel. DNAs were alkaline-transferred to nylon membranes as described [42], and 735 hybridized with probes to Bbrad51, -lactamase, centromeric, and hDHFR sequences. Probes 736 were generated by PCR amplification of pBACc3_Bbrad51wt sequences with primers EAM8 + 737 EAM11, DA290 + DA291, DA189 + DA190, or DA164 + DA165, etc., respectively. One g of 738 each amplicon was labeled with digoxigenin, and detected with anti-digoxigenin-HRP antibodies 739 and CSPD substrate, according to supplier’s instructions (Sigma Chemical; St. Louis, MO). Blots 740 were imaged with a FluorChemR instrument (Protein Simple; San Jose, CA) by detection of 741 luminescence. The stability of telomeric end lengths was tested by assessing the lengths of 742 BamHI restriction fragments of PmeI-linearized BbACc3 constructs used to transfect, and 743 BbACc3 constructs recovered after establishment of transformed parasites in culture. gDNAs 744 were cleaved with BamHI, and probed with the above-mentioned probes. pBACc3 digested with 745 PmeI to release stuffer sequences was used as control. Probes were labeled with 11-dUTP- 746 digoxigenin, following manufacturer’s instructions, and detected as described above. To observe 747 assembly of BbACc3 and BbACc3_Bbrad51wt into chromatin, nuclei were isolated from 748 transformed parasites, the chromatin partially digested with micrococcal nuclease (MNase), and 749 the fragments isolated as described [37]. To observe overall nucleosomal assembly and spacing, 750 digestion products were stained in-gel with SybrGold. Similar gels were also alkaline-blotted to 754 Acknowledgments. The authors thank Carlos Suarez and Shen-ichi Kawazu for 755 providing plasmids pGFP-Bsd and pDHFR-gfp-Bbcent2, respectively, and Kevin Brown and . CC-BY 4.0 International license a certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made available under The copyright holder for this preprint (which was not this version posted April 11, 2019. ; https://doi.org/10.1101/606590 doi: bioRxiv preprint . CC-BY 4.0 International license a certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made available under The copyright holder for this preprint (which was not this version posted April 11, 2019. ; https://doi.org/10.1101/606590 doi: bioRxiv preprint 756 Linda Bloom for providing critical early comments. 731 Observation of chromosome maintenance and chromatin assembly. We are indebted to Allison Vansickle for 757 assistance with bovine blood collection and maintenance of in vitro cultures. . CC-BY 4.0 International license a certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. 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Nature Methods. 939 2009;6:343-5. 940 70. . CC-BY 4.0 International license a certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made available under The copyright holder for this preprint (which was not this version posted April 11, 2019. ; https://doi.org/10.1101/606590 doi: bioRxiv preprint 758 References van den Hoff MJB, Moorman AFM, Lamers WH. Electroporation in 'intracellular' buffer 941 increases cell survival. Nucleic Acids Res. 1992;20:2902. 940 70. van den Hoff MJB, Moorman AFM, Lamers WH. Electroporation in 'intracellular' buffer 941 increases cell survival. Nucleic Acids Res. 1992;20:2902. 942 71. Conway AB, Lynch TW, Zhang Y, Fortin GS, Fung CW, Symington LS, et al. Crystal 943 structure of a Rad51 filament. Nat Struct Mol Biol. 2004;11(8):791-6. 942 71. Conway AB, Lynch TW, Zhang Y, Fortin GS, Fung CW, Symington LS, et al. Crystal 943 structure of a Rad51 filament. Nat Struct Mol Biol. 2004;11(8):791-6. 944 . CC-BY 4.0 International license a certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made available under The copyright holder for this preprint (which was not this version posted April 11, 2019. ; https://doi.org/10.1101/606590 doi: bioRxiv preprint . CC-BY 4.0 International license a certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made available under The copyright holder for this preprint (which was not this version posted April 11, 2019. ; https://doi.org/10.1101/606590 doi: bioRxiv preprint 945 Supporting Information Captions 946 S1 Fig.. Similarity of B. bovis RecA/RadA/Rad51 superfamily-related proteins to known or 947 putatively annotated Rad51 proteins from other species. Those proteins for which there is 948 experimental support for catalysis of canonical Rad51 functions are indicated in blue in Fig. 1. 949 The Walker A and Walker B motifs are indicated here by red and blue box overlays, 950 respectively. This alignment provided the basis for the phylogenetic tree shown in Fig. 1A. 946 S1 Fig.. Similarity of B. bovis RecA/RadA/Rad51 superfamily-related proteins to known or 947 putatively annotated Rad51 proteins from other species. Those proteins for which there is 948 experimental support for catalysis of canonical Rad51 functions are indicated in blue in Fig. 1. 949 The Walker A and Walker B motifs are indicated here by red and blue box overlays, 950 respectively. This alignment provided the basis for the phylogenetic tree shown in Fig. 1A. 0 respectively. This alignment provided the basis for the phylogenetic tree show 951 S2 Fig.. RT-PCR amplification of Bbrad51 and gfp-bsd transcripts. Total RNAs were 952 isolated from B. bovis CE11 subclones B8, C2, and C5, the initial uncloned CE11Bbrad51 953 knockout and three clonal lines derived from it (ko1C3, ko1H5, and ko1H6), and lines 954 CE11rad51ko2 and CE11rad51ko3. A. cDNAs made with oligo[dT] primer were amplified 955 with primers EAM8 and EAM11 [11, 51] for detection of Bbrad51 transcripts. B. cDNAs were 956 amplified with XW119 and XW121 [11, 51] for detection of gfp-bsd transcripts. Results 957 demonstrate Bbrad51 transcription by wild type lines but not by knockouts. Conversely, gfp-bsd 958 transcripts are present in knockout but not wild type lines. 959 S3 Fig.. Southern blot analysis of Bbrad51 locus. A. Schematic diagram of the Bbrad51 locus 960 in B. bovis CE11 wild type (bottom) and knockout (top) parasites, and the locations of restriction 961 endonuclease and primer binding sites. The tables provide the anticipated sizes (in bp) of specific 962 fragments detected with the indicated probes, based upon the genome sequence. B. Southern 963 blots of B. bovis CE11 wild type, and the initial CE11Bbrad51 knockout parasite population 964 both early and late (12 months later) in selection prior to cloning (left panel), after probing with 965 Bbrad51-specific oligonucleotide probe, EAM10. Initial B. bovis CE11Bbrad51 clonal lines 966 ko1C3, ko1H5, and ko1H6 are shown in the right panel. C. 945 Supporting Information Captions The same blots shown in panel B are 967 shown after being stripped and re-probed with oligonucleotide DA101R, specific for gfp 968 sequences. The numbers above the bands indicate the sizes of the bands in bp. 969 S4 Fig.. Titration of B. bovis CE11 wild type parasite sensitivity to MMS. In initial 970 experiments to determine useful concentrations of MMS to observe phenotype, parasites were 971 exposed to varying concentrations of MMS for 90 minutes at room temperature, then washed and 972 placed back into culture for 72h. Smears were made at 0, 48, and 72h post-treatment, fixed and 973 stained with Giemsa stain, and manually counted by microscopy in a blinded fashion. Growth is . CC-BY 4.0 International license a certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made available under The copyright holder for this preprint (which was not this version posted April 11, 2019. ; https://doi.org/10.1101/606590 doi: bioRxiv preprint . CC-BY 4.0 International license a ertified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made available under The copyright holder for this preprint (which was not this version posted April 11, 2019. ; https://doi.org/10.1101/606590 doi: bioRxiv preprint 974 indicated as percent parasitized erythrocytes (PPE). This experiment indicated that a range from 975 250 M to 1000 M MMS provided useful results. 974 indicated as percent parasitized erythrocytes (PPE). This experiment indicated that a range from 975 250 M to 1000 M MMS provided useful results. ructure of the pBbrad51wt_comp double crossover complementation plasmid. 977 This plasmid was designed to integrate into the genome by double-crossover homologous 978 recombination via sequences upstream and downstream to the Bbrad51 coding sequences. In 979 doing so, the strategy was to recreate the Bbrad51 locus, but with a short 3’-untranslated region 980 for regulation, followed by the hDHFR selection cassette. The plasmid was introduced into 981 parasites after NotI linearization, and selected for growth in the presence of pyrimethamine. This 982 occurred as intended in three of three attempts with wild type parasites, but failed in 11 attempts 983 with B. bovis ko1H5 parasites. 984 S6 Fig.. Identification of the B. bovis C9.1 chromosome 2 centromere. A. B. bovis T2Bo 985 isolate genomic sequences [25] were scanned with the “GC Content (%) with 2.5 SD Cutoff” 986 subroutine of Artemis v. 16.0.0 [68], using a sliding window of 1000 bp. Stretches of sequence > 987 2.5 standard deviations below the mean G+C content, and ≥ 2 Kbp in length, were identified. 988 Shown is the region of chromosome 3 identified by this strategy. Sequence comprising 989 nucleotides 2922285-2925108 of the B. bovis C9.1 line genome [36], corresponding to 990 nucleotides 29912-32736 of the B. bovis T2Bo isolate chromosome 3 [25], were recovered by 991 PCR and used in the construction of pBbACc3. B. Dot-plots of the putative B. bovis C9.1 line 992 chromosome 3 (left plot) and T2Bo isolate chromosome 2 (right plot) centromeres against 993 themselves. The internal repeat structure of each is apparent from the plots, with the 994 chromosome 2 centromere having a larger major repeat domain. The darkness of spots indicates 995 the degree of similarity, with the dark blue diagonal lines indicating identity. Values within the 996 range from 40-100% identity is shown. When plotted against one another there is no evidence for 997 any specific sequence relationship (not shown). 998 . CC-BY 4.0 International license a certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made available under The copyright holder for this preprint (which was not this version posted April 11, 2019. ; https://doi.org/10.1101/606590 doi: bioRxiv preprint . CC-BY 4.0 International license a certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. ructure of the pBbrad51wt_comp double crossover complementation plasmid. It is made available under The copyright holder for this preprint (which was not this version posted April 11, 2019. ; https://doi.org/10.1101/606590 doi: bioRxiv preprint certified by peer review) is the auth https://doi.or doi: bioRxiv preprint . CC-BY 4.0 International license a certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made available under The copyright holder for this preprint (which was not this version posted April 11, 2019. ; https://doi.org/10.1101/606590 doi: bioRxiv preprint . CC-BY 4.0 International license a certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made available under The copyright holder for this preprint (which was not this version posted April 11, 2019. ; https://doi.org/10.1101/606590 doi: bioRxiv preprint
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Directional properties of polar paramagnetic molecules subject to congruent electric, magnetic and optical fields
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This content has been downloaded from IOPscience. Please scroll down to see the full text. Download details: IP Address: 141.14.132.32 This content was downloaded on 11/05/2015 at 08:13 Please note that terms and conditions apply. Directional properties of polar paramagnetic molecules subject to congruent electric and optical fields View the table of contents for this issue, or go to the journal homepage for more 2015 New J. Phys. 17 045017 (http://iopscience.iop.org/1367-2630/17/4/045017) ome Search Collections Journals About Contact us My IOPscience Directional properties of polar paramagnetic molecules subject to congruent electric, magnetic and optical fields Home Search Collections Journals About Contact us My IOPscience Abstract Weshowthat congruent electric, magneticand non-resonant opticalfieldsactingconcurrentlyon a polar paramagnetic(and polarizable) moleculeoffer possibilitiesto both amplifyand controlthe directionalityoftheensuingmolecular statesthat surpassthoseavailablein double-field combinations or in singlefieldsalone.At thecoreofthesetriple-field effectsistheliftingofthedegeneracyofthe projection quantum number M bythemagneticfield superimposed on theopticalfield and a subsequent couplingofthemembersofthe‘doubled’ (for stateswith ≠ M 0) tunnelingdoubletsdue to theopticalfield byeven aweakelectrostaticfield. Directionalpropertiesofpolarparamagneticmoleculessubjectto congruentelectric,magneticandopticalfields Ketan Sharma and BretislavFriedrich1 Fritz-Haber-Institut der Max-Planck-Gesellschaft Faradayweg4-6,D-14195Berlin, Germany 1 Author to whom anycorrespondenceshould beaddressed. E-mail:ketan@fhi-berlin.mpg.deand bretislav.friedrich@fhi-berlin.mpg.de Keywords:Starkeffect, Zeeman effect,induced-dipoleinteraction, polarizability, nonresonant laser field,combined fields, alignment orientation Content from thiswork maybeused under the termsoftheCreative CommonsAttribution 3.0 licence. Directional properties of polar paramagnetic molecules subject to congruent electric, magnetic and optical fields This content has been downloaded from IOPscience. Please scroll down to see the full text. 2015 New J. Phys. 17 045017 (http://iopscience.iop.org/1367-2630/17/4/045017) This content has been downloaded from IOPscience. Please scroll down to see the full text. View the table of contents for this issue, or go to the journal homepage for more Download details: Please note that terms and conditions apply. NewJ.Phys.17(2015) 045017 doi:10.1088/1367-2630/17/4/045017 PAPER Directionalpropertiesofpolarparamagneticmoleculessubjectto congruentelectric,magneticandopticalfields Ketan Sharma and BretislavFriedrich1 Fritz-Haber-Institut der Max-Planck-Gesellschaft Faradayweg4-6,D-14195Berlin, Germany 1 Author to whom anycorrespondenceshould beaddressed. E-mail:ketan@fhi-berlin.mpg.deand bretislav.friedrich@fhi-berlin.mpg.de Keywords:Starkeffect, Zeeman effect,induced-dipoleinteraction, polarizability, nonresonant laser field,combined fields, alignment and OPENACCESS RECEIVED 16January2015 REVISED 12March 2015 ACCEPTEDFORPUBLICATION 20March 2015 PUBLISHED 24April2015 Content from thiswork maybeused under the termsoftheCreative CommonsAttribution 3.0 licence. Anyfurther distribution of thiswork must maintain attribution to the author(s) and thetitleof thework, journalcitation and DOI. Anyfurther distribution of thiswork must maintain attribution to the author(s) and thetitleof thework, journalcitation and DOI. ©2015IOP PublishingLtd and DeutschePhysikalischeGesellschaft 1.Introduction Herein weshowthat amagneticand opticaldouble field interaction with apolar paramagneticmolecule maycreatenear-degeneraciesofadditionallevelsthat can beeasilycoupled byeven aweak electricfield (magneticand opticaland electrictriple-field combination). Thereby,thetriple-field combination could, for instance,enablefast switchingofdipolar orientation and other dynamicaleffectsthat arenot availablein a doublemagneticand electricor opticaland electricfield combinationsalone(not to speak about thesingle fields).At thecoreofthesenoveltriple-field effectsistheliftingofthedegeneracyoftheprojection quantum number M bythemagneticfield superimposed on theopticalfield and asubsequent couplingofthemembersof the‘doubled’ (for stateswith ≠ M 0) tunnelingdoubletsdueto theopticalfield byaweakelectrostaticfield. Thispaper isorganized asfollows:In section 2weintroducetherotationalHamiltonian ofa Σ 2 polar moleculeaswellasitsmatrixrepresentation in theHund’scase(b) basisset. In sections2.1–2.3wepresent,in turn, thesingle-field Hamiltoniansfor theelectric, magnetic, and opticalpotentials. In section 2.4,wepresent thefullHamiltonian for theelectricand magneticand opticaltriple-field interaction. In section 3wepresent and discusstheresultsofour calculationsoftheeigenpropertiesofthepartialHamiltoniansaswellasofthefull triple-field combined Hamiltonian. Section 4surveysand summarizesour results. Theappendixliststhekey matrixelementsused in thecalculations, describestheproceduredeveloped to assign thestatesobtained bythe diagonalization oftheHamiltonian matrix, and liststheconversion factorsneeded to evaluatethedimensionless parametersused throughout thepaper in termsofcustomaryunits. Thispaper isorganized asfollows:In section 2weintroducetherotationalHamiltonian ofa Σ 2 polar moleculeaswellasitsmatrixrepresentation in theHund’scase(b) basisset. In sections2.1–2.3wepresent,in turn, thesingle-field Hamiltoniansfor theelectric, magnetic, and opticalpotentials. In section 2.4,wepresent thefullHamiltonian for theelectricand magneticand opticaltriple-field interaction. In section 3wepresent and discusstheresultsofour calculationsoftheeigenpropertiesofthepartialHamiltoniansaswellasofthefull triple-field combined Hamiltonian. Section 4surveysand summarizesour results. Theappendixliststhekey matrixelementsused in thecalculations, describestheproceduredeveloped to assign thestatesobtained bythe diagonalization oftheHamiltonian matrix, and liststheconversion factorsneeded to evaluatethedimensionless parametersused throughout thepaper in termsofcustomaryunits. 1.Introduction Interactionswith externalelectric, magneticor opticalfieldsprovidethechiefmeansto manipulatethe rotationaland translationalmotion ofneutralgas-phasemolecules[1]. Theseinteractionscreatedirectional statesin which themolecular multipolemomentsbecomenon-vanishingin thelaboratoryframeso that space- fixed fieldscan act upon them. Directionalstatesareat thecoreofnumerousapplicationsin molecular physics, such asorientation/alignment ofmolecules[2–30],deflection and focusingofmolecular translation [31–34], molecular trapping[35], attainingtime-resolved photoelectron angular distributions[36–38],diffraction- from-within [39],separation ofphotodissociation products[40–42],deracemization [43],high-order harmonicgeneration and orbitalimaging[44–50],quantum simulation [51,52] or quantum computing [53–59]. Herein, weexaminedirectionalstatescreated byatriple-combination ofcongruent (parallelor antiparallel) electric, magneticand non-resonant opticalfieldsactingconcurrentlyon linear polar paramagnetic(and polarizable) molecules. Whiletheelectricand magneticfieldsinteract, respectively, with thebody-fixed electric and magneticdipolemomentsofthemolecule,thenon-resonant opticalfield couplesto themolecular anisotropicpolarizabilitytensor. Themolecular effectsgenerated bythedouble-field combinations(electricand magnetic, electricand optical,magneticand optical) areallsui generisand amount to morethan thesum oftheir parts. And so doesthetriple-field combination (electricand magneticand optical) which not onlyoffersahigh efficiencyand flexibilityin amplifyingthedirectionalityofmolecular statesbut isalso offundamentalinterest per se,assupersymmetry[60–64] aswellasmonodromyand quantum chaos[65, 66] lurk behind the combined-field effects. Polar paramagneticmoleculesareofpotentialimportancefor many-bodyphysicssimulations, studiesof crossingsofStark and Zeeman molecular energylevels, and quantum computing. Amongthemost prominent examplesoflinear polar paramagneticmoleculesaretheubiquitous Σ 2 , Σ 3 , and Π 2 linear species, such asSrF, SO,and OH. Heteronuclear diatomicsor larger polar moleculesthat contain arare-Earth atom often exhibit much higher orbitaland spin electronicangular momenta (e.g., CeO isa Φ 3 2 moleculein itselectronicground state) and, therefore, correspondinglylarger magneticdipolemoments. Therecentlydiscovered LiHevan der Waalsmolecule[67, 68], apolar and paramagnetichalo species,would also benefit from thestudyofits ©2015IOP PublishingLtd and DeutschePhysikalischeGesellschaft NewJ.Phys.17(2015) 045017 NewJ.Phys.17(2015) 045017 KSharmaand BFriedrich propertiesin combined fields, asthiswould likelyrevealadditionalparticularsabout itsstructureand the dynamicsofitsformation.Asurveyoflinear polar paramagneticmoleculesalongwith their keypropertiesis availablein table3of[9].However,our treatment hereisgeneric, makinguseofreduced molecular interaction parameters,and thereforeapplicableto anypolar paramagneticand polarizablemoleculein agiven electronic state.For thepurposesofthepresent study, wechosemoleculesin a Σ 2 stateasaprototype. Directionalstatesofmoleculesmayexhibit either orientation (visualized asasingle-headed arrowlibrating about aspace-fixed axis) and/or alignment (visualized asadouble-headed arrowlibratingabout aspace-fixed axis).Themoredirectionalthestate,thetighter thelibrationalamplitudeofthearrowand themorecomplete theprojection ofthecorrespondingdipole(whether permanent or induced) on thespace-fixed axis. Sinceoriented statesmayonlybeofindefiniteparity—otherwisetheywould violatetheparityselection rule [69]—arecipefor creatingoriented statesisto mixstatesofoppositeparity.Thecoupling—or hybridization— ofoppositeparitystatescan begenerallyachieved bytheelectricdipoleinteraction, which isthemoreeffectivein couplingtheopposite-paritylevelsthecloser theylieto oneanother. Close-lyingoppositeparitystatescan be prepared for largeclassesoflinear moleculesbyeither opticalor magneticfields. In our previouswork aswellas that ofothers, it hasbeen shown that, for linear molecules, theopposite-paritystatesamenableto facileelectric- dipolecouplingareeither thequasi-degeneratemembersofthetunnelingdoubletscreated bytheinduced- dipoleinteraction with anon resonant opticalfield (combination ofelectricand opticalfields) [4,5, 16,21,22, 30] or theintersectingopposite-parityZeeman levelsthat becomeexactlydegenerateat their intersection points(combination ofelectricand magneticfields) [9,10,70,71]. Herein weshowthat amagneticand opticaldouble-field interaction with apolar paramagneticmolecule maycreatenear-degeneraciesofadditionallevelsthat can beeasilycoupled byeven aweak electricfield (magneticand opticaland electrictriple-field combination). Thereby,thetriple-field combination could, for instance,enablefast switchingofdipolar orientation and other dynamicaleffectsthat arenot availablein a doublemagneticand electricor opticaland electricfield combinationsalone(not to speak about thesingle fields).At thecoreofthesenoveltriple-field effectsistheliftingofthedegeneracyoftheprojection quantum number M bythemagneticfield superimposed on theopticalfield and asubsequent couplingofthemembersof the‘doubled’ (for stateswith ≠ M 0) tunnelingdoubletsdueto theopticalfield byaweakelectrostaticfield. 2.Rotational structureofa polar Σ 2 moleculein combined electric, magneticand optical fields Thephenomenologicalfield-freerotationalHamiltonian ofa Σ 2 moleculeisgiven by[72] (1) γ = + H BN N S · , (1) 0 2 γ = + H BN N S · , 0 2 where N and Sare, respectively, therotationaland electronicspin angular momenta, ≡  B I 2 istherotational constant,with I themolecule’smoment ofinertiain agiven vibrationalstatehosted bythe Σ 2 electronicstate, and γisthespin-rotation couplingconstant. Hamiltonian (1) neglectsnuclear spin aswellasthe(small) magneticmoment arisingdueto therotation ofthemolecule. 1 where N and Sare, respectively, therotationaland electronicspin angular momenta, ≡  B I 2 istherotational constant,with I themolecule’smoment ofinertiain agiven vibrationalstatehosted bythe Σ 2 electronicstate, and γisthespin-rotation couplingconstant. Hamiltonian (1) neglectsnuclear spin aswellasthe(small) magneticmoment arisingdueto therotation ofthemolecule. Whilefor a Σ 2 statetheelectronicspin angular momentum = S 1 2,theorbitalelectronicangular momentum isidenticallyzero and so isthespin–orbit coupling. A Σ 2 statethusexhibitsaHund’scase(b) couplingbetween therotationaland electronicangular momenta[72],with theprojectionsofthetotaland spin electronicangular momenta on themolecular axis(an axisofcylindricalsymmetry) Ω Σ = = 1 2, seefigure1. electronicangular momenta on themolecular axis(an axisofcylindricalsymmetry) Ω Σ = = 1 2, seefigure1. TheHund’scase(b) basisfunctionsarean equallyweighted linear combination ofHund’scase(a) basis functions, each aproduct ofasymmetrictop wavefunction 2 TheHund’scase(b) basisfunctionsarean equallyweighted linear combination ofHund’scase(a) basis functions, each aproduct ofasymmetrictop wavefunction D Ω π θ ϕ χ = − + Ω Ω − − − J M J , , ( 1) (2 1) 8 ( , , ) (2) M M J , (2) 2 NewJ.Phys.17(2015) 045017 KSharmaand BFriedrich Figure1. Definition ofEuler angles θ ϕ χ , , describingtherotation ofthemolecule-fixed coordinatesx, y, zwith respect to thespace- fixed coordinates X Y Z , , for adiatomicmoleculedepicted asabar-bell. Thegreen axisisthelineofnodes,perpendicular to both z and Z. Also shown aretherotational, N,electron spin, S, and total, J,angular momentaaswellastheprojectionsM and Ω Σ = of J on thespace- and body-fixed axis. Figure1. Definition ofEuler angles θ ϕ χ , , describingtherotation ofthemolecule-fixed coordinatesx, y, zwith respect to thespace- fixed coordinates X Y Z , , for adiatomicmoleculedepicted asabar-bell. Thegreen axisisthelineofnodes,perpendicular to both z and Z. Also shown aretherotational, N,electron spin, S, and total, J,angular momentaaswellastheprojectionsM and Ω Σ = of J on thespace- and body-fixed axis. Figure1. Definition ofEuler angles θ ϕ χ , , describingtherotation ofthemolecule-fixed coordinatesx, y, zwith respect to thespace- fixed coordinates X Y Z , , for adiatomicmoleculedepicted asabar-bell. Thegreen axisisthelineofnodes,perpendicular to both z and Z. Also shown aretherotational, N,electron spin, S, and total, J,angular momentaaswellastheprojectionsM and Ω Σ = of J on thespace- and body-fixed axis. 2.Rotational structureofa polar Σ 2 moleculein combined electric, magneticand optical fields and aspin function and aspin function Σ α β Σ Σ = + − Σ Σ + − S S S , ( ) !( )! (3) S S (3) with = ± J N Sthetotal(rotation and electron spin) angular momentum quantum number, M and Ωthe projectionsofthetotalangular momentum on, respectively, thespace-fixed Z-axisand thebody-fixed z-axis, D θ ϕ χ Ω( , , ) M J , theWigner matrix, with θ ϕ χ , , theEuler angles, and α β , thespin functions. Thusfor a Σ 2 state ( = S 1 2),therearetwo typesofHund’scase(b) basisfunctions ψ Ω Ω ± = ± − − ± N M S J M S J M 1 2 , 1 2 , 1 2 , , , 1 2 , , (4) ⎜ ⎟ ⎛ ⎝ ⎞ ⎠ ⎡ ⎣⎢ ⎤ ⎦⎥ (4) pertainingto = ± J N 1 2, with parity − ( 1)N.Thecorrespondingeigenenergiesare γ + = + + + E N M BN N N 1 2 , ( 1) 2 , (5) ⎜ ⎟ ⎛ ⎝ ⎞ ⎠ γ − = + − + − E N M BN N N 1 2 , ( 1) 2 ( 1). (6) ⎜ ⎟ ⎛ ⎝ ⎞ ⎠ (5) (6) The± statesofa Σ 2 moleculeareconventionallyreferred to asF1(when = + J N 1 2) and F2(when = − J N 1 2). Both Jand N but not Ωaregood quantum numbersfor afield-free Σ 2 molecule. The± statesofa Σ 2 moleculeareconventionallyreferred to asF1(when = + J N 1 2) and F2(when = − J N 1 2). Both Jand N but not Ωaregood quantum numbersfor afield-free Σ 2 molecule. 2.1.Interaction with an electricfield 2.1.Interaction with an electricfield 2.1.Interaction with an electricfield Theinteraction potentialfor alinear moleculewith an electricdipolemoment μel alongthemolecule-fixed z- axissubject to an electrostaticfield εS (aStark field) definingaspace-fixed Z-axis, seefigure2,isgiven by η θ = − V B cos , (7) el el (7) where η μ ε ≡ B (8) S el el (8) μ ≡ B el B isadimensionlessparameter characterizingthestrength oftheStark interaction. Wenotethat theattainable externalelectricfield εS ismuch weaker than theinternalelectricfield produced bythemolecule’sconstituent electronsand nucleiand thusitseffect on theelectronicstructureofthemoleculeisnegligible. In what follows wewilldealsolelywith theeffect oftheexternalfieldson themolecular rotationalstructure. isadimensionlessparameter characterizingthestrength oftheStark interaction. Wenotethat theattainable externalelectricfield εS ismuch weaker than theinternalelectricfield produced bythemolecule’sconstituent electronsand nucleiand thusitseffect on theelectronicstructureofthemoleculeisnegligible. In what follows wewilldealsolelywith theeffect oftheexternalfieldson themolecular rotationalstructure. The θ cos operator (arisingfrom thedirection cosinematrix, seeappendix) mixesHund’scase(b) basis functionswith thesameM but with ′ N s that differ by±1and thushaveoppositeparities.Asaresult, thestates created bytheStark interaction areofindefiniteparityand N (and J) ceaseto begood quantum numbers. The 3 NewJ.Phys.17(2015) 045017 KSharmaand BFriedrich Figure2. An electrostaticfield εS directed alongthespace-fixed Z-axisactingon apolar Σ + S 2 1 moleculewith an electricdipole moment μel alongthemolecule-fixed z-axis. Also shown aretherotational,spin,and totalangular momenta N, S,and J aswellasthe polar angleθbetween thespace- and molecule-fixed axesand theazimuthalangleϕ uniformlydistributed about theZ-axis.Seetext. Figure2. An electrostaticfield εS directed alongthespace-fixed Z-axisactingon apolar Σ + S 2 1 moleculewith an electricdipole moment μel alongthemolecule-fixed z-axis. Also shown aretherotational,spin,and totalangular momenta N, S,and J aswellasthe polar angleθbetween thespace- and molecule-fixed axesand theazimuthalangleϕ uniformlydistributed about theZ-axis.Seetext. Figure3. Amagneticfield  directed alongthespace-fixed Z-axisactingon a Σ + S 2 1 moleculewith amagneticdipolemoment μm alongtheelectronicspin vector S.Also shown aretherotationaland totalangular momenta N and J aswellasthepolar angleθ between thespace- and molecule-fixed axesand theazimuthalangleϕ uniformlydistributed about theZ-axis.Seetext. Figure3. Amagneticfield  directed alongthespace-fixed Z-axisactingon a Σ + S 2 1 moleculewith amagneticdipolemoment μm alongtheelectronicspin vector S.Also shown aretherotationaland totalangular momenta N and J aswellasthepolar angleθ between thespace- and molecule-fixed axesand theazimuthalangleϕ uniformlydistributed about theZ-axis.Seetext. onlygood quantum number istheprojection M of J on theZ-axis. N and Jcan, nevertheless, beused asadiabatic labelsofthestatesin thefield,in which casetheyarefurnished with atilde, η ∣ →〉→∣ 〉 N J M N J M ˜ , ˜, ; 0 , , el . onlygood quantum number istheprojection M of J on theZ-axis. N and Jcan, nevertheless, beused asadiabatic labelsofthestatesin thefield,in which casetheyarefurnished with atilde, η ∣ →〉→∣ 〉 N J M N J M ˜ , ˜, ; 0 , , el . 2.1.Interaction with an electricfield 2.2.Interaction with a magneticfield 2.2.Interaction with a magneticfield Theinteraction potentialfor a Σ 2 moleculesubject to amagneticfield  (aZeeman field) definingaspace-fixed Z-axis, seefigure3, isgiven by μ η = − = V B S , (9) m Z m m Z (9) μ η = − = V B S , m Z m m Z μ η = − = V B S , (9) m Z m m Z where  η μ ≡ B (10) m m where η μ ≡ B (10) m m (10) B NewJ.Phys.17(2015) 045017 KSharmaand BFriedrich Figure4. Anonresonant opticalfield εL polarized alongthespace-fixed Z-axisactingon apolar Σ + S 2 1 moleculewith paralleland perpendicular componentsofthemolecular polarizability α∥and α⊥with respect to themolecular z-axis. Also shown arethe rotational, spin,and totalangular momenta N, S,and J aswellasthepolar angleθbetween thespace- and molecule-fixed axesand theazimuthalangleϕ uniformlydistributed about theZ-axis. Seetext. Figure4. Anonresonant opticalfield εL polarized alongthespace-fixed Z-axisactingon apolar Σ + S 2 1 moleculewith paralleland perpendicular componentsofthemolecular polarizability α∥and α⊥with respect to themolecular z-axis. Also shown arethe rotational, spin,and totalangular momenta N, S,and J aswellasthepolar angleθbetween thespace- and molecule-fixed axesand theazimuthalangleϕ uniformlydistributed about theZ-axis. Seetext. with μ μ = g m S Btheelectronicmagneticdipolemoment ofthe Σ 2 molecule, ≅ g 2.0023 S theelectronic gyromagneticratio and μBtheBohr magneton. TheSZoperator couplesHund’scase(b) basisfunctionswith thesameM but with ′ N s that areeither the sameor differ by±2and hencehavethesameparity.Theselection ruleon N moreover ensuresthat the Hamiltonian matrixin theHund’scase(b) basisfor theZeeman interaction ofa Σ 2 moleculefactorsinto blocks that areno greater than 2× 2,renderingthecorrespondingZeeman energyat most quadraticin  .Apart from M, also parity − ( 1)N˜ isagood quantum number. 2.3.Interaction with an optical field 2.3.Interaction with an optical field Asfor anylinear species, thepolarizabilitytensor ofa Σ 2 moleculeisanisotropic, with theprincipalcomponent alongthemolecular axisexceedingthat perpendicular to theaxis, α α > ∥ ⊥.When subject to an electricfield εL of an electromagneticwaveofintensity  linearlypolarized alongthespace-fixed Z- axis, figure4,whose oscillation frequencyisfar removed from anymolecular resonance,themoleculeundergoesan interaction given bythepotential η θ η = − − ⊥ V B B cos , (11) opt opt 2 (11) where η η η ≡ − ∥ ⊥ (12) opt η η η ≡ − ∥ ⊥ (12) opt and  η πα ≡ ∥⊥ ∥⊥ Bc 2 (13) , , with  π ε = c 4 . (14) L 2 (12) η η η ≡ − ∥ ⊥ opt and and η πα ≡ ∥⊥ ∥⊥ Bc 2 (13) , , (13) with π ε = c 4 . (14) L 2 (14) TheVopt potentialisadouble-wellpotentialwith two equivalent minimaat θ = 0 and 180°,separated byan equatorialbarrier at θ = π 2. Asaresult, allstatesbound byVopt occur asdoublets, split bytunnelingthrough the equatorialbarrier. The θ cos2 operator ofVopt hybridizesfree-rotor statesofsameparityand so thestatescreated byVopt areofdefiniteparity, given by − ( 1)J˜.ThemembersofanyofthetunnelingdoubletshavesameM but ′J s ˜ that differ by±1and thusareofoppositeparity. Thetunnelingsplitting Δ η η ∝ − E ( ) exp( ) t opt opt 1 2 ,see[73]. 5 NewJ.Phys.17(2015) 045017 KSharmaand BFriedrich Figure5. Congruent electrostatic, magneticand opticalfields εS,  ,and εL directed alongthespace-fixed Z-axisactingon a polar Σ + S 2 1 moleculewith body-fixed electricand magneticdipolemoments μel and μm and polarizabilitycomponents α∥and α⊥.Also shown aretherotational, spin, and totalangular momenta N, S,and J aswellasthepolar angleθbetween thespace- and molecule- fixed axesand theazimuthalangleϕ uniformlydistributed about theZ-axis. Seetext. Figure5. Congruent electrostatic, magneticand opticalfields εS,  ,and εL directed alongthespace-fixed Z-axisactingon a polar Σ + S 2 1 moleculewith body-fixed electricand magneticdipolemoments μel and μm and polarizabilitycomponents α∥and α⊥.Also shown aretherotational, spin, and totalangular momenta N, S,and J aswellasthepolar angleθbetween thespace- and molecule- fixed axesand theazimuthalangleϕ uniformlydistributed about theZ-axis. Seetext. Figure6. Dependence oftheeigenenergiesofapolar Σ 2 moleculeon thepermanent electricdipoleinteraction parameter ηel.F1and F2levelsareshown,respectively, bydashed and fulllines.Red and bluecurvespertain, respectively, to stateswith = J˜ 1 2 and = J˜ 3 2. Notethat here η η = = 0 m opt . Figure6. Dependence oftheeigenenergiesofapolar Σ 2 moleculeon thepermanent electricdipoleinteraction parameter ηel.F1and F2levelsareshown,respectively, bydashed and fulllines.Red and bluecurvespertain, respectively, to stateswith = J˜ 1 2 and = J˜ 3 2. Notethat here η η = = 0 m opt . 2.3.Interaction with an optical field 2.4.Interaction with congruent electric, magnetic, and optical fields In thecongruent electric, magnetic, and opticalfields,thepotentialisgiven by 2.4.Interaction with congruent electric, magnetic, and optical fields In thecongruent electric, magnetic, and opticalfields,thepotentialisgiven by = + + V V V V (15) m m el, ,opt el opt (15) NewJ.Phys.17(2015) 045017 KSharmaand BFriedrich Figure7. Probabilitydensities, orientation and alignment cosinesofa Σ 2 moleculeasfunctionsofthepermanent electricdipole interaction parameter ηel.Valuespertainingto theF1and F2statesareshown, respectively, bydashed and fulllines.Blueand red curvespertain,respectively, to theleft (orientation) and right (alignment) ordinate. Notethat here η η = = 0 m opt . Figure7. Probabilitydensities, orientation and alignment cosinesofa Σ 2 moleculeasfunctionsofthepermanent electricdipole interaction parameter ηel.Valuespertainingto theF1and F2statesareshown, respectively, bydashed and fulllines.Blueand red curvespertain,respectively, to theleft (orientation) and right (alignment) ordinate. Notethat here η η = = 0 m opt . and thecorrespondingHamiltonian becomes and thecorrespondingHamiltonian becomes Theintegraloftheeigenfunction’ssquareover thespin variables Theintegraloftheeigenfunction’ssquareover thespin variables ∑ θ ϕ χ ψ ψ = f ( , , ) * (20) S ∑ θ ϕ χ ψ ψ = f ( , , ) * (20) S i lifi t (20) D D ∑ θ ϕ χ π θ ϕ χ θ ϕ χ δ = − × + ′ + × Σ Σ Σ Σ Σ Σ ′ ′ ′ ′ ′ ′ − ′ ′ ′ − − ′ ( )( ) f c c J J ( , , ) ( 1) 2 1 2 1 64 ( , , ) ( , , ) . (21) J M J M J M J M M K M K J M K J , , , , , , * , , 2 , , , D D θ ϕ χ θ ϕ χ δ × Σ Σ ′ ′ ′ − − ′ ( , , ) ( , , ) . (21) M K J M K J , , , (21) In order to visualizethedirectionalpropertiesofthemolecular statescreated,wepresent probability distributionsofthespatialvariables, θ ϕ , ,aspolar plotsof θ ϕ χ = f ( , , 0). In order to visualizethedirectionalpropertiesofthemolecular statescreated,wepresent probability distributionsofthespatialvariables, θ ϕ , ,aspolar plotsof θ ϕ χ = f ( , , 0). and thecorrespondingHamiltonian becomes (16) = + H H V . (16) m m el, ,opt 0 el, ,opt Themolecular axis,z,angular momenta, J, N, Sand thedipolemoments, μel, μm,and polarizability components, α∥, α⊥aswellasthespace-fixed Z-axisareshown in figure5. Themolecular axis,z,angular momenta, J, N, Sand thedipolemoments, μel, μm,and polarizability components, α∥, α⊥aswellasthespace-fixed Z-axisareshown in figure5. Themolecular axis,z,angular momenta, J, N, Sand thedipolemoments, μel, μm,and polarizability components, α∥, α⊥aswellasthespace-fixed Z-axisareshown in figure5. BydividingHamiltonian (16) through therotationalconstant Band makinguseofequations(1),(7),(9), and (11),weobtain thereduced Hamiltonian BydividingHamiltonian (16) through therotationalconstant Band makinguseofequations(1),(7),(9), and (11),weobtain thereduced Hamiltonian γ η θ η η θ η ≡ = + ′ − + − − ⊥ H B H S N N S · cos cos (17) m m Z el, ,opt 2 el opt 2 ≡ H B H m el, ,opt γ η θ η η θ η = + ′ − + − − ⊥ S N N S · cos cos (17) m Z 2 el opt 2 (17) with γ′ ≡ γ B. with γ′ ≡ γ B. Theeigenfunction ofthetriple-field Hamiltonian (17) can bewritten as γ B. Theeigenfunction ofthetriple-field Hamiltonian (17) can bewritten as B Theeigenfunction ofthetriple-field Hamiltonian (17) can bewritten as ∑ ψ η η η Σ ≡ = Σ Σ J N M c J K M S ˜, ˜ , ; , , , , , (18) m J J J N M el opt , , ˜, ˜ , (18) with anormalization with anormalization with anormalization = Σ c 1. (19) J J N M , ˜, ˜ , 2 = Σ c 1. (19) J J N M , ˜, ˜ , 2 = Σ c 1. J J N M , ˜, ˜ , 2 (19) NewJ.Phys.17(2015) 045017 KSharmaand BFriedrich Figure8. Dependence oftheeigenenergiesofapolar Σ 2 moleculeon themagneticdipoleinteraction parameter ηm.F1and F2levels areshown,respectively,bydashed and fulllinesin panelspertainingto signed valuesofthegood quantum number M.Red and blue curvespertain,respectively, to stateswith = J˜ 1 2 and = J˜ 3 2.Notethat here η η = = 0 el opt . Figure8. Dependence oftheeigenenergiesofapolar Σ 2 moleculeon themagneticdipoleinteraction parameter ηm.F1and F2levels areshown,respectively,bydashed and fulllinesin panelspertainingto signed valuesofthegood quantum number M.Red and blue curvespertain,respectively, to stateswith = J˜ 1 2 and = J˜ 3 2.Notethat here η η = = 0 el opt . 3.1.Single-field effects 3.1.Single-field effects 3.Results and discussion Theeigenenergiesand eigenvectorsofHamiltonian (17) wereobtained bynumericaldiagonalization ofthe matrixrepresentation oftheHamiltonian in Hund’scase(a) basis. For collinear fields,considered here,M isa good quantum number and so theHamiltonian matrixtakesablock-diagonalform for different valuesofM. For each M,theblock wastruncated at = J 15 2 to ensuretheconvergenceofeigenvaluesand eigenvectors. This leadsto formation ofblock matricesofrank 30. Each oftheseblockswasdiagonalized separately.The diagonalization wascarried out usingtheArmadillo C++ linear algebralibrary[74]. In order to track which stateiswhich astheinteraction parameters ηel, ηm,and ηopt werevaried, aprocedure termed adiabaticfollowingwasdeveloped. Instead oflookingat thedependenceon theinteraction parametersof thecomponentsoftheeigenvectors, wemonitored thescalar product ofthestatesbeforeand after a(small) 8 NewJ.Phys.17(2015) 045017 KSharmaand BFriedrich Figure9. Probabilitydensities, orientation and alignment cosinesofa Σ 2 moleculeasfunctionsofthemagneticdipoleinteraction parameter ηm. Valuespertainingto theF1and F2statesareshown,respectively,bydashed and fulllines. Blueand red curvespertain, respectively, to theleft (orientation) and right (alignment) ordinate.Notethat here η η = = 0 el opt . Figure9. Probabilitydensities, orientation and alignment cosinesofa Σ 2 moleculeasfunctionsofthemagneticdipoleinteraction parameter ηm. Valuespertainingto theF1and F2statesareshown,respectively,bydashed and fulllines. Blueand red curvespertain, respectively, to theleft (orientation) and right (alignment) ordinate.Notethat here η η = = 0 el opt . changeoftheinteraction parameter. Thescalar product wascalculated between agiven stateat theinitialvalueof theinteraction parameter(s) and alltheother statesat thealtered valueoftheinteraction parameter(s). The maximum ofthescalar product wasthen found and used to identifythestatethat makesthesmallest anglewith thegiven state. Allthecalculationsbelowwerecarried out for ageneric Σ 2 moleculewith avalueofthereduced spin- changeoftheinteraction parameter. Thescalar product wascalculated between agiven stateat theinitialvalueof theinteraction parameter(s) and alltheother statesat thealtered valueoftheinteraction parameter(s). The maximum ofthescalar product wasthen found and used to identifythestatethat makesthesmallest anglewith thegiven state. g Allthecalculationsbelowwerecarried out for ageneric Σ 2 moleculewith avalueofthereduced spin- rotation constant γ γ ′ ≡ = B 0.41(which pertains, e.g., to theNaO moleculein itsA Σ 2 state[75]). 3.1.1.PureStarkinteraction TheStarkpotential, equation (7),splitseach J˜ levelinto + J˜ 1 2 stateswith different valuesof∣ ∣ M but doesnot undo the ± M degeneracy. Figure6showsthedependenceoftheeigenenergiesofthelowest sixstateson the permanent electricdipoleinteraction parameter ηel,which isproportionalto theelectricfield strength. Note that at zero field, theenergylevelsaregiven byequations(5) and (6).AllStarkstatesbecomehigh-field seeking (i.e.,their eigenenergydecreaseswith increasingfield strength) at sufficientlyhigh field strengths. However, at a lowfield,wheretheStark potentialmerelyhindersmolecular rotation, Stark stateswith < + M J J ˜(˜ 1) 1 3 2 arefirst high-field seeking(i.e., their eigenenergyincreaseswith increasingfield strength), asexemplified bythe η ∣ = = ∣ ∣= 〉 J N M ˜ , ˜ 1, ; 3 2 1 2 el and η ∣= = ∣ ∣= 〉 J N M ˜ , ˜ 2, ; 3 2 1 2 el states.Thisbehavior resultsfrom thetilt angleoftheangular momentum (approximatelyconserved at lowfield strengths) with respect to thefield vector (space-fixed Z-axis). When theangular momentum isnearlyperpendicular to thefield vector, themoleculeacts likeaplanar rotor and spendsmost ofitstimeoriented oppositelyto thedirection oftheStark field, wherethe rotor-fixed electricdipolemoment interactswith thefield repulsively. Oncethefield strength becomessufficient for theStarkpotentialto confinethemolecular rotation and convert it into libration about thefield vector, the body-fixed dipolegetsoriented alongthefield vector wherebytheStark interaction becomesattractive. 9 NewJ.Phys.17(2015) 045017 KSharmaand BFriedrich Figure10. Dependenceoftheeigenenergiesofapolar Σ 2 moleculeon theanisotropicpolarizabilityinteraction parameter ηopt.F1 and F2levelsareshown,respectively, bydashed and fulllinesin panelspertainingto different valuesofthegood quantum number ∣ ∣ M . Red and bluecurvespertain, respectively,to stateswith = J˜ 1 2 and = J˜ 3 2.Notethat here η η = = 0 m el . Figure10. Dependenceoftheeigenenergiesofapolar Σ 2 moleculeon theanisotropicpolarizabilityinteraction parameter ηopt.F1 and F2levelsareshown,respectively, bydashed and fulllinesin panelspertainingto different valuesofthegood quantum number ∣ ∣ M . Red and bluecurvespertain, respectively,to stateswith = J˜ 1 2 and = J˜ 3 2.Notethat here η η = = 0 m el . Figure7showstheorientation and alignment ofthelowest sixstatesasafunction of ηel.Theorientation and alignment ofthemolecular axisischaracterized, respectively, bytheexpectation values θ 〈 〉 cos and θ 〈 〉 cos2 .In addition, thedirectionalityofthestatesand itsvariation with field strength isvisualized bythepolar diagrams displaying, at intervals, theprobabilitydensity, equation (22).In thesepolar diagramsand in thosethat areto followbelow, thefullrangeoftherainbowalwayscorrespondsto thefullrangeoftheprobability. Theabsolute directionalityofthestatesisreflected in theshapeofthepolar plotsand theplotted valuesofthealignment and orientation cosines. Asthemoleculebecomesoriented in the + Z direction, thelower lobeoftheprobabilitydistribution becomessmaller and theupper lobelarger. At high electricfield strengthsthelower lobeishardlyvisible. For a given J˜ and N˜ ,stateswith ∣ ∣= M J˜ havethelowest energyand exhibit thehighest orientation. Wenotethat, by theHellmann–Feynman theorem, θ 〈 〉= − η ∂ ∂ cos E B ( ) el ,and so onecan glean thiskeymeasureofdirectionality from theslopesoftheStark energies. 3.1.1.PureStarkinteraction Figure7also illustratesthevariation ofthedirectionalityofthe η ∣= = ∣ ∣= 〉 J N M ˜ , ˜ 1, ; 3 2 1 2 el and η ∣ = = ∣ ∣= 〉 J N M ˜ , ˜ 2, ; 3 2 1 2 el states, i.e.,the‘wrong-way’ orientation at lowfield strengthsand itsconversion to the‘right-way’ orientation at high field strengths, asdescribed above. Alessintuitiveeffect oftheelectricfield on thepolar Σ 2 moleculeisatransfer oftheprobabilitydensityfrom otationalto spin angular momentum, asreflected bytheincreaseofthesizeofthepolar plots. 3.1.2.PureZeemaninteraction TheZeeman potential, equation (9),undoesthe ± M degeneracyand splitseach J˜ levelinto + J 2˜ 1stateswith different signed valuesofM. Figure8showsthedependenceoftheeigenenergiesofa Σ 2 moleculefor thelowest twelvestateson the magneticdipoleinteraction parameter ηm,which isproportionalto themagneticfield strength. The eigenenergiesarelinear in ηm for stateswith = ± M J˜ (so called stretched states) and at most quadraticfor other states. In thestrong-field limit,F1statesarelow- or high-field seekingdependingon whether M ispositiveor negative,whereasF2statesareallhigh-field seeking. In thestrong-field (Paschen–Back) limit, theelectron spin and therotationalangular momentum uncoupleand thedependenceoftheZeeman levelson themagneticfield strength becomes η ≈M E B S m, where = ± MS 1 2 istheprojection ofthespin angular momentum Son theZ-axis. 10 NewJ.Phys.17(2015) 045017 KSharmaand BFriedrich Figure11. Probabilitydensities, orientation and alignment cosinesofa Σ 2 moleculeasfunctionsoftheanisotropicpolarizability interaction parameter ηopt.Valuespertainingto theF1and F2statesareshown,respectively, bydashed and fulllines.Blueand red curvespertain,respectively, to theleft (orientation) and right (alignment) ordinate. Notethat here η η = = 0 m el . Figure11. Probabilitydensities, orientation and alignment cosinesofa Σ 2 moleculeasfunctionsoftheanisotropicpolarizability interaction parameter ηopt.Valuespertainingto theF1and F2statesareshown,respectively, bydashed and fulllines.Blueand red curvespertain,respectively, to theleft (orientation) and right (alignment) ordinate. Notethat here η η = = 0 m el . ThePaschen–Back uncouplingsetson at η γ ≫ = ′ + − + − N ( ˜ ) m E E B 1 2 ,i.e.,at η ⪆1 m for themolecular example considered. Sinceparity, − ( 1)N˜ ,and M aregood quantum numbers, thenumerouscrossingsoftheZeeman levelsthat comeabout for a Σ 2 moleculearegenuine. Ofparticular interest arecrossingsoflevelswith sameM but opposite parity,seesection 3.2.2.Wenotethat it isthePaschen–Back effect that precludestheoccurrenceofavoided crossingsofthe Σ 2 Zeeman levels[9,10]. Figure9displaysthedirectionalpropertiesofa Σ 2 moleculesubject to amagneticfield. Sinceamagnetic field cannot orient themolecular axis, theorientation cosinevanishesidentically. However, theaxiscan be aligned.Thealignment cosine, concurrent for agiven statewith theexpectation valueofthemagneticdipole moment [10],increases/decreasesmonotonouslywith ηm onlyfor thestretched stateswith < > M M 0 0, whilefor therest it variesbetween ‘wrong-way’ (lessthan field-freevalue) and ‘right-way’ (morethan field-free value) alignment. In thePaschen–Back limit,thealignment tendsto aconstant value. 3.2.Double-field effects In thissection wewillprovidea summaryofhowtwo collinear fieldsaffect apolar and polarizable Σ 2 molecule. 3.1.3.Purepolarizabilityinteractionwithanoptical field LiketheStark potential, equation (7),theanisotropicpolarizabilityinteraction with anonresonant opticalfield, equation (11), splitseach J˜ levelinto + J˜ 1 2 stateswith different valuesof∣ ∣ M but doesnot undo the ± M degeneracy. Figure10showsthedependenceoftheeigenenergiesofthelowest sixstatesofa Σ 2 moleculeon the interaction parameter ηopt,which isproportionalto theopticalfield intensity .Onecan seetheformation of theopposite-paritytunnelingdoubletswith increasing ηopt,which becomequasi-degenerateat high fields.Note that themembersofagiven tunnelingdoublet havesame J˜.In contrast to theStark interaction, wherefor a 11 NewJ.Phys.17(2015) 045017 KSharmaand BFriedrich Figure12. Dependenceoftheeigenenergiesofa Σ 2 moleculeon theopticalfield strength parameter ηopt in thepresenceofan electric field. F1 and F2levelsareshown,respectively,bydashed and fulllinesin panelspertainingto different valuesofthegood quantum number ∣ ∣ M . Red and bluecurvespertain, respectively,to stateswith = J˜ 1 2 and = J˜ 3 2. Notethat here η = 5 el and η = 0 m . Figure12. Dependenceoftheeigenenergiesofa Σ 2 moleculeon theopticalfield strength parameter ηopt in thepresenceofan electric field. F1 and F2levelsareshown,respectively,bydashed and fulllinesin panelspertainingto different valuesofthegood quantum number ∣ ∣ M . Red and bluecurvespertain, respectively,to stateswith = J˜ 1 2 and = J˜ 3 2. Notethat here η = 5 el and η = 0 m . given J˜, stateswith lower ∣ ∣ M haveahigher eigenenergy, theeigenenergyofstatescreated bytheanisotropic polarizabilityinteraction increaseswith increasing∣ ∣ M . Figure11displaysthedirectionalpropertiesofa Σ 2 moleculesubject to an opticalfield.Theopticalfield doesnot orient themoleculebut greatlyenhancesitsalignment. Notethat thealignment ofthemembersofa given tunnelingdoublet becomesthesameastheir eigenenergiesbecomeexponentiallyquasi-degenerateas η ∝ − exp( ) opt 1 2 .Thisbehavior followsfrom theHellmann–Feynman theorem, accordingto which θ 〈 〉= − η ∂ ∂ cos2 ( ) E B opt . Wenotethat thealignment ofthestatethat becomesthehigher member ofatunneling ˜ ˜ doublet (and so hasahigher valueof N˜ ) alwaysexceedsthat ofthelower member (with alower valueof N˜ ). Interestingly, for apair ofStark stateswith same J˜, it istheonewith lower N˜ that hasthelarger alignment ofthe two.Theopticalfield leadsto aconsiderabletransfer oftheprobabilitydensityfrom therotationalto thespin angular momentum, asreflected bytheincreasein thesizeofthepolar plotswith increasinginteraction parameter ηopt. 3.2.1.Congruent electricandoptical fields Figure12showsthedependenceoftheeigenenergiesofthelowest sixstatesofa Σ 2 moleculeon theinteraction parameter ηopt in thepresenceofan electricfield such that thecorrespondinginteraction parameter η = 5 el . Compared with figure10,weseethat theopposite-paritytunnelingdoubletsthat werequasi-degeneratein the absenceoftheelectricfield havebeen readilysplit dueto thecouplingbytheelectricdipoleinteraction. The tunnelingsplittingin thecombined fieldsat agiven ηopt isproportionalto ηel, Δ η η η = ∝ E ( const ., ) t opt el el [5,62]. Figure12showsthedependenceoftheeigenenergiesofthelowest sixstatesofa Σ 2 moleculeon theinteraction parameter ηopt in thepresenceofan electricfield such that thecorrespondinginteraction parameter η = 5 el . Compared with figure10,weseethat theopposite-paritytunnelingdoubletsthat werequasi-degeneratein the absenceoftheelectricfield havebeen readilysplit dueto thecouplingbytheelectricdipoleinteraction. The tunnelingsplittingin thecombined fieldsat agiven ηopt isproportionalto ηel, Δ η η η = ∝ E ( const ., ) t opt el el [5,62]. [ , ] Figure13displaysthedirectionalpropertiesofa Σ 2 moleculesubject to an opticalfield in thepresenceofan electricfield.Theseexhibit quiteafewdistinct features, such asthesudden back-and-forth variationsofthe [ , ] Figure13displaysthedirectionalpropertiesofa Σ 2 moleculesubject to an opticalfield in thepresenceofan electricfield.Theseexhibit quiteafewdistinct features, such asthesudden back-and-forth variationsofthe 12 NewJ.Phys.17(2015) 045017 KSharmaand BFriedrich Figure13. Probabilitydensities, orientation and alignment cosinesofa Σ 2 moleculeasfunctionsoftheanisotropicpolarizability interaction parameter ηopt in thepresenceofan electricfield.Valuespertainingto theF1and F2 statesareshown, respectively, by dashed and fulllines.Blueand red curvespertain,respectively, to theleft (orientation) and right (alignment) ordinate.Notethat here η = 5 el and η = 0 m . Figure13. Probabilitydensities, orientation and alignment cosinesofa Σ 2 moleculeasfunctionsoftheanisotropicpolarizability interaction parameter ηopt in thepresenceofan electricfield.Valuespertainingto theF1and F2 statesareshown, respectively, by dashed and fulllines.Blueand red curvespertain,respectively, to theleft (orientation) and right (alignment) ordinate.Notethat here η = 5 el and η = 0 m . orientation and alignment cosineswith ηopt.Most ofthesefeaturesareconnected with themutual‘repelling’ of thelevelswithin agiven tunnelingdoublet—which lendsthecorrespondingstatesopposite-wayorientation— and with intersectionsofthoselevelswith levelsofsame∣ ∣ M but pertainingto different tunnelingdoublets. 1 1 and with intersectionsofthoselevelswith levelsofsame∣ ∣ M but pertainingto different tunnelingdoublets. So, for instance,likeitstunneling-doublet partner, the∣= = ∣ ∣= 〉 J N M ˜ , ˜ 1, 1 2 1 2 stateisinitiallyright-way oriented but flipsitsorientation, at η ≈23 opt ,dueto itsinteraction with the∣= = ∣ ∣= 〉 J N M ˜ , ˜ 1, 3 2 1 2 state. Thisisreflected in thepolar plotsoftheprobabilitydensitiesaswellin that theupper lobevanishesand thelower lobebecomeshuge, portendingthewrongwayorientation ofthemolecular state. Likewise, the ∣= = ∣ ∣= 〉 J N M ˜ , ˜ 1, 3 2 1 2 state, which isinitiallywrong-wayoriented, flipsitsorientation at η ≈23 opt dueto its ˜ ˜ 1 1 Thisisreflected in thepolar plotsoftheprobabilitydensitiesaswellin that theupper lobevanishesand thelower lobebecomeshuge, portendingthewrongwayorientation ofthemolecular state. 3.2.1.Congruent electricandoptical fields Likewise, the ∣= = ∣ ∣= 〉 J N M ˜ , ˜ 1, 3 2 1 2 state, which isinitiallywrong-wayoriented, flipsitsorientation at η ≈23 opt dueto its interaction with the∣= = ∣ ∣= 〉 J N M ˜ , ˜ 1, 1 2 1 2 stateand acquiresaright-wayorientation. The ∣= = ∣ ∣= 〉 J N M ˜ , ˜ 1, 3 2 1 2 state, which isinitiallywrong-wayoriented, flipsitsorientation at η ≈23 opt dueto its interaction with the∣= = ∣ ∣= 〉 J N M ˜ , ˜ 1, 1 2 1 2 stateand acquiresaright-wayorientation. The ∣= = ∣ ∣= 〉 J N M ˜ , ˜ 2, 3 2 1 2 stateundergoestheflip twice, wherebythefirst flip isdueto theinteraction with the ∣= = ∣ ∣= 〉 J N M ˜ , ˜ 1, 3 2 1 2 stateand thesecond flip comesabout becauseofthestate’sinteraction with the ∣= = ∣= 〉 J N M ˜ , ˜ 2, 5 2 1 2 stateat η ≈28 opt (ahigher-lyingstatenot shown here).Thestateisright-wayoriented between thesetwo flipsand iswrong-wayoriented in thehigh field region. Apart from that, thereis,asexpected, aprobabilitydensitytransfer from therotationalangular momentum to thespin angular momentum. = = ∣ ∣= 〉 J N M ˜ , ˜ 2, 3 2 1 2 stateundergoestheflip twice, wherebythefirst flip isdueto theinteraction with the = = ∣ ∣= 〉 J N M ˜ , ˜ 1, 3 2 1 2 stateand thesecond flip comesabout becauseofthestate’sinteraction with the ˜ ˜ 5 1 Theseflipsin theorientation ofthemoleculeareofparticular importancesincenot onlydo theseprovidethe meansfor switchingtheorientation ofthemolecule, but, aswewillseein section 3.3, thevaluesoftheinteraction parameter wheretheflipstakeplacecan becontrolled byintroducingathird field. Theseflipsin theorientation ofthemoleculeareofparticular importancesincenot onlydo theseprovidethe meansfor switchingtheorientation ofthemolecule, but, aswewillseein section 3.3, thevaluesoftheinteraction parameter wheretheflipstakeplacecan becontrolled byintroducingathird field. 13 NewJ.Phys.17(2015) 045017 KSharmaand BFriedrich Figure14. Dependenceoftheeigenenergiesofapolar Σ 2 moleculeon themagneticdipoleinteraction parameter ηm in thepresenceof an electricfield.F1and F2levelsareshown, respectively, bydashed and fulllinesin panelspertainingto signed valuesofthegood quantum number M.Red and bluecurvespertain, respectively, to stateswith = J˜ 1 2 and = J˜ 3 2.Notethat here η = 5 el and η = 0 opt . Figure14. Dependenceoftheeigenenergiesofapolar Σ 2 moleculeon themagneticdipoleinteraction parameter ηm in thepresenceof an electricfield.F1and F2levelsareshown, respectively, bydashed and fulllinesin panelspertainingto signed valuesofthegood quantum number M.Red and bluecurvespertain, respectively, to stateswith = J˜ 1 2 and = J˜ 3 2.Notethat here η = 5 el and η = 0 opt . 3.2.2.Congruent electricandmagneticfields Figure14showsthedependenceoftheeigenenergiesofthelowest twelvestatesofa Σ 2 moleculeon the interaction parameter ηm in thepresenceofan electricfield such that thecorrespondinginteraction parameter η = 5 el .Compared with figure8, weseethat thegenuineintersection in theabsenceoftheelectricfield ofthe opposite-paritylevelshavebecomeavoided crossingsdueto thecouplingbytheelectricdipoleinteraction. This transformsthelow-field seekingstatesinto high-field seekersand viceversa. Theconcomitant directionalpropertiesareexemplified in figure15.For instance, the = = = − J N M ˜ , ˜ 1, 1 2 1 2 statechangesitsshapefrom an oriented torusto an oriented double-lobed form whilethecrossing = = = − J N M ˜ , ˜ 1, 3 2 1 2 statechangesfrom an oriented double-lobeto an oriented torus. Wenotethat sincetheintersectinglevelsareexactlydegenerateat thecrossingpoint,even asmallelectricfield can mixthem and thusgenerateorientation. For η η ⩾ m el,themaximum valueoftheorientation cosineis determined just bytheintersectingpurelyZeeman statesand isindependent of ηel,see[9, 10]. Wenotethat sincetheintersectinglevelsareexactlydegenerateat thecrossingpoint,even asmallelectricfield can mixthem and thusgenerateorientation. For η η ⩾ m el,themaximum valueoftheorientation cosineis determined just bytheintersectingpurelyZeeman statesand isindependent of ηel,see[9, 10]. Wenotethat sincetheintersectinglevelsareexactlydegenerateat thecrossingpoint,even asmallelectricfield can mixthem and thusgenerateorientation. For η η ⩾ m el,themaximum valueoftheorientation cosineis determined just bytheintersectingpurelyZeeman statesand isindependent of ηel,see[9, 10]. 3.2.3.Congruent magneticandoptical fields In light ofthefact that themagneticdipoleinteraction onlycouplesstateswith sameparity, theopposite-parity membersofthetunnelingdoubletscreated bythepolarizabilityinteraction with theopticalfield remain uncoupled in thepresenceofthemagneticfield. However, themagneticfield liftsthe ± M degeneracyofthe good quantum number ∣ ∣ M that characterizeseach tunnelingdoublet in theopticalfield aloneand thus,for ∣ ∣> M 0,doublesthenumber ofthetunnelingdoublets. Thisisillustrated in figure16, which showsthedependenceoftheeigenenergiesofa Σ 2 moleculeon the opticalfield in thepresenceofamagneticfield such that η = 2.5 m .Thekeyfeatureofthe‘doubled’ tunneling doubletsisthat theyallremain quasi-degenerateat high ηopt.However, thestatescreated bytheanisotropic polarizabilityinteraction with theopticalfield arealso affected bythepresenceofthemagneticfield in other waysthan removingthe ± M degeneracy. In particular, sincethemagneticfield movesthelevelsofa paramagneticmoleculearound,seesection 3.1.2,someofthetunnelingdoubletsundergo a flip ofthepartner levels:what wasalower member ofadoublet becomesahigher member and viceversa. 14 NewJ.Phys.17(2015) 045017 KSharmaand BFriedrich Figure15. Probabilitydensities, orientation and alignment cosinesofa Σ 2 moleculeasfunctionsofthemagneticdipoleinteraction parameter ηm in thepresenceofan electricfield. Valuespertainingto theF1and F2statesareshown, respectively, bydashed and full lines. Blueand red curvespertain,respectively, to theleft (orientation) and right (alignment) ordinate. Notethat here η = 5 el and η = 0 opt . Figure15. Probabilitydensities, orientation and alignment cosinesofa Σ 2 moleculeasfunctionsofthemagneticdipoleinteraction parameter ηm in thepresenceofan electricfield. Valuespertainingto theF1and F2statesareshown, respectively, bydashed and full lines. Blueand red curvespertain,respectively, to theleft (orientation) and right (alignment) ordinate. Notethat here η = 5 el and η = 0 opt . Figure17showsthedirectionalpropertiesofa Σ 2 moleculeasafunction ofan opticalfield in thepresenceof amagneticfield.Aswehaveseen in figure9, themagneticfield doesnot alter thedirectionalpropertiesofa molecular stateascreated bytheopticalfield unlessthestateencountersanother statethat couplesto it. Since neither amagneticnor an opticalfield can orient a molecule, θ 〈 〉 cos vanishesidenticallyfor allstatescreated by thesefields. Finally, weobservethat theopticalfield leadsto asignificant transfer ofprobabilitydensityfrom rotationalto spin angular momentum in thecombined magneticand opticalfields. p In thissection westudytheeffectsofallthreefieldsactingon apolar and polarizable Σ 2 molecule simultaneously. p In thissection westudytheeffectsofallthreefieldsactingon apolar and polarizable Σ 2 molecule simultaneously. Variationoftheelectricfield.Figure18showsthedependenceoftheeigenenergiesofthelowest sixstateson theelectricdipoleinteraction parameter ηel in thepresenceofconstant magnetic (η = 2.5 m ) and opticalfields (η = 15 opt ).Sincethepresenceofthemagneticfield liftsthe ± M degeneracy, thefigureissplit into four panels, each pertainingto agiven valueofM, asstateswith > M 0 behavedifferentlyfrom stateswith < M 0. Wesee that thestatesarepaired up at η →0 el dueto theformation ofthequasi-degeneratetunnelingdoubletsbythe opticalfield. For η > 0 el thedoubletsareincreasinglycoupled bytheelectricdipoleinteraction and split up asa result.Themagneticfield bringsabout arelativeshift ofthedoublet levelswhich leadsto avoided crossings. Figure19showsthedirectionalpropertiesofa Σ 2 moleculeasafunction oftheelectricinteraction parameter at constant magneticand opticalfields.Weagain observeabrupt changesin thesenseofthemolecular axisorientation. However, thefield strengthsat which theseabrupt changestakeplacecan becontrolled by 15 NewJ.Phys.17(2015) 045017 KSharmaand BFriedrich Figure16. Dependenceoftheeigenenergiesofapolar Σ 2 moleculeon theanisotropicpolarizabilityinteraction parameter ηopt in the presenceofamagneticfield.F1and F2levelsareshown,respectively, bydashed and fulllinesin panelspertainingto signed valuesof thegood quantum number M. Red and bluecurvespertain, respectively,to stateswith = J˜ 1 2 and = J˜ 3 2. Notethat here η = 2.5 m and η = 0 el . Figure16. Dependenceoftheeigenenergiesofapolar Σ 2 moleculeon theanisotropicpolarizabilityinteraction parameter ηopt in the presenceofamagneticfield.F1and F2levelsareshown,respectively, bydashed and fulllinesin panelspertainingto signed valuesof thegood quantum number M. Red and bluecurvespertain, respectively,to stateswith = J˜ 1 2 and = J˜ 3 2. Notethat here η = 2.5 m and η = 0 el . tuningthevalueofthesuperimposed magneticfield.For instance, the∣= = ∣ ∣= 〉 J N M ˜ , ˜ 1, 1 2 1 2 statein the absenceofthemagneticfield changesitsorientation at η ≈4 el ;herethechangetakesplaceat ahigher valueof theelectricfield for the∣ = = = −〉 J N M ˜ , ˜ 1, 1 2 1 2 state (η ≈6 el at η = 2.5 m ) and for the ∣= = = 〉 J N M ˜ , ˜ 1, 1 2 1 2 stateat alower valueofelectricfield (η ≈2 el at η = 2.5 m ). ∣= = = 〉 J N M ˜ , ˜ 1, 1 2 1 2 stateat alower valueofelectricfield (η ≈2 el at η = 2.5 m ). 2 2 In general, for stateswith < M 0,thehigher thevalueofthemagneticfield,thegreater istheelectricfield strength required to flip theorientation ofthestate–and viceversa for stateswith > M 0:thehigher thevalueof magneticfield, thelower istheelectricfield strength required to flip theorientation. Thishappensbecausethe avoided crossingsformed that lead to aflip in orientation ariseat alower electricfield for stateswith < M 0 and ahigher electricfield for stateswith > M 0 asthemagneticfield strength isincreased.For < M 0, thelower of thetwo statesformingtheavoided crossingishigh-field seekingand thehigher stateislow-field seekingunder themagneticfield alone. p In thissection westudytheeffectsofallthreefieldsactingon apolar and polarizable Σ 2 molecule simultaneously. Thisresultsin an increasein theenergysplittingbetween thesetwo statesasthe magneticfield isincreased. This,in turn, leadsto theformation ofavoided crossings, and henceto theflippingof theorientation ofthestateat ahigher electricfield. On theother hand,for > M 0 states,thehigher ofthetwo statesformingtheavoided crossingishigh-field seekingand thelower stateislow-field seekingunder the magneticfield alone.Thisresultsin adecreaseoftheenergysplittingbetween thesetwo statesasthemagnetic field isincreased and theformation ofavoided crossings, and henceto theflippingoftheorientation ofthestates at alower electricfield. Theabovefeatureofthetriple-field interaction lendsitselfasameansto controlthesense ofthemolecular axisorientation with thesuperimposed magneticfield asacontrolparameter. Variationofthemagneticfield.Figure20displaysthedependenceoftheeigenenergiesofa Σ 2 moleculeon themagneticfield in thepresenceofan electricfield (η = 5 el ) and opticalfield (η = 15 opt ). Aswecan see, the tunnelingdoubletsareno longer quasi-degenerateastheyaresplit bytheelectricfield. Figure20bearsa similaritywith figure14;however, dueto thelevelshiftsbrought about bytheopticalfield, theenergysplittingat theavoided crossingof, e.g.,the∣= = = −〉 J N M ˜ , ˜ 1, 1 2 1 2 and ∣= = = −〉 J N M ˜ , ˜ 1, 3 2 1 2 statesislessthan in theabsenceoftheopticalfield. Thisleadsto a much moreabrupt variation oftheorientation cosinein the vicinityofthecrossing, ascan beseen in figure21.In addition,bycomparingfigures21and 15, weseethat the 16 NewJ.Phys.17(2015) 045017 KSharmaand BFriedrich Figure17. Probabilitydensities, orientation and alignment cosinesofa Σ 2 moleculeasfunctionsoftheanisotropicpolariuzability interaction parameter ηopt in thepresenceofamagneticfield.Valuespertainingto theF1and F2statesareshown, respectively, by dashed and fulllines.Blueand red curvespertain,respectively, to theleft (orientation) and right (alignment) ordinate.Notethat here η = 2.5 m and η = 0 el . Figure17. Probabilitydensities, orientation and alignment cosinesofa Σ 2 moleculeasfunctionsoftheanisotropicpolariuzability interaction parameter ηopt in thepresenceofamagneticfield.Valuespertainingto theF1and F2statesareshown, respectively, by dashed and fulllines.Blueand red curvespertain,respectively, to theleft (orientation) and right (alignment) ordinate.Notethat here η = 2.5 m and η = 0 el . Figure17. Probabilitydensities, orientation and alignment cosinesofa Σ 2 moleculeasfunctionsoftheanisotropicpolariuzability interaction parameter ηopt in thepresenceofamagneticfield.Valuespertainingto theF1and F2statesareshown, respectively, by dashed and fulllines.Blueand red curvespertain,respectively, to theleft (orientation) and right (alignment) ordinate.Notethat here η = 2.5 m and η = 0 el . presenceoftheopticalfield can lead to ahigher orientation ofthestates(i.e.,greater valuesof θ ∣〈 〉∣ cos .The flippingoftheorientation can beconvenientlycontrolled bymakinguseoftheopticalfield asacontrol parameter. For stateswith < M 0,thehigher theopticalfield,thelower isthemagneticfield required to flip the orientation and viceversafor stateswith > M 0.Thisisbecausetheelectricfield couplesthetunnelingdoublets formed bytheopticalfield. For < M 0 states,thelowest statefor everyM isahigh-field seekingstatewhich, therefore, doesnot haveanypointsofinflection.Theavoided crossings, wheretheflippingoftheorientation takesplace, areformed between statesofdifferent J˜. Theenergysplittingbetween thesestatesdecreaseswith increasingopticalfield asthetunnelingdoubletsformed bytheopticalfield arecoupled bytheelectricfield.This leadsto adecreasein themagneticfield strength required to flip theorientation ofthestatewith increasing opticalfield. On theother hand,for stateswith > M 0,thelowest statefor each M isalow-field seekingstate under themagneticfield interaction. So theavoided crossingswheretheflip in orientation takesplacearewithin thesametunnelingdoublet. Theenergysplittingbetween thetwo statesincreaseswith increasingopticalfield becausethetunnelingdoubletsarecoupled bytheelectricfield, therebyrequiringagreater magneticfield to flip theorientation. Variationof theoptical field. p In thissection westudytheeffectsofallthreefieldsactingon apolar and polarizable Σ 2 molecule simultaneously. Figure22showsthedependenceoftheeigenenergieson theopticalfield strength parameter ηopt in thepresenceofelectric (η = 5 el ) and magnetic (η = 2.5 m ) fields.Likein figure16,weseethat thetunnelingdoubletssplit bytheelectricdipoleinteraction. However, dueto theZeeman shifts, someofthe levelshaveinterchanged their order. So thelower member ofthe = = = J M N ˜ , , ˜ 0, 1 1 2 1 2 tunnelingdoublet hasbecomethehigher member and thehigher member hasbecomethelower member. Such an interchangein theorder ofthestatesoccursbecausethetwo statesgenuinelycrosseach other under theeffect ofmagneticfield, seefigure8. 17 NewJ.Phys.17(2015) 045017 KSharmaand BFriedrich Figure18. Dependenceoftheeigenenergiesofapolar Σ 2 moleculeon theelectricdipoleinteraction parameter ηel in thepresenceofa magneticand an opticalfield.F1and F2 levelsareshown,respectively, bydashed and fulllinesin panelspertainingto signed valuesof thegood quantum number M. Red and bluecurvespertain, respectively,to stateswith = J˜ 1 2 and = J˜ 3 2. Notethat here η = 2.5 m and η = 15 opt . Figure18. Dependenceoftheeigenenergiesofapolar Σ 2 moleculeon theelectricdipoleinteraction parameter ηel in thepresenceofa magneticand an opticalfield.F1and F2 levelsareshown,respectively, bydashed and fulllinesin panelspertainingto signed valuesof thegood quantum number M. Red and bluecurvespertain, respectively,to stateswith = J˜ 1 2 and = J˜ 3 2. Notethat here η = 2.5 m and η = 15 opt . Thisisdetailed in figure23which showsthedependenceon theopticalfield ηopt ofthetunnelingsplitting, ΔE B t,between the∣ = = ∣ ∣= 〉 J N M ˜ , ˜ 0, 1 2 1 2 and ∣ = = ∣ ∣= 〉 J N M ˜ , ˜ 1, 1 2 1 2 states(thelowest tunnelingdoublet) in theabsence(red curve) and presence(bluecurves) ofthemagneticfield.Themagneticfield separatesthe tunnelingdoublet into two, with each pertainingeither to = M 1 2 or = − M 1 2.Anegativetunnelingsplitting correspondsto a reversaloftheorder ofthemembersofthetunnelingdoublets. Wenotethat thetunneling doubletsdepicted becomequasi-degeneratein thehigh field region, followingthe η ∝ − Δ exp( ) E B opt t 1 2 asymptotic dependence. Figure24detailswhat happenswhen an electricfield (correspondingto η = 5 el ) issuperimposed. Thered curveshowsthetunnelingsplitting ΔE B t in theabsenceofthemagneticfield.Sincetheelectricfield couplesthe ∣= = ∣ ∣= 〉 J N M ˜ , ˜ 0, 1 2 1 2 and ∣= = ∣ ∣= 〉 J N M ˜ , ˜ 1, 1 2 1 2 statesin question, they‘repel’ each other asaresult. Initially,thetunnelingsplittingrapidlyincreaseswith theopticalfield onlyto taper offin thehigh field region. When themagneticfield isswitched on,thistunnelingdoublet dividesinto two separatetunnelingdoublets, one with = − M 1 2 and another with = M 1 2.Thetunnelingsplittingofthetwo tunnelingdoubletsformed isshown bythedashed blueline. Whilethedependenceon ηopt ofthetunnelingsplittingofthedoublet pertainingto = − M 1 2 resemblesthat ofthetunnelingdoublet in theabsenceofthemagneticfield, thetunnelingsplitting keepson increasingwith theopticalfield strength for thedoublet pertainingto = M 1 2. exchanged their labels;in thislatter caseit isthestatewith lower N˜ that exhibitsawrongwayorientation at high opticalfields. exchanged their labels;in thislatter caseit isthestatewith lower N˜ that exhibitsawrongwayorientation at high opticalfields. p In thissection westudytheeffectsofallthreefieldsactingon apolar and polarizable Σ 2 molecule simultaneously. Notethat in thelatter case, themembersofthetunnelingdoublet interchanged their order, asreflected in thechangeofsign of ΔE B t from positiveto negative. Figure25showsthedirectionalpropertiesofa Σ 2 moleculeasafunction oftheopticalfield in thepresence ofboth electric (η = 5 el ) and magnetic (η = 2.5 m ) fields. Thepresenceofthemagneticfield can beused to controltheopticalfield strength at which theorientation cosinechangessign.Theopticalfield strength required to flip theorientation decreaseswith increasingmagneticfield for stateswith < M 0 and viceversafor states with > M 0.Finally,wenotethat thetunnelingdoublet member with greater N˜ exhibitsawrongway orientation in thehigh field region, except for thecasewhen thetwo membersofthetunnelingdoublet have 18 NewJ.Phys.17(2015) 045017 KSharmaand BFriedrich Figure19. Probabilitydensities, orientation and alignment cosinesofa Σ 2 moleculeasfunctionsoftheelectricdipoleinteraction parameter ηel in thepresenceofamagneticand an opticalfield.Valuespertainingto theF1and F2statesareshown, respectively, by dashed and fulllines.Blueand red curvespertain,respectively, to theleft (orientation) and right (alignment) ordinate.Notethat here η = 2.5 m and η = 15 opt . Figure19. Probabilitydensities, orientation and alignment cosinesofa Σ 2 moleculeasfunctionsoftheelectricdipoleinteraction parameter ηel in thepresenceofamagneticand an opticalfield.Valuespertainingto theF1and F2statesareshown, respectively, by dashed and fulllines.Blueand red curvespertain,respectively, to theleft (orientation) and right (alignment) ordinate.Notethat here η = 2.5 m and η = 15 opt . 4.Conclusions Weexamined theeigenpropertiesofpolar, paramagnetic, and polarizablelinear moleculesin congruent electric, magnetic, and nonresonant opticalfieldsbynumericaldiagonalization ofthecorrespondingHamiltonian matrix.Wefound that thedirectionalityofthemolecular stateswhich can beachieved with thetriple-field combination supersedes—in itsmagnitudeaswellascontrollability—that obtained bythedouble-field combinationsor singlefields,asstudied previously.Thehighlydirectionalmolecular statescreated bythetriple- field combination can bethen acted upon byspacefixed fields,permittingto manipulatereadilyand efficiently both therotation and translation ofthemolecules. Possibleapplicationsabound, but herewewould liketo emphasizethepotentialfor manipulatingcold molecules. Not onlyarecold moleculesgenerallymore susceptibleto manipulation byexternalfieldsdueto their lowtranslationalenergy, but someoftheir applications, such asquantum computing[76] or thesearch for theelectricdipolemoment oftheelectron [70], havealreadyenvisioned theuseofcombined fieldsfor both trappingand probing. Thepresent paper expandson what theuseofthetriple-field combination would entail. Thecombination ofan opticaland amagneticfield createsamultitudeofdegenerateor quasi-degenerate statesofoppositeparitythat can undergo afacilecouplingbyasuperimposed (weak) electricfield. Thisisthe essenceoftheeffectsofthethreecongruent fieldsand thebasisfor their synergy. That oneofthefields—the opticalone—can bevaried or switched on and offat timescaleson theorder oftherotationalperiod would lend 19 NewJ.Phys.17(2015) 045017 KSharmaand BFriedrich Figure20. Dependenceoftheeigenenergiesofapolar Σ 2 moleculeon themagneticdipoleinteraction parameter ηm in thepresenceof an electricand an opticalfield. F1and F2levelsareshown,respectively,bydashed and fulllinesin panelspertainingto signed valuesof thegood quantum number M. Red and bluecurvespertain, respectively,to stateswith = J˜ 1 2 and = J˜ 3 2. Notethat here η = 5 el and η = 15 opt . Figure20. Dependenceoftheeigenenergiesofapolar Σ 2 moleculeon themagneticdipoleinteraction parameter ηm in thepresenceof an electricand an opticalfield. F1and F2levelsareshown,respectively,bydashed and fulllinesin panelspertainingto signed valuesof thegood quantum number M. Red and bluecurvespertain, respectively,to stateswith = J˜ 1 2 and = J˜ 3 2. Notethat here η = 5 el and η = 15 opt . amanipulation techniquebased on thetriple-field effectsadegreeofcontrollabilitythat isneeded for such applicationsasstereo-dynamicalcollisionalstudiesor quantum computing. In our forthcomingwork wewillexaminethenon adiabaticeffectsthat areexpected [30] to arisewhen the opticalfield isvaried at timescalesshorter than therotationalperiod ofthemolecule.Also worthyofexploringis thedependenceofthetriple-field effectson thetilt anglesamongthethreefield vectors[21,71].Relevant to both isthetopologyoftheeigenenergysurfacesspanned bythe ηel, ηm,and ηopt interaction parametersthat mayresult in conicalintersections[63,64], another subject ofour forthcomingstudy. Acknowledgments Discussionswith Dr Burkhard Schmidt, Prof. John Doyleand Dr MikeTarbutt aswellassupport bytheDFG through grant FR3319/3-1 aregratefullyacknowledged. AppendixA. Direction cosinematrixelements in thesymmetrictop basis Thenon-vanishingelementsofthedirection cosinematrix, ϕI j, used in thiswork aregiven by Ω ϕ Ω Ω Ω ′ ′ ′ = ′ ′ ′ × ′ ′ ( ) ( ) J M J M f J J g J J h J M J M , , , , , , , , ( , , , ) (22) I j j I (22) with ′ f J J ( , ), Ω Ω ′ ′ g J J ( , , , ) j ,and ′ ′ h J M J M ( , , , ) I listed in tablesA1 –A5,see[72]. with ′ f J J ( , ), Ω Ω ′ ′ g J J ( , , , ) j ,and ′ ′ h J M J M ( , , , ) I listed in tablesA1 –A5,see[72]. with ′ f J J ( , ), Ω Ω ′ ′ g J J ( , , , ) j ,and ′ ′ h J M J M ( , , , ) I listed in tablesA1 –A5,see[72]. A.1. Matrixelements in Hund’s case(a) basis A.1. Matrixelements in Hund’s case(a) basis For theelectricfield interaction,weneed matrixelementsoftheoperator θ cos which arelisted in tableA6 . For theopticalfield interaction, weneed matrixelementsoftheoperator θ cos2 ,which arelisted in tableA7 . ( ) For theelectricfield interaction,weneed matrixelementsoftheoperator θ cos which arelisted in tableA6 For theopticalfield interaction, weneed matrixelementsoftheoperator θ cos2 ,which arelisted in tableA7 20 NewJ.Phys.17(2015) 045017 KSharmaand BFriedrich Figure21. Probabilitydensities, orientation and alignment cosinesofa Σ 2 moleculeasfunctionsofthemagneticdipoleinteraction parameter ηm in thepresenceofamagneticand an opticalfield.Valuespertainingto theF1and F2statesareshown, respectively, by dashed and fulllines.Blueand red curvespertain,respectively, to theleft (orientation) and right (alignment) ordinate.Notethat here η = 5 el and η = 15 opt . Figure21. Probabilitydensities, orientation and alignment cosinesofa Σ 2 moleculeasfunctionsofthemagneticdipoleinteraction parameter ηm in thepresenceofamagneticand an opticalfield.Valuespertainingto theF1and F2statesareshown, respectively, by dashed and fulllines.Blueand red curvespertain,respectively, to theleft (orientation) and right (alignment) ordinate.Notethat here η = 5 el and η = 15 opt . AppendixA. Direction cosinematrixelements in thesymmetrictop basis For themagneticfield interaction, weneed thematrixelementsoftheSZoperator For themagneticfield interaction, weneed thematrixelementsoftheSZoperator ϕ ϕ ϕ = + + + − − + ( ) S S S S 1 2 , (23) Z Z Z Z z z (23) wherethesuperscriptspertain to thebody-fixed and thesubscriptsto thespace-fixed frame.Theelectron spin matrixelementsare wherethesuperscriptspertain to thebody-fixed and thesubscriptsto thespace-fixed frame.Theelectron spin matrixelementsare wherethesuperscriptspertain to thebody-fixed and thesubscriptsto thespace-fixed frame.Theelectron spin matrixelementsare Ω Ω ± ± = ± S J M S S J M , 1 2 , , , 1 2 , , 1, (24) Ω Ω ± ∓ = S J M S S J M , 1 2 , , , 1 2 , , 1 2 . (25) z (24) (25) AppendixB. Conversion factors Dependenceofthetunnelingsplitting Δ = − = = ∣ ∣= = = ∣ ∣= E B E E B ( ) t J N M J N M ˜ , ˜ 1, ˜ , ˜ 0, 1 2 1 2 1 2 1 2 on theopticalfield interaction parameter ηopt for η = 5 el (red curve) and η = 2.5 m (bluecurves). Figure25. Probabilitydensities, orientation and alignment cosinesofa Σ 2 moleculeasfunctionsoftheanisotropicpolarizability interaction parameter ηopt in thepresenceofamagneticand an electricfield.Valuespertainingto theF1and F2statesareshown, respectively, bydashed and fulllines.Blueand red curvespertain, respectively,to theleft (orientation) and right (alignment) ordinate. Notethat here η = 5 el and η = 2.5 m . Figure24. Dependenceofthetunnelingsplitting Δ = − = = ∣ ∣= = = ∣ ∣= E B E E B ( ) t J N M J N M ˜ , ˜ 1, ˜ , ˜ 0, 1 2 1 2 1 2 1 2 on theopticalfield interaction parameter ηopt for η = 5 el (red curve) and η = 2.5 m (bluecurves). Figure24. Dependenceofthetunnelingsplitting Δ = − = = ∣ ∣= = = ∣ ∣= E B E E B ( ) t J N M J N M ˜ , ˜ 1, ˜ , ˜ 0, 1 2 1 2 1 2 1 2 on theopticalfield interaction parameter ηopt for η = 5 el (red curve) and η = 2.5 m (bluecurves). Figure25. Probabilitydensities, orientation and alignment cosinesofa Σ 2 moleculeasfunctionsoftheanisotropicpolarizability interaction parameter ηopt in thepresenceofamagneticand an electricfield.Valuespertainingto theF1and F2statesareshown, respectively, bydashed and fulllines.Blueand red curvespertain, respectively,to theleft (orientation) and right (alignment) ordinate. Notethat here η = 5 el and η = 2.5 m . Figure25. Probabilitydensities, orientation and alignment cosinesofa Σ 2 moleculeasfunctionsoftheanisotropicpolarizability interaction parameter ηopt in thepresenceofamagneticand an electricfield.Valuespertainingto theF1and F2statesareshown, respectively bydashed and fulllines Blueand red curvespertain respectively to theleft (orientation) and right (alignment) ordinate Figure25. Probabilitydensities, orientation and alignment cosinesofa Σ 2 moleculeasfunctionsoftheanisotropicpolarizability interaction parameter ηopt in thepresenceofamagneticand an electricfield.Valuespertainingto theF1and F2statesareshown, respectively, bydashed and fulllines.Blueand red curvespertain, respectively,to theleft (orientation) and right (alignment) ordinate Notethat here η = 5 el and η = 2.5 m . TableA1.The ′ f J J ( , ) term ofthe direction cosinematrix, equation (22). AppendixB. Conversion factors With quantitiesexpressin customaryunits, thedimensionlessinteraction parametersaregiven by: η μ ε = − − B 0.0168 (Debye) (kV cm ) (cm ), S el el 1 1   η = − B 0.9347 (Tesla) (cm ), m 1   η Δα = × Å − − − B 1.05 10 [ ] (W cm ) (cm ). opt 11 3 2 1 η μ ε = − − B 0.0168 (Debye) (kV cm ) (cm ), S el el 1 1   η = − B 0.9347 (Tesla) (cm ), m 1 3 η Δα = × Å − − − B 1.05 10 [ ] (W cm ) (cm ). opt 11 3 2 1 η Δα = × Å − − − B 1.05 10 [ ] (W cm ) (cm ). opt 11 3 2 1 21 NewJ.Phys.17(2015) 045017 KSharmaand BFriedrich Figure22. Dependenceoftheeigenenergiesofapolar Σ 2 moleculeon theanisotropicpolarizabilityinteraction parameter ηopt in the presenceofan electricand amagneticfield.F1and F2levelsareshown,respectively, bydashed and fulllinesin panelspertainingto signed valuesofthegood quantum number M.Red and bluecurvespertain,respectively, to stateswith = J˜ 1 2 and = J˜ 3 2.Notethat here η = 2.5 m and η = 5 el . Figure22. Dependenceoftheeigenenergiesofapolar Σ 2 moleculeon theanisotropicpolarizabilityinteraction parameter ηopt in the presenceofan electricand amagneticfield.F1and F2levelsareshown,respectively, bydashed and fulllinesin panelspertainingto signed valuesofthegood quantum number M.Red and bluecurvespertain,respectively, to stateswith = J˜ 1 2 and = J˜ 3 2.Notethat here η = 2.5 m and η = 5 el . Figure23. Dependenceofthetunnelingsplitting Δ = = ∣ ∣= − = = = ∣ ∣= E B E N M E B ( ˜ 1, t J J N M ˜ , 1 2 ˜ , ˜ 0, ) 1 2 1 2 1 2 on theopticalfield interaction parameter ηopt for η = 0 m (red curve) and η = 2.5 m (bluecurves). Figure23. Dependenceofthetunnelingsplitting Δ = = ∣ ∣= − = = = ∣ ∣= E B E N M E B ( ˜ 1, t J J N M ˜ , 1 2 ˜ , ˜ 0, ) 1 2 1 2 1 2 on theopticalfield interaction parameter ηopt for η = 0 m (red curve) and η = 2.5 m (bluecurves). TableB1 showswhat valuestheinteraction parameters ηel, ηm, and ηopt takeat particular valuesofthe respectivefield strengthsfor choice Σ 2 molecules. 22 NewJ.Phys.17(2015) 045017 KSharmaand BFriedrich Figure24. AppendixB. Conversion factors ′ ′ h J M J M ( , ; , ) Z ′ = + J J 1 + + − + J M J M 2 ( 1)( 1) ′ = J J M 2 ′ = − J J 1 + − J M J M 2 ( )( ) Table A5.The ′ ′ ± h J M J M ( , 1; , ) X and ± ′ ′ ± h J M J M i ( , 1; , ) Y termsofthedirection cosinematrix,equation (22). ′ ′ ± h J M J M ( , 1; , ) X or ± ′ ′ ± h J M J M i ( , 1; , ) Y ′ = + J J 1 ∓ ± + ± + J M J M ( 1)( 2) ′ = J J ∓ ∓ + J M J M ( )( 1) ′ = − J J 1 ∓ ∓ ∓ − J M J M ( )( 1) Table A6. Non-vanishingmatrixelementsofthe θ cos operator in thesymmetrictop basisset. Ω θ Ω ′ ∣ ∣ J M J M , , cos , , ′ = + J J 1 Ω Ω + + − + + + − + + + + J J J M J M J J J ( 1)( 1)( 1)( 1) ( 1) (2 1)(2 3) ′ = J J Ω + M J J( 1) ′ = − J J 1 Ω Ω + − + − + − J J J M J M J J J ( )( )( )( ) (2 1)(2 1) Table A7. Nonvanishingelementsofthe θ cos2 operator in thesymmetrictop basisset. AppendixB. Conversion factors ′ f J J ( ; ) ′ = + J J 1 + + + J J J 1 4( 1) (2 1)(2 3) ′ = J J + J J 1 4 ( 1) ′ = − J J 1 + + − J J J 1 4( 1) (2 1)(2 1) 23 NewJ.Phys.17(2015) 045017 KSharmaand BFriedrich NewJ.Phys.17(2015) 045017 TableA2. The Ω Ω ′ ′ g J J ( , ; , ) z term ofthe direction cosinematrix, equation (22). Ω Ω ′ ′ g J J ( , ; , ) z ′ = + J J 1 Ω Ω + + − + J J 2 ( 1)( 1) ′ = J J Ω 2 ′ = − J J 1 Ω Ω + − J J 2 ( )( ) TableA2. The Ω Ω ′ ′ g J J ( , ; , ) z term ofthe direction cosinematrix, equation (22). Ω Ω ′ ′ g J J ( , ; , ) z ′ = + J J 1 Ω Ω + + − + J J 2 ( 1)( 1) ′ = J J Ω 2 ′ = − J J 1 Ω Ω + − J J 2 ( )( ) TableA3.The Ω Ω ′ ′ ± g J J ( , 1; , ) x and Ω Ω ∓ ′ ′ ± g J J i ( , 1; , ) y termsofthedirection cosinematrix,equation (22). Ω Ω ′ ′ ± g J J ( , 1; , ) x or Ω Ω ∓ ′ ′ ± g J J i ( , 1; , ) y ′ = + J J 1 Ω Ω ∓ ± + ± + J J ( 1)( 2) ′ = J J Ω Ω ∓ ∓ + J J ( )( 1) ′ = − J J 1 Ω Ω ∓ ∓ ∓ − J J ( )( 1) TableA3.The Ω Ω ′ ′ ± g J J ( , 1; , ) x and Ω Ω ∓ ′ ′ ± g J J i ( , 1; , ) y termsofthedirection cosinematrix,equation (22). Table A4.The ′ ′ h J M J M ( , ; , ) Z term ofthe direction cosinematrix, equation (22). AppendixB. Conversion factors Ω θ Ω ′J M J M cos2 ′ = + J J 2 Ω Ω Ω Ω + + + + − + − + + + + + − + − + + + + + + J J J J J M J M J M J M J J J J J ( 2)( 1)( 2)( 1)( 2)( 1)( 2)( 1) ( 1)( 2)(2 3) (2 1)(2 5) ′ = + J J 1 + Ω Ω Ω + + − + + + − + + + + + M J J J M J M J J J J J ( 1)( 1)( 1)( 1) ( 1) (2 1)(2 3) 1 1 2 2 ⎡ ⎣ ⎤ ⎦ ′ = J J + + Ω Ω Ω − − − + + − + − + + − J J M J J M J J J J M J J ( )( ) (4 1) ( 1) (( 1) )(( 1) ) ( 1) (4( 1) 1) 2 2 2 2 2 2 2 2 2 2 2 2 2 2 2 2 ′ = − J J 1 + Ω Ω Ω + − + − + − − + M J J J M J M J J J J J ( )( )( )( ) (2 1)(2 1) 1 1 1 1 2 ⎡ ⎣ ⎤ ⎦ ′ = − J J 2 Ω Ω Ω Ω + + − − − − + + − − − − − − + − J J J J J M J M J M J M J J J J J ( )( 1)( )( 1)( )( 1)( )( 1) ( 1)(2 1) (2 1)(2 3) Table A7. Nonvanishingelementsofthe θ cos2 operator in thesymmetrictop basisset. 24 NewJ.Phys.17(2015) 045017 NewJ.Phys.17(2015) 045017 KSharmaand BFriedrich TableB1.Rotationalconstants, B,spin-rotation constants, γ,electricdipolemoments, μel,polarizabilityanisotropies, Δα,and valuesof thedimensionlessinteraction parameters ηel, ηm,and ηopt at electric, magnetic, and opticalfieldsof100 kV cm−1,1Tesla, and 1012 W cm−2, respectively,for choice Σ 2 molecules, seetext.Dipolemomentsand polarizabilitiesgiven in parenthesesareestimates. Compi- lation based on [5,77–86] and own calculations. [39] LandersAet al 2001Phys.Rev.Lett.87 [39] LandersAet al 2001Phys.Rev.Lett. 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Financeirização da moradia e segregação socioespacial: Minha Casa, Minha Vida em São José dos Campos, Taubaté e Jacareí/SP
Urbe. Revista Brasileira de Gestão Urbana
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Financeirização da moradia e segregação socioespacial: Minha Casa, Minha Vida em São José dos Campos, Taubaté e Jacareí/SP Housing financing and socio-spatial segregation: “Minha Casa Minha Vida” in São José dos Campos, Taubaté and Jacareí/SP Daniela das Neves Alvarenga[a], Paulo Romano Reschilian[a] [a] Universidade do Vale do Paraíba (Univap), Instituto de Pesquisa e Desenvolvimento (IP&D), São José dos Campos, SP, Brasil Resumo O presente artigo aborda o processo de internacionalização do capital financeiro no Brasil após a década de 1990, no período denominado neoliberal, e a política pública habitacional na fase neodesenvolvimentista, entre 2002 e 2016, por intermédio da produção de moradia vinculada ao Programa Federal Minha Casa, Minha Vida (MCMV). A discussão foi feita a partir do entendimento da forma pela qual a moradia de mercado se tornou um ativo financeiro, uma nova fronteira de ganhos para o mercado de capitais no espaço urbano, evidenciando a sofisticação do Sistema Financeiro Imobiliário (SFI) criado em 1997, e seu distanciamento do conceito da universalização da habitação enquanto direito. O objetivo do trabalho é demonstrar que as unidades habitacionais produzidas nas cidades de São José dos Campos, Taubaté e Jacareí, situadas na Região Metropolitana do Vale do Paraíba e Litoral Norte (RMVPLN), no Estado de São Paulo, no período de 2009 a 2014, passaram a atender a esse novo mercado. Palavras-chave: Financeirização. Política habitacional. Espaço urbano. Segregação socioespacial. MCMV. DNA é mestre, e-mail: danielaneves.arq@gmail.com PRR é doutor, e-mail: pauloromano@univap.br DOI: 10.1590/2175-3369.010.003.AO01 ISSN 2175-3369 Licenciado sob uma Licença Creative Commons DOI: 10.1590/2175-3369.010.003.AO01 ISSN 2175-3369 Licenciado sob uma Licença Creative Commons urbe. Revista Brasileira de Gestão Urbana (Brazilian Journal of Urban Management), 2018 set./dez., 10(3), 473-484 Introdução O Programa Minha Casa, Minha Vida (MCMV), instituído em 2009 com base em um Sistema Nacional de Habitação (SNH) focado no mercado, foi responsável pela produção de moradia por meio do financiamento ao setor privado. Segundo Maricato (2015), a retomada da visão empresarial da política habitacional, com a finalidade explícita de enfrentamento à crise econômica de 2008, foi determinante não só para construção de novas casas, mas também pelo boom imobiliário ocorrido no Brasil. Contribuiu também para o aumento do preço da terra, resultando na expulsão dos pobres para a periferia, além do espraiamento das cidades. Ferreira (2015) define o MCMV como um programa de financiamento, e não uma política urbana e habitacional, focado, portanto, no lucro do setor imobiliário e das construtoras, mas distante do interesse público. Segundo Santos (2004), a lógica do mercado global se impõe ao mercado interno. A ciência, a tecnologia e a informação, vetores fundamentais do processo social, são a base da produção, da utilização e do funcionamento do espaço, cada vez mais equipado para facilitar a circulação da informação. Esse nexo informacional, presente no processo de globalização, é representado por capitais fixos (estradas, pontes etc.), constantes (maquinário, veículos etc.) e pelos fluxos, inclusive financeiro, que atuam na requalificação dos espaços para atender aos interesses dos atores hegemônicos da economia, da política e da cultura. Da mesma forma que a incorporação imobiliária, o capital financeiro imobiliário busca moldar o espaço urbano. Os lobbies imobiliários sobre o executivo e o legislativo, no início do que se configurou como fase neodesenvolvimentista, foram decisivos para o desenho do Programa MCMV, discutido fora do Ministério das Cidades, sem a participação do Conselho das Cidades ou da Habitação, diretamente na Casa Civil. O entusiasmo neoliberal, principalmente a partir da criação do Sistema Financeiro Imobiliário (SFI) em 1997, foi responsável por incorporar a racionalidade das finanças na organização do financiamento da política habitacional, por meio de instrumentos financeiros, como os créditos imobiliários, distanciando-se do conceito da universalização da moradia enquanto direito (Royer, 2009). De acordo com Fix (2011), a financeirização da política habitacional carrega a lógica da habitação como frente da acumulação capitalista, e não como um direito social, colaborando para a construção de espaços urbanos cada vez mais distanciados do direito à cidade. Introdução Estado e um mercado fortes, por meio de uma política nacional focada no crescimento econômico, atrelada ao aumento da renda da população e ao fortalecimento do mercado de massas (inclusão pelo consumo). A Política Nacional de Desenvolvimento Regional (PNDR), criada em 2003 no Governo Lula, foi responsável pela instituição de programas de transferência de renda e de infraestrutura social, especialmente, para as regiões Norte e Nordeste do país, com destaque para o Programa Territórios da Cidadania criado em 2008, que trabalhava com o conceito de territórios rurais sustentáveis. O Programa de Aceleração do Crescimento (PAC), criado em 2007, responde aos princípios da Política Nacional de Habitação (PNH) de 2004 e promoveu a integração tecnológica e econômica, abrindo novos territórios ao mercado (Senra, 2011). Estado e um mercado fortes, por meio de uma política nacional focada no crescimento econômico, atrelada ao aumento da renda da população e ao fortalecimento do mercado de massas (inclusão pelo consumo). A Política Nacional de Desenvolvimento Regional (PNDR), criada em 2003 no Governo Lula, foi responsável pela instituição de programas de transferência de renda e de infraestrutura social, especialmente, para as regiões Norte e Nordeste do país, com destaque para o Programa Territórios da Cidadania criado em 2008, que trabalhava com o conceito de territórios rurais sustentáveis. O Programa de Aceleração do Crescimento (PAC), criado em 2007, responde aos princípios da Política Nacional de Habitação (PNH) de 2004 e promoveu a integração tecnológica e econômica, abrindo novos territórios ao mercado (Senra, 2011). No Brasil, o período denominado neoliberal (décadas de 1990-2000) ocorreu em um contexto de redemocratização do país, dentro de um cenário global de avanços tecnológicos nas áreas de computação e de transportes, possibilitando a internacionalização do capital financeiro por meio de uma conexão direta e imediata do global com o local, baseado em um modelo de acumulação flexível, ao mesmo tempo que as desigualdades entre países centrais e periféricos se acentuavam. Apoiado nos princípios do Consenso de Washington, especialmente na liberalização financeira, na liberalização do comércio exterior e na eliminação de restrições ao capital externo, durante o governo de Fernando Henrique Cardoso (1994-2002) houve uma forte desestatização da economia, baseada em uma política pública fundamentada na criação dos Eixos Nacionais de Integração e Desenvolvimento em 1996, com o objetivo de conectar espaços produtivos nacionais aos mercados internacionais (Senra, 2011). Abstract This article discusses the post 1990’s process of internationalization of financial capital in Brazil, in the so-called neoliberal period, and public housing policy in the neodevelopmental phase, between 2002 and 2016, through the production of housing linked to the federal program “Minha Casa Minha Vida” (MCMV - My House My Life). The discussion was based on the understanding of how the market housing became a financial asset, a new frontier of gains for the capital market in urban space. We evidence the sophistication of the Housing Financial System created in 1997, and its distancing from the concept of the universalization of housing as a right. The objective of this work is to demonstrate that the housing units produced in the cities of São José dos Campos, Taubaté and Jacareí, located in the Metropolitan Region of the Vale do Paraíba e Litoral Norte (RMVPLN) - SP, from 2009 to 2014, began to fulfill this market. Keywords: Financialization. Housing policy. Urban space. Socio-spatial segregation. MCMV. 474 Alvarenga, D. N., & Reschilian, P. R. urbe. Revista Brasileira de Gestão Urbana (Brazilian Journal of Urban Management), 2018 set./dez., 10(3), 473-484 Introdução Para este trabalho, foram obtidos dados primários do MCMV, referentes às construtoras e à localização dos empreendimentos contratados na Região Metropolitana do Vale do Paraíba e Litoral Norte (RMVPLN), no Estado de São Paulo, a partir da Caixa Econômica Federal, bem como dados secundários fornecidos pelo Ministério das Cidades, compilados no site deepAsk. A fase neodesenvolvimentista ocorrida no Brasil, a partir de 2000, é marcada pela presença de um urbe. Revista Brasileira de Gestão Urbana (Brazilian Journal of Urban Management), 2018 set./dez., 10(3), 473-484 475 Financeirização da moradia e segregação socioespacial De acordo com Fix (2011), a política habitacional estava pautada na criação de fluxos contínuos, por meio do FGTS e do SBPE, bases financeiras para constituição do circuito imobiliário. No entanto, a única opção pós-1964 foi a oferta da casa própria, em benefício da indústria da construção, principalmente no subsetor de edificações. Mesmo com a construção de quatro milhões de unidades habitacionais, o cenário urbano nacional que se formava, sobretudo com a crise de 1980, era de crescimento das favelas nas médias e grandes cidades. com, relativos ao número de unidades contratadas e concluídas e aos recursos aplicados nas três faixas do programa, no período de 2009 a 2014. O objetivo desta pesquisa é analisar a financeirização da habitação e seu efeito no espaço urbano a partir dos municípios de São José dos Campos, Taubaté e Jacareí, maiores déficits habitacionais da RMVPLN, no ano de 2010. A hipótese é de que os empreendimentos produzidos tenham atendido ao mercado de capitais, reafirmando a lógica do MCMV enquanto programa de financiamento, focado no lucro do setor imobiliário e das construtoras, acentuando a segregação socioespacial. O processo de internacionalização do capital financeiro, especialmente a partir da década de 1980, induziu diversos países em desenvolvimento a adotar políticas neoliberais, inclusive o Brasil, que reduziu despesas do Estado, promovendo a liberação da economia e ampliando as exportações para pagamento da dívida externa. Após 1994, com Fernando Henrique Cardoso na presidência, foram priorizadas políticas públicas destinadas à promoção do aumento da competitividade das empresas nacionais, para posterior inserção no mercado internacional (Senra, 2011). Surgimento e configuração do circuito imobiliário no Brasil Após a Segunda Guerra Mundial e, principalmente, a partir da década de 1970, o mercado global vem se consolidando com base no desenvolvimento da ciência, da tecnologia e da informação, em que os espaços atendem, sobretudo, aos interesses dos atores hegemônicos da economia, da cultura e da política (Santos, 2014). Segundo Senra (2011), a fase neoliberal foi acompanhada também pela globalização, com os avanços tecnológicos de comunicação e transportes, conectando o global ao local. As políticas públicas de desenvolvimento regional implementadas na época tinham o objetivo de diminuir as desigualdades regionais e promover a ocupação do território. O Programa dos Eixos Nacionais de Integração e Desenvolvimento foi uma das frentes adotadas pelo Governo FHC. A crescente penetração de práticas financeiras em todas as relações econômicas e sociais alcança então a produção do espaço urbano, em um processo em que as instituições financeiras passam a buscar novas fontes de lucratividade. O mercado imobiliário é então absorvido pela dinâmica financeira, responsável pela definição do capitalismo mais recente (Sanfelici, 2013). Foi nesse contexto que o Plano Plurianual 1996/1999 introduziu a proposta de sete Eixos Nacionais de Integração e Desenvolvimento, considerada a política de desenvolvimento regional mais relevante do período, que teve como principal objetivo conectar espaços produtivos nacionais aos mercados internacionais (Senra, 2011, p. 181). Para entender como esse fluxo financeiro transformou a moradia de mercado em uma nova fronteira de ganhos para o mercado de capitais no espaço urbano brasileiro, faz-se necessário retornar ao período da ditadura militar, analisando a montagem do sistema de financiamento habitacional, a partir da criação do Banco Nacional de Habitação (BNH). O desenvolvimento do crédito imobiliário no Brasil foi construído a partir de 1964, quando da criação do BNH, do Sistema Financeiro de Habitação (SFH), do Fundo de Garantia do Tempo de Serviço (FGTS), em 1967, e do Sistema Brasileiro de Poupança e Empréstimos (SBPE). O SFH, funding composto por recursos públicos, privados e paraestatais, evidenciava o papel do Estado na dinamização do setor imobiliário e das políticas públicas de habitação durante o regime militar (Royer, 2009). Segundo Rolnik (2015), o Governo FHC não se preocupou em respeitar os direitos sociais estabelecidos pela Constituição Federal de 1988, focando no retraimento da intervenção estatal, no ajuste e na consolidação de instituições financeiras internacionais, como o Banco Mundial, o Fundo Monetário Internacional (FMI) e a Organização Mundial do Comércio (OMC). urbe. Revista Brasileira de Gestão Urbana (Brazilian Journal of Urban Management), 2018 set./dez., 10(3), 473-484 Surgimento e configuração do circuito imobiliário no Brasil A partir da carência de investimentos públicos, o capital privado passou a apresentar suas propostas, baseadas nos pontos estabelecidos no Consenso 476 Alvarenga, D. N., & Reschilian, P. R. de Washington, privatizando os serviços públicos, desregulamentando os contratos de trabalho e flexibilizando os direitos sociais (Maricato, 2013). do caráter de mercadoria da habitação, dentro de um contexto mundial, que afetou de modo estrutural a universalização do direito à moradia digna. A política habitacional no Governo FHC ficou subordinada à Secretaria de Política Urbana do Ministério do Planejamento e Orçamento. O Programa Pró-Moradia e o Programa de Arrendamento Residencial (PAR) foram seguidos pela instituição do SFI em 1997, com a finalidade de promover o financiamento imobiliário em geral. Segundo Royer (2009), instituiu-se também a racionalidade das finanças na organização do financiamento da política de crédito imobiliário e habitacional, por intermédio dos instrumentos financeiros, uma vez que o SFI, diferentemente do SFH, procurava captar fundos no mercado de capitais. A fase neodesenvolvimentista ocorrida a partir de 2000 foi responsável pela definição de políticas sociais, que possibilitaram uma nova geração de políticas públicas regionais, fomentando a competitividade entre sistemas regionais de produção, com a finalidade de promover o crescimento econômico e o desenvolvimento social. Nesse cenário de competição em um sistema global de produção, foram criadas, em 2003, a PNDR e as mesorregiões diferenciadas, além da política dos Territórios da Cidadania, a fim de evitar uma fragmentação ainda maior no território brasileiro, promovendo a adoção de políticas sociais com impacto territorial, como os programas de transferência de renda e de infraestrutura social, como o Bolsa Família e o Programa Luz Para Todos, que privilegiaram as regiões Norte e Nordeste, as mais vulneráveis do Brasil (Senra, 2011). Somando-se aos instrumentos financeiros já existentes, como as Debêntures, as Letras Hipotecárias e as Cédulas de Crédito Bancário, foram criados os Certificados de Recebíveis Imobiliários (CRI), as Cédulas de Crédito Imobiliário (CCI) e as Letras de Crédito Imobiliário (LCI), fazendo com que o SFI se configurasse com uma nova arquitetura do financiamento imobiliário, fundada na crença de que o mercado de capitais seria a solução para a provisão de fundos para o sistema imobiliário. A construção da política habitacional do início do Governo Lula foi inspirada no Projeto Moradia — formulado no Instituto Cidadania, em 2000 —, sendo já prevista a criação do Ministério das Cidades. urbe. Revista Brasileira de Gestão Urbana (Brazilian Journal of Urban Management), 2018 set./dez., 10(3), 473-484 O MCMV e a produção do espaço urbano na Região Metropolitana do Vale do Paraíba e Litoral Norte (RMVPLN) O ano de 2006 foi marcado pela influência de grandes incorporadoras, como a Cyrela, a PDG e a MRV, as quais levantavam capital na bolsa de valores, estabelecendo vínculo com o mercado de capitais, configurando o poder crescente das finanças (Sanfelici, 2013). O Programa MCMV, na sua fase 1, de 2009 a 2011, previa um investimento total de R$ 28 bilhões a serem destinados para faixa 1, modalidade FAR, FDS (Entidades) e Rural, além das faixas 2 e 3, modalidade FGTS, para construção de um milhão de unidades habitacionais. Na fase 2, até 2014, o valor desse investimento subiu para R$ 125 bilhões, a serem direcionados, principalmente, para a construção de moradias para as famílias com renda de zero a três salários mínimos (60% de todo investimento), prevendo a construção de dois milhões de unidades habitacionais (Amore, 2015). Segundo Rolnik (2015), quando estourou a crise hipotecária e financeira nos Estados Unidos no final de 2008, os empresários atingidos passaram a intensificar o lobby no Ministério da Fazenda para a implementação de um pacote habitacional. Como forma de salvar as empresas e, ao mesmo tempo, funcionar como medida contracíclica para garantir empregos e crescimento econômico, o presidente Lula propôs a construção de um milhão de moradias. Rolnik (2015) ressalta que Em 2010, os maiores déficits habitacionais absolutos da RMVPLN estavam presentes nos municípios de São José dos Campos, Taubaté e Jacareí, com 21.910, 8.969 e 7.053 unidades, respectivamente (Vieira, 2015). Segundo dados da Fundação SEADE (2016) São José dos Campos apresentava a maior população, com 660.008 habitantes, seguida por Taubaté, com 296.449, e Jacareí, com 221.650 habitantes. [...] de pacote de salvamento de incorporadoras financeirizadas, o MCMV transformou-se na política habitacional do país, baseado no modelo único de promoção de casa própria, acessada via mercado e crédito hipotecário (Rolnik, 2015, p. 309). Desde 2009, os três municípios localizados no eixo da Rodovia Presidente Dutra, conforme Figura 1, passaram a receber um grande aporte de recursos para construção de unidades habitacionais contratadas nas faixas 1, 2 e 3 do Programa MCMV. Até 2014, foram contratadas 15.367 unidades em São José dos Campos, 8.725 em Taubaté e 6.317 na cidade de Jacareí (DeepAsk, 2016). O Programa MCMV, criado em 2009, foi elaborado sob a liderança da Casa Civil e do Ministério da Fazenda, promovendo “[...] um diálogo direto com representantes da construção, e sem a participação popular [...]” (Fix, 2011, p. 140). Surgimento e configuração do circuito imobiliário no Brasil A ampliação dos recursos e subsídios para a habitação de interesse social teve como partida a Resolução nº 460 do Conselho Gestor do FGTS em 2005, seguida da aprovação do Fundo Nacional de Habitação de Interesse Social (FNHIS) no mesmo ano, resultado de um projeto de lei de iniciativa popular. Como se pode depreender dos principais instrumentos e da forma como foi estruturado, o SFI constitui na verdade um marco regulatório da participação e da operação de instituições financeiras e correlatas no financiamento imobiliário e no mercado de capitais, diferente do SFH, que buscava constituir um aparato estatal para o financiamento de habitação (Royer, 2009, p. 102). Em síntese, o volume de recursos cresceu significativamente combinando diversas fontes, como o Orçamento Geral da União, o Fundo de Amparo ao Trabalhador (FAT), o Fundo de Arrendamento Residencial (FAR), o Fundo de Desenvolvimento Social (FDS), o Tesouro Nacional e, principalmente, o FGTS (Fix, 2011, p. 134). Fix (2011) comenta que, embora o objetivo do SFI fosse integrar o crédito imobiliário ao mercado de capitais, a caderneta de poupança, o FGTS e o binômio SFH/BNH permaneceram como base de suas operações. A criação do Ministério das Cidades, inspirada nas reivindicações trazidas pelo Fórum Nacional da Reforma Urbana (FNRU), contribuiu para redução das desigualdades sociais e urbanas, principalmente a partir da Resolução nº 460, que permitiu a redução do custo dos financiamentos com recursos do FGTS, e por meio da criação do FNHIS, que passou a fornecer subsídios diretos para habitação de interesse social. Como ressaltam Cardoso & Aragão (2013), essas duas medidas demonstraram o compromisso do governo Shimbo (2016) ressalta a forma capitalista de produção de habitação a partir dos anos 2000, por meio do alinhamento entre Estado, finanças e construção civil, responsável pela mobilização de diversos agentes produtores — proprietários de terras, construtoras subcontratadas, investidores internacionais e agentes financeiros —, pela centralização do capital e pela concentração de terras urbanas nos denominados land banks. Segundo a autora, ocorreu a exacerbação urbe. Revista Brasileira de Gestão Urbana (Brazilian Journal of Urban Management), 2018 set./dez., 10(3), 473-484 477 Financeirização da moradia e segregação socioespacial federal em subsidiar a produção de moradia para as camadas de mais baixa renda. urbe. Revista Brasileira de Gestão Urbana (Brazilian Journal of Urban Management), 2018 set./dez., 10(3), 473-484 O MCMV e a produção do espaço urbano na Região Metropolitana do Vale do Paraíba e Litoral Norte (RMVPLN) O pacote proposto direcionava os recursos para o Fundo de Arrendamento Residencial (FAR), em que o FGTS e o Orçamento Geral da União (OGU) tornaram-se as fontes de financiamento. A Figura 2 traz o número de unidades contratadas pelo programa na faixa 1 entre 2009 e 2014, evidenciando a contratação de empreendimentos de interesse social já em 2009 no município de Jacareí, a partir de 2012 em Taubaté e desde 2013 em São José dos Campos – esta última cidade com o maior número de unidades contratadas (4.914). Segundo Fix (2011), o Programa MCMV carrega a lógica do BNH, em que a habitação é tratada como mercadoria a ser produzida em modelos capitalistas, favorecendo, especialmente, as grandes empresas que passaram a atuar no “segmento econômico” (faixas de quatro a dez salários mínimos). Em que pesem as dimensões não equacionadas pelo Programa MCMV, relativas principalmente ao privilégio concedido ao setor privado no combate ao déficit habitacional, a proposta trazida pelo governo previa a destinação de 40% de todo investimento às famílias com renda de até três salários mínimos. Tratava-se, portanto, de um subsídio jamais visto em programas anteriores, seja por meio do Fundo de Arrendamento Residencial (FAR), seja pelo Fundo de Desenvolvimento Social (FDS), no caso da modalidade Entidades. De acordo com Suriano (2012), o processo de expansão das periferias e a consequente segregação socioespacial se consolidaram em São José dos Campos no período de 1997 a 2011, por meio de planos e programas habitacionais da Prefeitura Municipal em parceria com a Caixa Econômica Federal ou com a Companhia de Desenvolvimento Habitacional e Urbano (CDHU). Conforme dados da Caixa Econômica Federal (CEF, 2017), das 4.914 unidades contratadas pelo Programa MCMV na faixa 1, 1.318 estavam urbe. Revista Brasileira de Gestão Urbana (Brazilian Journal of Urban Management), 2018 set./dez., 10(3), 473-484 478 Alvarenga, D. N., & Reschilian, P. R. Figura 1 - Mapa da RMVPLN, municípios e sub-regiões Fonte: elaboração própria, a partir de EMPLASA (2012). Figura 2 - Unidades contratadas pelo Programa MCMV na faixa 1, de 2009 a 2014 Fonte: elaboração própria, a partir de DeepAsk (2016). localizadas na Região Leste da cidade, sob a forma de condomínio, a 18 Km do centro. urbe. Revista Brasileira de Gestão Urbana (Brazilian Journal of Urban Management), 2018 set./dez., 10(3), 473-484 O MCMV e a produção do espaço urbano na Região Metropolitana do Vale do Paraíba e Litoral Norte (RMVPLN) O único loteamento aprovado dentro do programa foi o “Pinheirinho dos Palmares II”, destinado às famílias removidas Sudeste, totalmente desarticulado da malha urban próximo à Rodovia Carvalho Pinto, a quase 20 Km d Região Central, e foi construído para 1.461 família Da mesma forma que programas anteriores, as nov Figura 1 - Mapa da RMVPLN, municípios e sub-regiões Fonte: elaboração própria, a partir de EMPLASA (2012). Figura 2 - Unidades contratadas pelo Programa MCMV na faixa 1, de 2009 a 2014 Fonte: elaboração própria, a partir de DeepAsk (2016). urbe. Revista Brasileira de Gestão Urbana (Brazilian Journal of Urban Management), 2018 set./dez., 10(3), 473-484 478/484 Figura 2 - Unidades contratadas pelo Programa MCMV na faixa 1, de 2009 a 2014 Fonte: elaboração própria, a partir de DeepAsk (2016). localizadas na Região Leste da cidade, sob a forma de condomínio, a 18 Km do centro. O único loteamento aprovado dentro do programa foi o “Pinheirinho dos Palmares II”, destinado às famílias removidas do antigo Pinheirinho. O loteamento fica na Região Sudeste, totalmente desarticulado da malha urbana, próximo à Rodovia Carvalho Pinto, a quase 20 Km da Região Central, e foi construído para 1.461 famílias. Da mesma forma que programas anteriores, as novas localidades da moradia de interesse social no município Figura 2 - Unidades contratadas pelo Programa MCMV na faixa 1, de 2009 a 2014 Fonte: elaboração própria, a partir de DeepAsk (2016). Figura 2 - Unidades contratadas pelo Programa MCMV na faixa 1, de 2009 a 2014 Fonte: elaboração própria, a partir de DeepAsk (2016). Sudeste, totalmente desarticulado da malha urbana, próximo à Rodovia Carvalho Pinto, a quase 20 Km da Região Central, e foi construído para 1.461 famílias. Da mesma forma que programas anteriores, as novas localidades da moradia de interesse social no município localizadas na Região Leste da cidade, sob a forma de condomínio, a 18 Km do centro. O único loteamento aprovado dentro do programa foi o “Pinheirinho dos Palmares II”, destinado às famílias removidas do antigo Pinheirinho. O loteamento fica na Região urbe. Revista Brasileira de Gestão Urbana (Brazilian Journal of Urban Management), 2018 set./dez., 10(3), 473-484 479 Financeirização da moradia e segregação socioespacial A Figura 3 aponta o número de unidades habitacionais contratadas nas faixas 2 e 3 (segmento econômico), atingindo famílias com renda de quatro a dez salários mínimos. O MCMV e a produção do espaço urbano na Região Metropolitana do Vale do Paraíba e Litoral Norte (RMVPLN) Considerando o gráfico relativo à faixa 2 do programa, destaca-se, novamente, o município de São José dos Campos, com 7.353 unidades contratadas. Taubaté e Jacareí aparecem juntas, com aproximadamente 4 mil unidades contratadas no MCMV. O gráfico relativo à faixa 3 reflete o maior número de unidades contratadas em São José dos Campos (3.100 unidades habitacionais), seguida por Taubaté (2.231) e Jacareí (859). de São José dos Campos refletem a negação do direito à cidade para 2.779 famílias com renda de até três salários mínimos, em oposição aos princípios do Plano Local de Habitação de Interesse Social (PLHIS), que prevê a garantia do direito à moradia digna como vetor de inclusão social. Como ressalta Rolnik (2015), os conjuntos habitacionais voltados para as famílias enquadradas na faixa 1 continuam ocorrendo nas periferias, próximos às margens das cidades, reproduzindo a segregação socioespacial de programas habitacionais anteriores. No caso de Jacareí, de acordo com Vieira (2015), os sete condomínios financiados pelo Programa MCMV, na faixa 1, foram construídos na Região Sul, em uma área de franja urbana do município, apesar da diretriz constante no PLHIS de 2010, de se construir moradia para a população de mais baixa renda nos vazios urbanos e em áreas centrais. A localização dos empreendimentos construídos para 1.248 famílias sob a forma de condomínio revela a dinâmica segregadora e o espraiamento da cidade, visto também em São José dos Campos. Segundo Sanfelici (2013), a lógica estabelecida enormemente a partir de 2000, com a integração das incorporadoras imobiliárias à dinâmica da Bolsa de Valores, é a da maximização da apropriação de rendas: A oferta de grandes empreendimentos, somente tornada possível pela capacidade de levantar somas assombrosas no mercado de capitais, funciona como uma espécie de economia de escala, por intermédio da qual as incorporadoras podem ampliar a diferença entre a renda do solo atual urbe. Revista Brasileira de Gestão Urbana (Brazilian Journal of Urban Management), 2018 set./dez., 10(3), 473-484 479/484 Figura 3 - Unidades contratadas pelo Programa MCMV nas faixas 2 e 3, de 2009 a 2014 Fonte: elaboração própria, a partir de DeepAsk (2016). a 3 Unidades contratadas pelo Programa MCMV nas faixas 2 e 3 de 2009 a 2014 Figura 3 - Unidades contratadas pelo Programa MCMV nas faixas 2 e 3, de 2009 a 2014 Fonte: elaboração própria, a partir de DeepAsk (2016). urbe. O MCMV e a produção do espaço urbano na Região Metropolitana do Vale do Paraíba e Litoral Norte (RMVPLN) Revista Brasileira de Gestão Urbana (Brazilian Journal of Urban Management), 2018 set./dez., 10(3), 473-484 480 Alvarenga, D. N., & Reschilian, P. R. (aquela paga ou permutada com o proprietário do terreno) e a renda do solo potencial, colocada pela localização do terreno na totalidade do tecido urbano (Sanfelici, 2013, p. 41). Para Maricato (2013), o problema da moradia persiste por estar vinculado a um pedaço de solo que se valoriza de acordo com os atributos do entorno, estando relacionado então à renda da terra. De acordo com Ferreira (2015), a ordem fundiária e urbana vigente não foi alterada pelo Programa MCMV. A lógica de produzir segregação e desigualdade permanece. Segundo o autor, a negação do direito à cidade e a responsabilidade pelo impacto urbano-ambiental passam pelas construtoras, mas é, essencialmente, culpa dos governos locais, responsáveis pelas políticas territorial, urbana e habitacional, capazes de As formas assumidas pelos empreendimentos são inúmeras, segundo o autor, passando por empreendimentos multifuncionais de alto padrão, mesclando o uso residencial com os usos comerciais e de serviços, até condomínios de baixa renda nas franjas dos tecidos metropolitanos e loteamentos de unidades unifamiliares no âmbito do Programa MCMV, contribuindo para construção de um espaço urbano cada vez mais fragmentado. “[...] alterar o equilíbrio de forças políticas, de enfrentar de fato a desigualdade sócio espacial e mexer no poder dos proprietários fundiários e das elites urbanas [...]” (Ferreira, 2015, p. 8). O espraiamento urbano, segregação socioespacial, consolidação de grandes vazios urbanos, bairros monofuncionais, formação de áreas isoladas ou desarticuladas da malha urbana, sem diversidade de usos e grupos sociais, caracterizam padrão de produção das cidades que já se mostrou insustentável do ponto de vista ambiental, econômico e social (Ferreira, 2012, p. 64). Com relação aos recursos aplicados nos três municípios, o valor total dos financiamentos e subsídios contratados para a aquisição ou produção das unidades habitacionais do Programa MCMV destaca a cidade de São José dos Campos, com um valor total de R$ 1.311.302.935,24, conforme a Figura 4, seguida por Taubaté, com R$ 637.744.677,63, e Jacareí, com R$ 473.476.409,94. A Figura 5 apresenta o valor dos financiamentos contratados nos três municípios, sem considerar os subsídios da União. São José dos Campos também aparece com o maior valor de financiamento contratado, R$ 783.360.975,69, seguida de Taubaté, com R$ 402.484.050,16, e Jacareí, com R$ 304.246.655,67. O MCMV e a produção do espaço urbano na Região Metropolitana do Vale do Paraíba e Litoral Norte (RMVPLN) Os diferentes agentes da construção civil têm por isso obrigação de assumir corresponsabilidades quanto à sustentabilidade e efeitos futuros da urbanização que promovem, em relação à qualidade da arquitetura que bela 1 - São José dos Campos, Taubaté e Jacareí: unidades contratadas por faixa de renda – Programa MCMV (2009-2014) Tabela 1 - São José dos Campos, Taubaté e Jacareí: unidades contratadas por faixa de renda – Programa MCMV (2009-2014) Tabela 1 - São José dos Campos, Taubaté e Jacareí: unidades contratadas por faixa de renda – Programa MCMV (2009-2014) Município Faixa 1 Faixa 2 Faixa 3 Total nas 3 faixas (0 a 3 SM) (3 a 6 SM) (6 a 10 SM) São José dos Campos 4.914 7.353 3.100 15.367 Taubaté 2.124 4.370 2.231 8.725 Jacareí 1.248 4.210 859 6.317 Total 8.286 15.933 6.190 30.049 Fonte: elaboração própria, a partir de DeepAsk (2016). urbe. Revista Brasileira de Gestão Urbana (Brazilian Journal of Urban Management), 2018 set./dez., 10(3), 473-484 482/484 Tabela 1 - São José dos Campos, Taubaté e Jacareí: unidades contratadas por faixa de renda – Programa MCMV (2009-2014) Município Faixa 1 Faixa 2 Faixa 3 Total nas 3 faixas (0 a 3 SM) (3 a 6 SM) (6 a 10 SM) São José dos Campos 4.914 7.353 3.100 15.367 Taubaté 2.124 4.370 2.231 8.725 Jacareí 1.248 4.210 859 6.317 Total 8.286 15.933 6.190 30.049 Fonte: elaboração própria, a partir de DeepAsk (2016). Tabela 2 - São José dos Campos, Taubaté e Jacareí: relação entre o déficit habitacional e o número de unidades concluídas por meio do Programa MCMV (2009-2014) Município Déficit absoluto 2010 Unidades concluídas PMCMV Déficit absoluto restante Redução do déficit relativo (%) (Faixas 1, 2 e 3) São José dos Campos 21.910 9.661 12.249 45 Taubaté 8.969 4.235 4.734 47 Jacareí 7.053 6.186 867 88 Fonte: elaboração própria, a partir de DeepAsk (2016). Fonte: elaboração própria, a partir de DeepAsk (2016). O MCMV e a produção do espaço urbano na Região Metropolitana do Vale do Paraíba e Litoral Norte (RMVPLN) Esse padrão de urbanização que tem marcado o período neoliberal, a partir de 2000, reflete o poder financeiro dos grandes investidores globais por trás dos agentes imobiliários, contribuindo para a continuidade das dinâmicas segregadoras no território nacional. Mais uma vez os interesses particulares prevalecem nas recentes transformações do espaço, impedindo a realização da vida urbana e de uma sociedade mais justa. A Figura 6 aponta o valor dos subsídios da União para os empreendimentos MCMV oriundos do b R i B il i d G ã U b (B ili J l f U b M ) 2018 /d 10(3) 473 484 480/484 Figura 4 - Valor total dos financiamentos e subsídios do Programa MCMV, de 2009 a 2014 Fonte: elaboração própria, a partir de DeepAsk (2016). Figura 4 - Valor total dos financiamentos e subsídios do Programa MCMV, de 2009 a 2014 Fonte: elaboração própria, a partir de DeepAsk (2016). urbe. Revista Brasileira de Gestão Urbana (Brazilian Journal of Urban Management), 2018 set./dez., 10(3), 473-484 urbe. Revista Brasileira de Gestão Urbana (Brazilian Journal of Urban Management), 2018 set./dez., 10(3), 473-484 481 Financeirização da moradia e segregação socioespacial Figura 5 - Valor dos financiamentos contratados, de 2009 a 2014 Fonte: elaboração própria, a partir de DeepAsk (2016). Figura 5 - Valor dos financiamentos contratados, de 2009 a 2014 Fonte: elaboração própria, a partir de DeepAsk (2016). Figura 5 - Valor dos financiamentos contratados, de 2009 a 2014 Fonte: elaboração própria, a partir de DeepAsk (2016). Figura 5 - Valor dos financiamentos contratados, de 2009 a 2014 Fonte: elaboração própria, a partir de DeepAsk (2016). b R i t B il i d G tã U b (B ili J l f U b M t) 2018 t /d 481/484 Figura 6 - Valor dos subsídios da União e do FGTS, de 2009 a 2014 Fonte: elaboração própria, a partir de DeepAsk (2016). urbe Revista Brasileira de Gestão Urbana (Brazilian Journal of Urban Management) 2018 set /dez 481/484 Figura 6 - Valor dos subsídios da União e do FGTS, de 2009 a 2014 Fonte: elaboração própria, a partir de DeepAsk (2016). Figura 6 - Valor dos subsídios da União e do FGTS, de 2009 a 2014 Fonte: elaboração própria, a partir de DeepAsk (2016). urbe. Revista Brasileira de Gestão Urbana (Brazilian Journal of Urban Management), 2018 set./dez., 10(3), 473-484 482 Alvarenga, D. N., & Reschilian, P. R. O MCMV e a produção do espaço urbano na Região Metropolitana do Vale do Paraíba e Litoral Norte (RMVPLN) produzem, em relação ao espaço público e à cidade (Ferreira, 2012, p. 52). produzem, em relação ao espaço público e à cidade (Ferreira, 2012, p. 52). Orçamento Geral da União (OGU) e o valor dos subsídios aplicados na aquisição das unidades habitacionais oriundos do FGTS. Observa-se um salto no valor dos subsídios da União no caso de São José dos Campos a partir de 2013, refletindo a contratação de unidades habitacionais voltadas para a faixa 1, verificada na Figura 2, totalizando 4.914 moradias para a população de baixíssima renda. Os subsídios oriundos do OGU representam, aproximadamente, 30% do valor total dos financiamentos e subsídios do programa MCMV em São José dos Campos, 25% no caso de Taubaté e 18% na cidade de Jacareí. Segundo dados da Caixa Econômica Federal (CEF, 2017), as construtoras Gafisa, MRV, Rossi, Tenda, Rodobens, PDG, Goldfarb e Cury passaram a atuar na RMVPLN a partir de 2009. De acordo com a Tabela 1, o número total de unidades contratadas na faixa 1 do programa, considerando os três municípios analisados, é de 8.286 unidades; já nas faixas 2 e 3, o total sobe para 22.123 unidades habitacionais contratadas em São José dos Campos, Jacareí e Taubaté, no período de 2009 a 2014. Os dados relativos à contratação nas faixas 2 e 3 revelam a atuação das grandes empresas no chamado segmento econômico, em detrimento da moradia de interesse social. Ferreira (2012) destaca a urgência na conscientização das construtoras e incorporadoras quanto à qualidade da inserção urbana dos novos empreendimentos, principalmente no acesso à rede de infraestrutura e transportes, uma vez que, ao atuar no MCMV, o setor privado se beneficia de importantes fundos públicos. Com relação ao déficit habitacional absoluto nos três municípios, nota-se uma significativa redução do déficit relativo, principalmente na cidade de Jacareí, conforme Tabela 2. No entanto, de acordo com Vieira (2015), o déficit habitacional relativo à faixa de renda de zero a três salários mínimos ainda não foi equacionado no município, uma vez que o déficit dessa faixa era igual 3.849 unidades em 2010, e foram construídas 1.248 moradias até o ano de 2014. Por isso, a responsabilidade social de sua atividade deve ser-lhe imputada. O MCMV e a produção do espaço urbano na Região Metropolitana do Vale do Paraíba e Litoral Norte (RMVPLN) Tabela 2 - São José dos Campos, Taubaté e Jacareí: relação entre o déficit habitacional e o número de unidades concluídas por meio do Programa MCMV (2009-2014) Município Déficit absoluto 2010 Unidades concluídas PMCMV Déficit absoluto restante Redução do déficit relativo (%) (Faixas 1, 2 e 3) São José dos Campos 21.910 9.661 12.249 45 Taubaté 8.969 4.235 4.734 47 Jacareí 7.053 6.186 867 88 Tabela 2 - São José dos Campos, Taubaté e Jacareí: relação entre o déficit habitacional e o número de unidades concluídas por meio do Programa MCMV (2009-2014) Tabela 2 - São José dos Campos, Taubaté e Jacareí: relação entre o déficit habitacional e o número de unidades concluídas por meio do Programa MCMV (2009-2014) urbe. Revista Brasileira de Gestão Urbana (Brazilian Journal of Urban Management), 2018 set./dez., 10(3), 473-484 482/484 Tabela 2 - São José dos Campos, Taubaté e Jacareí: relação entre o déficit habitacional e o número de unidades concluídas por meio do Programa MCMV (2009-2014) Município Déficit absoluto 2010 Unidades concluídas PMCMV Déficit absoluto restante Redução do déficit relativo (%) (Faixas 1, 2 e 3) São José dos Campos 21.910 9.661 12.249 45 Taubaté 8.969 4.235 4.734 47 Jacareí 7.053 6.186 867 88 Fonte: elaboração própria, a partir de DeepAsk (2016). 483 Financeirização da moradia e segregação socioespacial Embora o problema habitacional no Brasil demonstre a dependência dos municípios em relação aos recursos do governo federal, é necessário retomar alternativas baseadas no fortalecimento da gestão pública, na autogestão, individual ou coletiva, e na produção de habitação de interesse social. Há de se pensar arranjos que preveem a melhoria de habitação e núcleos existentes, como discutem as assessorias técnicas nos movimentos de moradia, uma vez que o financiamento dessas melhorias atenderiam a uma demanda muito maior do que aquela por empreendimentos novos. É preciso fortalecer a discussão na proposta de ocupação de imóveis construídos vagos, para que se cumpra a função social da propriedade, uma vez que muitas vezes são unidades providas de infraestrutura completa, como ressaltam Fix & Arantes (2009). regulação e apropriação, manteve-se inalterada. A política pública habitacional do Governo Lula priorizou a construção de moradia como resposta ao déficit habitacional no país, por meio da criação do Programa MCMV. O pacote habitacional lançado em 2009 foi adotado como medida contracíclica para garantir empregos e crescimento econômico, após a crise financeira de 2008. Conclusão O desenvolvimento nas áreas de telecomunicação e de logística foi determinante para configuração de uma relação direta e imediata do global com local, assinalando o rápido crescimento do processo de internacionalização do capital financeiro após a década de 1980, no período neoliberal. Estratégias políticas federais foram adotadas no Governo FHC, conectando espaços produtivos nacionais aos mercados internacionais, consolidando o papel das instituições financeiras internacionais no país. A criação do SFI, em 1997, incorporou a racionalidade das finanças na organização do financiamento da política habitacional, integrando o crédito imobiliário ao mercado de capitais, por meio da criação de novos instrumentos financeiros, os CRI, as CCI e as LCI. O MCMV e a produção do espaço urbano na Região Metropolitana do Vale do Paraíba e Litoral Norte (RMVPLN) Apesar do grande investimento direcionado ao programa, principalmente para a faixa 1, o espaço urbano vem se desenhando distanciado do ideal de cidade no seu valor de uso. A reforma urbana não ocorreu. A continuidade do processo de segregação e de periferização na formação dos novos tecidos urbanos reforça a cidade no seu valor de troca, evidenciando o favorecimento aos interesses particulares, em uma escala internacional, em detrimento do interesse público. A financeirização do espaço urbano aparece como um A financeirização do espaço urbano aparece como um A financeirização da moradia, discutida por intermédio do número de unidades habitacionais produzidas e dos recursos aplicados nas três faixas do Programa MCMV, no período de 2009 a 2014, nas cidades de São José dos Campos, Jacareí e Taubaté, situadas na RMVPLN, revela a predominância da produção de moradia focada no mercado, para famílias com renda mensal de quatro a dez salários mínimos, o então chamado “segmento econômico”. O processo de financeirização da moradia e do solo urbano marca o triunfo dos mercados e promove a continuidade das dinâmicas segregadoras, especialmente para a população de baixíssima renda, para as quais o direito à moradia não se encontra associado à promoção do direito à cidade. “[...] rearranjo escalar que articula, de forma inédita, as escalas global e local no cerne do processo de urbanização, com todas as tensões e contradições que esse processo engendra [...]” (Sanfelici, 2013, p. 43), “[...] rearranjo escalar que articula, de forma inédita, as escalas global e local no cerne do processo de urbanização, com todas as tensões e contradições que esse processo engendra [...]” (Sanfelici, 2013, p. 43), evidenciando o triunfo dos mercados nessa nova ordem global e a posição do Estado como servo das corporações multinacionais (Santos, 2014). evidenciando o triunfo dos mercados nessa nova ordem global e a posição do Estado como servo das corporações multinacionais (Santos, 2014). urbe. Revista Brasileira de Gestão Urbana (Brazilian Journal of Urban Management), 2018 set./dez., 10(3), 473-484 Referências Amore, C. S. (2015). “Minha Casa Minha Vida” para iniciantes. In C. S. Amore, L. Z. Shimbo & M. B. C. Rufino (Eds.), Minha casa... e a cidade? Avaliação do programa minha casa minha vida em seis estados brasileiros. Rio de Janeiro: Letra Capital. Caixa Econômica Federal – CEF. (2017). Gerência de Habitação. São José dos Campos: CEF. Cardoso, A. L., & Aragão, T. A. (2013). Do fim do BNH ao Programa Minha Casa Minha Vida: 25 anos da política habitacional no Brasil. In A. L. Cardoso (Eds.), O programa Minha Casa Minha Vida e seus efeitos territoriais (pp. 17-65). Rio de Janeiro: Letra Capital. A despeito da perspectiva neodesenvolvimentismo e sua abordagem social, não se observa ruptura com questões estruturais da política urbana habitacional, tendo em vista que a questão nodal, a terra, sua urbe. Revista Brasileira de Gestão Urbana (Brazilian Journal of Urban Management), 2018 set./dez., 10(3), 473-484 484 Alvarenga, D. N., & Reschilian, P. R. 240:financeirizacao-da-politica-habitacional&catid= 24:comentario 240:financeirizacao-da-politica-habitacional&catid= 24:comentario DeepAsk: O mundo e as cidades através de gráficos e mapas. (2016). Minha Casa Minha Vida: veja quantidade de imóveis entregues e recursos aplicados por cidade do Brasil. Recuperado em 8 de dezembro de 2016, de http://www. deepask.com/goes?page=Programa-Minha-Casa-Minha- Vida:-Veja-quantidade-de-imoveis-entregues-e-recursos- aplicados-por-municipio-do-Brasil Maricato, E. (2015). Para entender a crise urbana (1a ed.). São Paulo: Expressão Popular. Rolnik, R. (2015). Guerra dos lugares: a colonização da terra e da moradia na era das finanças. (1a ed.). São Paulo: Boitempo. Empresa Paulista de Planejamento Metropolitano, S.A. – EMPLASA. (2012). Atlas de Unidade de Informações Territorializadas (UITs) da Região Metropolitana do Vale do Paraíba e Litoral Norte. São Paulo: Emplasa. Recuperado em 20 de janeiro de 2016, de https://bibliotecavirtual. emplasa.sp.gov.br/CatalogoEmplasa.aspx Royer, L. O. (2009). Financeirização da Política Habitacional: limites e perspectivas (Tese de doutorado). Faculdade de Arquitetura e Urbanismo, Universidade de São Paulo, São Paulo. Sanfelici, D. (2013). Financeirização e a produção do espaço urbano no Brasil: uma contribuição ao debate. Revista EURE. Santiago do Chile, 39(118), 27-46. Ferreira, J. S. W. (2012). Produzir casas ou construir cidades? Desafios para um novo Brasil urbano. Parâmetros de qualidade para a implementação de projetos habitacionais e urbanos (1a ed.). São Paulo: FUPAM. Santos, M. (2014). A natureza do espaço: técnica e tempo, razão e emoção (4a ed.). São Paulo: Editora da Universidade de São Paulo. Ferreira, J. S. W. (2015). Notas sobre a responsabilidade coletiva de um desastre urbano. In Anais do XVI ENAMPUR (pp. urbe. Revista Brasileira de Gestão Urbana (Brazilian Journal of Urban Management), 2018 set./dez., 10(3), 473-484 Referências 1-11). Belo Horizonte: ANPUR. Suriano, A. L. (2012). Urbanização, habitação e segregação socioespacial. Revista Univap, 18(32), 190-202. Fix, M. (2011). Financeirização e transformações recentes no circuito imobiliário no Brasil (Tese de doutorado). Instituto de Economia, Universidade Estadual de Campinas, Campinas. Senra, K. V. (2011). Cinquenta anos de políticas públicas federais de desenvolvimento regional no Brasil. In R. Faria & B. Schvarsberg (Eds.), Políticas Urbanas e Regionais no Brasil (pp. 168-194). Brasília: Universidade de Brasília, Faculdade de Arquitetura e Urbanismo. Fix, M., & Arantes, P. F. (2009). Como o governo Lula pretende resolver o problema da habitação (Relatório). São Paulo: Correio da Cidadania. Shimbo, L. (2016). Sobre os capitais que produzem habitação no Brasil. Revista Novos Estudos Cebrap, 35(2), 119-133. Fundação Sistema Estadual de Análise de Dados – SEADE. (2016). IMP - Informações dos Municípios Paulistas - 2016. São Paulo: SEADE. Recuperado em 11 de janeiro de 2017, de http://www.imp.seade.gov.br Fundação Sistema Estadual de Análise de Dados – SEADE. (2016). IMP - Informações dos Municípios Paulistas - 2016. São Paulo: SEADE. Recuperado em 11 de janeiro de 2017, de http://www.imp.seade.gov.br Vieira, I. G. (2015). Novas formas de urbanização, estudo da tipologia de condomínios fechados para o programa Minha Casa, Minha Vida, Jacareí, São Paulo (Dissertação de mestrado). Instituto de Pesquisa e Desenvolvimento, Universidade do Vale do Paraíba, São José dos Campos. Maricato, E. (2013). Financeirização da política habitacional. In L. O. Royer (Eds.), Financeirização da política habitacional: limites e perspectivas. São Paulo: Annablume Editora. Recuperado em 8 de janeiro de 2017, de http://annablume.com.br/ index.php?option=com_content&view=article&id= Recebido: Fev. 17, 2017 Aprovado: Jan. 29, 2018 urbe. Revista Brasileira de Gestão Urbana (Brazilian Journal of Urban Management), 2018 set./dez., 10(3), 473-484
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Building a DNA barcode library for the freshwater fishes of Bangladesh
Scientific reports
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Md. Mizanur Rahman1, Michael Norén2, Abdur Rob Mollah1 & Sven O. Kullander We sequenced the standard DNA barcode gene fragment in 694 newly collected specimens, representing 243 species level Operational Barcode Units (OBUs) of freshwater fishes from Bangladesh. We produced coi sequences for 149 out of the 237 species already recorded from Bangladesh. Another 83 species sequenced were not previously recorded for the country, and include about 30 undescribed or potentially undescribed species. Several of the taxa that we could not sample represent erroneous records for the country, or sporadic occurrences. Species identifications were classified at confidence levels 1(best) to 3 (worst). We propose the new term Operational Barcode Unit (OBU) to simplify references to would-be DNA barcode sequences and sequence clusters. We found one case where there were two mitochondrial lineages present in the same species, several cases of cryptic species, one case of introgression, one species yielding a pseudogene to standard barcoding primers, and several cases of taxonomic uncertainty and need for taxonomic revision. Large scale national level DNA barcode prospecting in high diversity regions may suffer from lack of taxonomic expertise that cripples the result. Consequently, DNA barcoding should be performed in the context of taxonomic revision, and have a defined, competent end-user. Received: 21 November 2018 Accepted: 3 June 2019 Published online: 28 June 2019 Fish and fisheries play an important role in Bangladesh’s economy, nutrition and culture. With 47 609 km2 of inland water bodies, it is the third inland fish producing country in the world after China and India1. The annual fish production from inland waters is estimated to be 3,496,958 mt, of which 1,163,606 mt comes from inland open waters1. Fish is second staple food in Bangladesh and alone supplements about 60% of animal protein in the daily dietary requirement2. Open water fishery is still the primary source of food fishes for the larger population. The Hilsa (Tenualosa ilisha) capture fishery alone contributes about 12% of the fish production1 and provides outcome for 2.5 million people2. p p Bangladesh sits in between the biologically rich and diverse Indo-Burma and Eastern Himalaya regions, and is traversed by three of Asia’s largest rivers, the Ganga, Brahmaputra, and Meghna, which reach the Bay of Bengal in Bangladesh. Nonetheless, Bangladesh has one of the most incompletely known national freshwater fish faunas in Asia. As with other tropical countries, diversity estimates for Bangladeshi freshwater fishes are uncertain. 1University of Dhaka, Department of Zoology, Dhaka, Dhaka, 1000, Bangladesh. 2Swedish Museum of Natural History, Department of Zoology, SE-104 05, Stockholm, Sweden. Correspondence and requests for materials should be addressed to S.O.K. (email: sven.kullander@nrm.se) www.nature.com/scientificreports www.nature.com/scientificreports www.nature.com/scientificreports C d bl h C Corrected: Publisher Correction Corrected: Publisher Correction Scientific Reports | (2019) 9:9382 | https://doi.org/10.1038/s41598-019-45379-6 Building a DNA barcode library for the freshwater fishes of Bangladesh Md. Mizanur Rahman1, Michael Norén2, Abdur Rob Mollah1 & Sven O. Kullander Md. Mizanur Rahman1, Michael Norén2, Abdur Rob Mollah1 & Sven O. Kullander Estimates of freshwater fish species vary from 2373 to 2474, 2605, or 2676, but those numbers include migrating and estuarine species; an estimate of riverine species only gives 104 species5. From several books and review papers on Bangladeshi fishes4,6,7, it is evident, however, that the taxonomy used is outdated and not harmonized with the taxonomy employed globally or even in neighbour countries. Apparently, considering the economic importance of inland fishery and the expected richness of the fish fauna, and in the absence of an expert based taxonomy, DNA barcoding may be an important component in biological conservation and management of bio- diversity and fishery of Bangladeshi freshwater fishes. yi y gi DNA barcoding is a tool based on the observation and premise that each species is genetically distinct — has a unique DNA. Unique sequences of DNA from expert-identified specimens enable construction of a library of species-specific DNA sequences, “DNA barcodes”, against which unidentified samples can be matched8,9. DNA barcodes are useful for identification of both fresh specimens and market products such as frozen fish10,11. The standard barcode sequence, a fragment of the mitochondrial cytochrome c subunit I gene (coi), is also frequently used in phylogeographic and phylogenetic analyses12,13, but concerns have also been raised that the utility of bar- coding has been overstated and that dependency on single markers may lead to deficient taxonomy14,15.i i Here, we present the results and lessons from sequencing freshwater fishes collected in markets and natural habitats in Bangladesh, 2014–2016, in a study strictly aimed at a complete coi-based DNA barcode reference 1University of Dhaka, Department of Zoology, Dhaka, Dhaka, 1000, Bangladesh. 2Swedish Museum of Natural History, Department of Zoology, SE-104 05, Stockholm, Sweden. Correspondence and requests for materials should be addressed to S.O.K. (email: sven.kullander@nrm.se) Scientific Reports | (2019) 9:9382 | https://doi.org/10.1038/s41598-019-45379-6 1 www.nature.com/scientificreports/ www.nature.com/scientificreports/ Figure 1. Map of collecting sites in Bangladesh, 2014–2016. A symbol may cover more than one collecting site. The map was constructed in QGIS 2.18.1 (QGIS Development Team, 2016. QGIS Geographic Information System. Open Source Geospatial Foundation. http://qgis.org), using free vector and raster map data made available by Natural Earth (http://naturalearthdata.com). All maps are in the public domain (http://www. naturalearthdata.com/about/terms-of-use/). Figure 1. Map of collecting sites in Bangladesh, 2014–2016. A symbol may cover more than one collecting site. The map was constructed in QGIS 2.18.1 (QGIS Development Team, 2016. QGIS Geographic Information System. Open Source Geospatial Foundation. Md. Mizanur Rahman1, Michael Norén2, Abdur Rob Mollah1 & Sven O. Kullander http://qgis.org), using free vector and raster map data made available by Natural Earth (http://naturalearthdata.com). All maps are in the public domain (http://www. naturalearthdata.com/about/terms-of-use/). library of Bangladeshi freshwater fish species, introduce a new term for species level DNA sequence clusters interpreted as candidates for DNA barcodes: the Operational Barcode Unit (OBU). Results h Fish specimens were obtained from 150 collecting events and additional ad hoc sampling, including both markets and natural habitats in Bangladesh (Fig. 1). Phylogenetic and mPTP trees based on the Bayesian tree are shown in Figs 2 and 3. OBUs/species distinguished by the mPTP analysis are listed in Table S1. Metadata for sequenced specimens are summarized in Table S2.h p The mPTP analysis found 243 OBUs (Table S1), representing 53 families. Most of these OBUs relate to single species verified by morphological examination. Using a scale 3 (lowest) to 1 (highest), the species determination expertise levels for the vouchers were estimated to be 3 in 82 OBUs, 2 in 17, and 1 in 46; 99 determinations were classed as unqualified. i Correcting for disagreement with the trees (Figs 2 and 3), and consideration of morphological analyses, suggests recognition of 237 candidate species. The majority of the OBUs are uncontroversial, representing well-known, common species. There was no indication of contamination. Five species descriptions were already published based on the present material16–20. Two species failed sequencing (Taenioides cirratus, Johnius coitor). One probable nuclear pseudogene (NUMT) was found in Pseudapocryptes elongatus (OBU 14). One species had two mitochondrial lineages without morpho- logical or nuclear DNA divergence (Brachygobius nunus OBU 10–11), and one species had two distinct popula- tions separated by highly divergent mitochondrial and nuclear sequences but without detected morphological divergence (Danio rerio OBUs 160–161). Introgression was found in Devario anomalus (OBUs 152–153). Other OBUs may lack morphological support, as detailed in the discussion. y p g pp Several cases of putative cryptic species were recorded, as detailed below. Here we consider as putative cryptic species OBUs that are genetically distinct, but not reciprocally diagnosable morphologically. Several of the puta- tive cryptic OBU pairs consist of one OBU in the Meghna River drainage and another in the Karnafuli and Sangu River drainages. Discussion OBUs in the Bayesian tree (Fig. 2) and the mPTP analysis (Fig. 3, Table S1) were monophyletic at higher levels, except that the trees came out unresolved or with OBUs in unexpected position, as an artefact of the wide sam- pling (spanning the entire Actinopterygii) with several long branches without close relationship to other taxa in the tree. This affects, e.g., Psilorhynchus (Psilorhynchidae), nested within the Cyprinidae, and Butis humeralis (Eleotridae), within the Gobiidae). The objective of the tree-based analysis is, however, not phylogenetic relation- ships but species delimitation by branch clusters. The mPTP analysis reported 243 “species” but there are discord- ances with the tree. The “species” in the mPTP output includes potential or probable cases of excess taxonomic Scientific Reports | (2019) 9:9382 | https://doi.org/10.1038/s41598-019-45379-6 2 www.nature.com/scientificreports/ www.nature.com/scientificreports/ www.nature.com/scientificreports/ 3 19) 9:9382 | https://doi.org/10.1038/s41598-019-45379-6 Figure 2. Phylogram of relationships of Bangladeshi fish species, based on Bayesian analysis of the standard DNA barcode fragment of the mitochondrial coi gene. The scale bar indicates expected number of substitutions per site. Terminals are identified by NRM or DU tissue sample collection identifiers (metadata, including GenBank Accession Numbers, in Supplementary Table S2). Figure 2. Phylogram of relationships of Bangladeshi fish species, based on Bayesian analysis of the standard DNA barcode fragment of the mitochondrial coi gene. The scale bar indicates expected number of substitutions per site. Terminals are identified by NRM or DU tissue sample collection identifiers (metadata, including GenBank Accession Numbers, in Supplementary Table S2). Figure 2. Phylogram of relationships of Bangladeshi fish species, based on Bayesian analysis of the standard DNA barcode fragment of the mitochondrial coi gene. The scale bar indicates expected number of substitutions per site. Terminals are identified by NRM or DU tissue sample collection identifiers (metadata, including GenBank Accession Numbers, in Supplementary Table S2). Figure 2. Phylogram of relationships of Bangladeshi fish species, based on Bayesian analysis of the standard DNA barcode fragment of the mitochondrial coi gene. The scale bar indicates expected number of substitutions per site. Terminals are identified by NRM or DU tissue sample collection identifiers (metadata, including GenBank Accession Numbers, in Supplementary Table S2). Scientific Reports | (2019) 9:9382 | https://doi.org/10.1038/s41598-019-45379-6 3 www.nature.com/scientificreports/ www.nature.com/scientificreports/ www.nature.com/scientificreports/ 4 9) 9:9382 | https://doi.org/10.1038/s41598-019-45379-6 Figure 3. mPTP phylogram of Bangladeshi freshwater fish species based on the standard barcode fragment of the mitochondrial coi gene. The scale bar indicates expected number of substitutions per site. www.nature.com/scientificreports/ www.nature.com/scientificreports/ splitting and lumping, but also a number of ghost OBUs. In the following, OBU numbers are based on the mPTP species numbers from the analysis based on the Bayesian tree.l p y y To investigate the influence of the choice of method for creating the phylogenetic tree used as indata for the mPTP species delimitation analysis, we used two different Maximum Likelihood (ML) software, RAxML and PHYML, to create Single Most Likely Trees, and analysed those as for the Bayesian tree. All three methods of anal- ysis resulted in the same OBUs, with the exception of Rasbora rasbora (OBU 149) and Microphis cuncalus (OBU 25), which were recovered as single OBUs from the BI tree, but as two OBUs each from the ML trees. The genetic variation within the two putative species was intermediate between typical intra- and inter-specific variation, the two M. cuncalus (from Padma and Karnafuli) and three R. rasbora (from Padma and Meghna) sequences having an uncorrected furthest pairwise-p distance of 1.7% and 1.2%, respectively. Morphological examination did not find support for recognizing more than single species within OBUs 25 and 149.i i pp g g g p A GenBank BLAST search of OBUs 4–6 identified them all as Glossogobius giuris, but they represent three genetically and morphologically distinct species. The common G. giuris morphology was represented by OBU 5, whereas OBU 4 is more similar to G. aureus from Hong Kong. OBU 6 remains unidentified. Our identification of OBU 4 is highly tentative.i g y OBUs 10 and 11 represent a single species, Brachygobius nunus. Morphologically identified specimens of Brachygobius nunus from the Turag River had distinctly different coi sequences; i.e., there were two mitochondrial lineages within a single population of B. nunus. g g p p Species of Pseudolaguvia, OBUs 57–62, showed up as six distinct lineages in the mPTP tree (Fig. 3), but got split into seven species (Table S1), which is probably unwarranted. Unfortunately, it was not straightforward to identify some of the included lineages with named species.h y g p Three specimens of Pseudapocryptes elongatus (OBU 14) were sequenced, and all three sequences had a unique 1 basepair (thymine, T) insertion at position 622 in the complete coi sequence (corresponding to posi- tion 6142 in the reference genome of Parapocryptes serperaster, GenBank accession number NC_029455). Scientific Reports | (2019) 9:9382 | https://doi.org/10.1038/s41598-019-45379-6 www.nature.com/scientificreports/ This insertion creates a TAG stop-codon and frame shift, which would render the coi protein nonfunctional, strongly suggesting that the sequences are not of mitochondrial coi, but of a nuclear pseudogene (NUMT). Resequencing the specimens with different primers produced the same result. There are 12 coi sequences of P. elongatus depos- ited at GenBank, but only two, AF391394 and KT124739, are long enough to include the insertion. AF391394 is identical to our sequences, with the exception that there is no T insertion at position 622. KT124739 contains nine differences to our sequences, out of which four are unique insertions, but also the T insertion at position 622. It is possible that our sequences are a chimera of the coi gene and a nuclear pseudogene, although nothing suggesting this is apparent in the raw data. However, this is the sequence one will obtain from Bangladeshi P. elongatus when using standard barcode primers. It is unique for the species, serving as a DNA barcode.i g p q p g Two specimens identified morphologically as Chaca chaca were analyzed, and turn out as distinct OBUs, 81 and 82. If representing distinct species, it cannot be decided at this time which one, if either, is the Chaca chaca of Hamilton (1822). There is one other potentially valid species described from Bengal, Chaca lophioides Cuvier & Valenciennes, 1832. Likewise, OBU 155–156 separates two specimens of Chela cachius, but there is no morpho- logical support for this.i g pp OBU 32 represents Trichopsis vittata. It is the first record of a feral population of an introduced species orig- inating in South East Asia, which is not also an aquaculture species and the barcode is specific for the aquarium population derived from a specific region in Thailand21.i p pi gh OBUs 83–85 represent three distinct species of Olyra, of which OBU 84 identified as Olyra longicaudata Remaining two OBUs require more analysis before a final determination can be made. g yi OBUs 101–102 refer to two species of Badis, which are nearly indistinguishable morphologically, but have complementary geographical distribution and distinct coi sequences and were considered as cryptic sister species20. g yi OBUs 101–102 refer to two species of Badis, which are nearly indistinguishable morphologically, but have complementary geographical distribution and distinct coi sequences and were considered as cryptic sister species20. Discussion The clusters marked with red were resolved as species by the mPTP analysis. Terminals are identified by NRM or DU tissue sample collection identifiers (metadata, including GenBank Accession Numbers, in Supplementary Table S2). Figure 3. mPTP phylogram of Bangladeshi freshwater fish species based on the standard barcode fragment of the mitochondrial coi gene. The scale bar indicates expected number of substitutions per site. The clusters marked with red were resolved as species by the mPTP analysis. Terminals are identified by NRM or DU tissue sample collection identifiers (metadata, including GenBank Accession Numbers, in Supplementary Table S2). Figure 3. mPTP phylogram of Bangladeshi freshwater fish species based on the standard barcode fragment of the mitochondrial coi gene. The scale bar indicates expected number of substitutions per site. The clusters marked with red were resolved as species by the mPTP analysis. Terminals are identified by NRM or DU tissue sample collection identifiers (metadata, including GenBank Accession Numbers, in Supplementary Table S2). Figure 3. mPTP phylogram of Bangladeshi freshwater fish species based on the standard barcode fragment of the mitochondrial coi gene. The scale bar indicates expected number of substitutions per site. The clusters marked with red were resolved as species by the mPTP analysis. Terminals are identified by NRM or DU tissue sample collection identifiers (metadata, including GenBank Accession Numbers, in Supplementary Table S2). Scientific Reports | (2019) 9:9382 | https://doi.org/10.1038/s41598-019-45379-6 Scientific Reports | (2019) 9:9382 | https://doi.org/10.1038/s41598-019-45379-6 www.nature.com/scientificreports/ p OBUs 109–111 refer to Pethia guganio, a little studied small cyprinid species in the Ganga and Brahmaputra basins, but also present in the Karnafuli and Sangu drainages. The mPTP analysis recovered three OBUs initially identified as one species. Pethia guganio is probably a species complex that cannot be resolved taxonomically here. OBU 109, however, was distinguished by the presence of barbels, not otherwise known from P. guganio.f g y p g g OBUs 126, Osteobrama cotio, and 127, O. cf. cotio, differed in coi and mt-rnr2 sequences22. Samples from the Karnafuli and Sangu drainages were slightly different from those from the Meghna drainage, including published sequences from the Barak River. No morphological differences were found and they might be cryptic species22. Because O. cotio is a widespread species, it should be re-analyzed with inclusion of representation of the entire geographical distribution23. The mPTP analysis here recognizes the Meghna and Karnafuli + Sangu OBUs as two species, for which there is no morphological support.i OBUs 126, Osteobrama cotio, and 127, O. cf. cotio, differed in coi and mt-rnr2 sequences22. Samples from the Karnafuli and Sangu drainages were slightly different from those from the Meghna drainage, including published sequences from the Barak River. No morphological differences were found and they might be cryptic species22. Because O. cotio is a widespread species, it should be re-analyzed with inclusion of representation of the entire geographical distribution23. The mPTP analysis here recognizes the Meghna and Karnafuli + Sangu OBUs as two species, for which there is no morphological support.i p p g pp OBU 136 was identified morphologically as Labeo rohita, but disagrees with the other coi sequence of L. rohita, and is identical with GenBank sequences identified as Labeo dyocheilus and L. pangusia, with a considerably dif- ferent morphology. It may be a case of introgression in aquaculture conditions, but L. dyocheilus and L pangusia are large mountain species, unlikely candidates for hybridization in aquaculture. p p g pp OBU 136 was identified morphologically as Labeo rohita, but disagrees with the other coi sequence of L. rohita, and is identical with GenBank sequences identified as Labeo dyocheilus and L. pangusia, with a considerably dif- ferent morphology. It may be a case of introgression in aquaculture conditions, but L. dyocheilus and L pangusia are large mountain species, unlikely candidates for hybridization in aquaculture. www.nature.com/scientificreports/ Scientific Reports | (2019) 9:9382 | https://doi.org/10.1038/s41598-019-45379-6 5 www.nature.com/scientificreports/ OBU 177 contains a single small specimen from the Sangu drainage, similar to Barilius barila in the Meghna and Padma drainages, but assessment of its taxonomic status requires revision of Barilius. g q OBU 185 represents a morphologically distinct species of Lepidocephalichthys. g q OBU 185 represents a morphologically distinct species of Lepidocephalichthys. p p g y p p p y OBU 202 represents two morphologically distinct, but similar species of Schistura, S. sijuensis from the Garo Hills, and a possibly undescribed species from the Sangu River. It seems likely that these two taxa may be sister species. p OBUs 186 and 189–190 represent Lepidocephalichthys guntea morphologically, but separate in three distinct OBUs with tentative morphological support. OBU 189 probably represents the true L. guntea. p g pp p y p g OBU 202 combines two morphologically distinct taxa incompletely separated in two drainages.i p g y p y p g OBUs 222–226 suggest (Table S1) five species of Oryzias, including O. dancena, but the tree indicates only four (Fig. 2). Only two species of Oryzias have been reported from Bangladesh24 and the Oryzias sequences maybe excessively split in the mPTP analysis.hi y p y The identification of OBU 240 presents a nomenclatural problem. Authorship and name of this species is uncertain. It was described as a new species, Parambassis ornatus by Geetakumari & Vishwanath, 201225, and also, but with a different holotype, as P. bistigmata by Geetakumari, 201226. The latter was published in May, 2012, but the date of publication of Geetakumari & Vishwanath’s book25 was not stated in the publication itself, only the year. The valid name, authorship and date needs more research. yh p All OBUs reported here, occur in freshwater, and the majority, 2001 (Table S1), may be considered to be exclusively or predominantly freshwater species. Classification by salinity level, however, is not straightforward. Species of the Gobiidae and Eleotridae, Oryzias, Hyporhamphus limbatus, may be common far inland, but may also be present in coastal waters and estuaries. www.nature.com/scientificreports/ The major component of the Bangladeshi fish fauna, however, is ostariophysan, and as such only contains freshwater species.i p y y p We obtained coi sequences from 149 out of the 237 freshwater fish species already recorded from Bangladesh3, including some euryhaline or estuarine species that are common in inland waters; and 116 out of 162 classed as strictly freshwater. Of the 237, 31 species represented taxa erroneously recorded for the country27, or sporadic occurrences, and several more may be considered to be marine rather than freshwater5. Nine of our sequenced species represented the 12 species of aquaculture or aquarium releases recorded from natural habitats. We further obtained 83 OBUs representing species that were not previously recorded for the country, including an estimated about 30 species that may be undescribed or not yet identified. Calculations of “unknowns” are uncertain insofar as they may represent previously misidentified species or synonyms to be re-validated.h y y p p yi p y y The present study is weak in coverage in the southwestern and western parts of Bangladesh, principally the Sundarbans and a portion of the Ganga River and tributaries. Expectations of additional taxa from that area, are low, however. The coverage of the Chittagong Division, including Karnafuli and Sangu Rivers and the Cox’s Bazar region provides for considerable new ocurrences and new species16,18–20, but also strong affinity to the adjacent western Rakhine in Myanmar, e.g., in the shared distribution of Laubuka tenella19. Most of the samples, from the Meghna, Jamuna and Padma tributaries, reflect a common fish fauna with adjacent India, the samples from the Pyain River, draining the Garo Hills, provide a distinct representation belonging to the Eastern Himalaya Region28. Numerous fish species have been described from the Barak River in Manipur29, draining to the Surma and Meghna Rivers in Bangladesh. That fauna is not reflected in our material from Bangladesh, however. Also, the taxa shared with the Kaladan River30 are only species with very wide distribution in Bangladesh and India.h The above examples demonstrate that DNA barcoding is not trivial. Contamination, introgression, multi- ple mitochondrial DNA lineages, cryptic species, introductions and NUMTs do occur and may be difficult to detect. The examples in our data set are very limited, however. To this comes lack of modern revisions, and short- age of taxonomic expertise for qualified identification of voucher specimens. www.nature.com/scientificreports/ p p g pp OBU 136 was identified morphologically as Labeo rohita, but disagrees with the other coi sequence of L. rohita, and is identical with GenBank sequences identified as Labeo dyocheilus and L. pangusia, with a considerably dif- ferent morphology. It may be a case of introgression in aquaculture conditions, but L. dyocheilus and L pangusia are large mountain species, unlikely candidates for hybridization in aquaculture. g p y y q OBU 152 is complicated, as it includes specimens of Devario anomalus with D. aequipinnatus mitochondrial genome18. Devario coxi is here nested with D. aequipinnatus from which it was diagnosed morphologically and by mitochondrial and nuclear genes18. Intra-OBU variation suggests inclusion of three distinct haplogroups.ih g p y y q OBU 152 is complicated, as it includes specimens of Devario anomalus with D. aequipinnatus mitochondrial genome18. Devario coxi is here nested with D. aequipinnatus from which it was diagnosed morphologically and by mitochondrial and nuclear genes18. Intra-OBU variation suggests inclusion of three distinct haplogroups.ih g p y y q OBU 152 is complicated, as it includes specimens of Devario anomalus with D. aequipinnatus mitochondrial genome18. Devario coxi is here nested with D. aequipinnatus from which it was diagnosed morphologically and by mitochondrial and nuclear genes18. Intra-OBU variation suggests inclusion of three distinct haplogroups. g gg p g p OBUs 160–161 represent distinct OBUs, morphologically identified as Danio rerio. The two D. rerio mitotypes were already reported23 based on mitochondrial cytochrome b sequences and SNPs. Intraspecific variation in D. rerio needs additional analysis, but at this time there is no information to support species distinction. g gg p g p OBUs 160–161 represent distinct OBUs, morphologically identified as Danio rerio. The two D. rerio mitotypes were already reported23 based on mitochondrial cytochrome b sequences and SNPs. Intraspecific variation in D. rerio needs additional analysis, but at this time there is no information to support species distinction. y pp p OBUs 164–165, 171, 181–183, 187–88, 191–192 may represent cryptic species, or overlooked distinct spe- cies, or even populations of the same species excessively split by mPTP, but no indication was found of species differences. y pp p OBUs 164–165, 171, 181–183, 187–88, 191–192 may represent cryptic species, or overlooked distinct spe- cies, or even populations of the same species excessively split by mPTP, but no indication was found of species differences. Scientific Reports | (2019) 9:9382 | https://doi.org/10.1038/s41598-019-45379-6 www.nature.com/scientificreports/ Very few of the South Asian fish genera or species groups have been revised by specialists, e.g., Psilorhynchus31, Lepidocephalichthys32, Oryzias24, Paracanthocobitis33,34, and Sperata35. , p Our results provide relevant aspects on large-scale/country-wide DNA barcoding, end-use of barcodes, and limits of application of the DNA barcode concept, as detailed below.h The original expectations of DNA Barcoding were relatively modest: establishment of a database of distinct vouchered reference sequences against which unidentified sequences could be compared, and which would pro- vide a name (identification) of the organism from which the unidentified sample was taken8,15. coi, however, has assumed additional roles, namely for building phylogenetic trees, and for species discovery15. Studies using DNA barcodes as a tool for species discovery15,36 were called “molecular parataxonomy” by Collins & Cruickshank15, but are perhaps better referred to as barcode or DNA prospecting. The principle of species recognition in DNA prospecting is based on the barcode gap convention8,15, and not on a species concept or a prior hypothesis of diagnostic characters or phylogenetic relationships8. The barcode gap, however, is a distance measure that only shows probability of reproductive isolation between two a priori determined samples. Consequently, we prefer the term suggested here, Operational Barcode Unit (OBU), which is akin to the ‘species-like units’ of Collins & Cruickshank15, for raw standard barcode coi sequences, with the caveat that identity may be the result of intro- gression. Dissimilar coi sequences indicate reduced interbreeding, which to differing degrees may also occur between demes or populations, not only between species, considered as independent evolutionary lineages37.i p p y p p y g OBUs can be referred to without the need for relation to a particular species, but need specialist verification to serve as markers for species. The expectation of species delimitation methods is that they should provide automatic species recognition8,37, but a coi sequence distinct from others or not, is not a species marker, but only exhibiting levels of sequence divergence that uggestive species status38. As exemplified in our trees, units delin- eated by mPTP may conflict with species identification in several aspects, primarily dependent on the expertise of the determiner, but also reflecting introgression and sibling species, species concepts37, and availability of a taxonomic platform, requiring taxonomic validation using independent criteria. Scientific Reports | (2019) 9:9382 | https://doi.org/10.1038/s41598-019-45379-6 6 www.nature.com/scientificreports/ DNA barcoding projects frequently report cryptic species as well as discovery of previously unrecognized species in fishes39,40. www.nature.com/scientificreports/ Commonly, the analysis stops there, and the recognition of cryptic species in DNA barcoding remains a by-product of molecular prospecting39. Putative cryptic species may represent widely different evolu- tionary processes, however41. Species may be considered cryptic because of insufficient morphological analysis, i.e., a shortcoming of morphological analysis and thus a temporary state of identification (provisionally cryptic42). Mitochondrial genome substitution due to hybridization in a particular population may also be mistaken for cryptic species. Consequently, “cryptic OBUs” require taxonomic assessment, and evolutionary analysis, and in the meantime remain ghost OBUs. Whereas many of our sequences cannot be linked to a particular species, only few cases of sibling or cryptic species were demonstrated in cyprinids and badids so far18,20,22, and the unidentified OBUs should not be assumed to represent cryptic species or undescribed species in the absence of taxonomic analysis. Nation-wide or regional barcoding surveys, have been proposed to accelerate DNA barcode coverage43, and some have been attempted, such as the one reported here. Another project was started in 2013 to barcode Swedish vertebrates44. Despite Sweden’s relatively species-poor and fully known vertebrate fauna, this barcode project does still not contain all species of vertebrates in Sweden (78.1% barcoded), or even fishes (72% bar- coded)44. It seems unlikely that similar projects in species-rich tropical countries will be successful in achieving completeness. DNA barcodes are taxonomic statements. Each OBU translated to a species name expresses infor- mation that requires taxonomic expertise, sequencing and sampling skills, permanent voucher repositories, and constant revision of the taxonomic background. It also takes competence in using a proposed barcode, because similar OBUs may have different evolutionary explanations. We therefore consider that DNA barcoding should be done on a taxonomic, and not a geographical, or national basis.i g g p Fish DNA barcodes serve a purpose, e.g., in the fishery industry for control of product authenticity and ori- gin and for clearing potentially harmful imports, as explicit in several projects10,11,43,45,46. Hence, trade and con- sumption species are priority targets, and the fishery and biohazard agencies are most likely the institutions that have the means to sample and blast sequences against openly available digital sequence providers such as BOLD and GenBank, but which do not maintain a private DNA barcode library. In the context of such use, the overall critical factor is that users have an unknown tissue to check against a barcode library. Methods Sampling. Fish specimens were collected using a 5 mm mesh size beach seine, or, for commercial species, by purchase directly from fishermen or at local fish markets. Specimens were obtained from 150 collecting events and additional ad hoc sampling including both markets and natural habitats (Fig. 1). The result was 3278 whole specimens yielding 2514 tissue samples (whole specimens or fin clips) with considerable redundance. Based on morphological criteria and representation of different river basins, 697 samples from Bangladesh were selected for sequencing. coi sequencing failed in two the two specimens svailable of Johnius coitor (Sciaenidae) and the single specimen of Taenioides cirratus Gobiidae), resulting in 692 specimens for analysis.ii g p g p y Whole fish or fin clips were stored in 95% ethanol at −80 °C. Fin-clipped whole specimens and excess spec- imens for morphological analyses were fixed in 10% formalin and eventually transferred to 70% ethanol for permanent storage. Tissue samples and voucher specimens were deposited in the fish collection of the Swedish Museum of Natural History (NRM), and the Department of Zoology, University of Dhaka (DU). Taxonomic identification. Specimens were sorted and identified morphologically before sequencing. Identification was made by using comparative material identified by experts in the NRM collection, specialist publications, and monographs. Several experts assisted in determinations based on high-resolution photographs, or with comments on taxonomic status. The FISH-BOL collaborator’s protocol9 provides a graded classification of determination quality, which we employ here, with the modification that we report confidence per specimens in the respective OBU, and not per OBU or individual sequence, and disregard their levels 4–5 which do not produce a species determination at all. “Level 1: highly reliable identification-specimen identified by (1) an internationally recognized authority of the group, or (2) a specialist that is presently studying or has reviewed the group in the region in question”.iii The FISH-BOL collaborator’s protocol9 provides a graded classification of determination quality, which we employ here, with the modification that we report confidence per specimens in the respective OBU, and not per OBU or individual sequence, and disregard their levels 4–5 which do not produce a species determination at all. www.nature.com/scientificreports/ The role of the end user is commonly underestimated or ignored in attempts at DNA barcoding, which then becomes barcode or DNA prospecting without any explicit end user.hi p p g y p This might mean less stress on taxonomic hair-splitting over the taxonomy of small-size fish species, which is relevant for many of the species in our analysis. In southern Asia, however small-size fishes are a significant com- ponent of the nutrition47, and identification protocols for small-size fish species may be relevant for monitoring purposes.ti In conclusion, we have lifted some of the curtain to Bangladeshi fish coi sequences in the present report, but also exposed some of the challenges and critical aspects of DNA barcoding from a taxonomic perspective. It sug- gests that the decisive component of DNA barcoding, are end-user capacity, and taxonomic revision dealing with identification, nomenclature, species delimitation, introgression, multiple mitochondrial lineages, and more, We have been successful in generating numerous coi sequences and correlate them with species, but it turns out that was only the start of the barcoding effort. Scientific Reports | (2019) 9:9382 | https://doi.org/10.1038/s41598-019-45379-6 Methods The cleaned PCR product was sent to Macrogen (Amstelween, The Netherlands) for sequencing. Reads were assembled and proofread in Geneious version 1049, and a GenBank BLAST (megablast) search of the GenBank nr database was used as a first screening for misidentified or contaminated sequences. To detect and visualize con- taminations and problems with identification, sequences were aligned in Geneious and phylogenetic hypotheses were constructed in MrBayes version 3.250, with the following settings: GTR + I + G model, 15 million genera- tions, with the first 25% discarded as burn-in, then sampled every 1000 generations. Convergence was checked with Tracer51. Sequences which passed quality control were uploaded to the Barcode of Life Database (BOLD52 and pub- lished to GenBank53. A total of 694 barcode-compliant sequences were produced on material from Bangladesh. An additional two sequences from Bangladeshi specimen sequences were not barcode compliant, due to being pseudogenes (NUMTs). Species delimitation. OBU detection and delimitation by multi-rate Poisson Tree Process method was investigated based on the Bayesian tree, using mPTP version54, with the following settings: 15 separate runs, minimum branch length estimated from alignment (0.0053), one lambda for the coalescent, starting with random species assignments, 40 million generations, the first 20 milllion generations discarded as burn-in, then sampled every 50 thousand generations. Convergence was checked by examining mPTP’s log files. i Single Most Likely Trees were constructed using the RAxML55 and PHYML56 plug-ins for Geneious. RAxML analysis was performed with GTR + G model, rapid hill climbing algorithm, seven starting trees, parsimony random seed 1, and Maximum Likelihood search convergence criterion. PHYML analysis was performed with GTR + I + G model, proportion of invariable sites and gamma estimated in analysis, NNI topology search. The mPTP analysis was performed as for the Bayesian. Terminology. Gene names and symbols follow ZFIN nomenclature conventions57, except that we use here the universally common synonym coi for the mt-co1). We reserve the term DNA barcode for a ~655 bp Folmer region coi sequence from a vouchered specimen identified as a particular species, i.e., quality levels 1–39. We use barcoding/barcoded for the process/accomplishment of establishing a DNA barcode for a particular species. We use the short coi for the standard DNA barcode part of the mt-co1 gene unless stated otherwise. We do not consider coi sequences as necessarily representing species or even taxonomic units. Each such sequence needs further analysis to assess it status. Methods “Level 1: highly reliable identification specimen identified by (1) an internationally recognized authority of g p p p y y g g p g q “Level 2: identification made with high degree of confidence at all levels-specimen identified by a trained iden- tifier who had prior knowledge of the group in the region or used available literature to identify the specimen”.iii i p g g p g y p Level 3: (modified) “identification made with high confidence to genus but less so to species—specimen iden- tified by (1) a trained identifier who was confident of its generic placement but did not substantiate their species identification using the literature”. Scientific Reports | (2019) 9:9382 | https://doi.org/10.1038/s41598-019-45379-6 7 www.nature.com/scientificreports/ Dataset limitation. The dataset reported is limited to the data assembled by the project, 2014–2016. DNA barcode sequencing. Approximately 1 mm3 tissue was taken from the alcohol preserved fish or fin clip, and DNA extracted using either a Kingfisher Duo (Thermo Scientific) DNA extraction robot, with recom- mended protocol, or DNEasy Blood & Tissue Kit (QIAGEN) spin columns, with recommended protocol.h p , y (Q ) p , p The standard barcode sequence, 655 bp from the 5′ end of coi, was sequenced with the primers Fish Fish-F2, Fish-R1, Fish-R248 PCR was performed using illustra PuReTaq RTG PCR Beads (GE Healthcare), with 2 µL DNA extract, 0.5 µL of each primer and adding water for a 25 µL reaction PCR cycling was: 94 °C 5′, 35 * (54 °C 30″, 72 °C 30″), 72 °C 7′. In a few cases where molecular and morphological analyses conflicted (i.e., where there was reason to suspect hybridization or that the coi sequence was a nuclear pseudogene), an additional mitochondrial gene (mt-rnr2) and a nuclear gene rag1 exon 3) were sequenced. g g q mt-rnr2 was sequenced using the primers 16S_arLm2 (CCTCGCCTGTTTACCAAAAACA) and 16S_brHm (CTCCGGTCTGAACTCAGATCACGT), with PCR cycling as for COI except with 57 °C annealing tempera- ture. rag1 exon 3 was sequenced using the primers R1_Dan1f (TGGCCATAAGGGTMAACAC) and R1_4078r (TGAGCCTCCATGAACTTCTGAAGRTAYTT), with PCR cycling as for coi. ( ) y g The PCR product was cleaned by adding 5 μL of a mix of 20% Exonuclease I (EXO) and 80% FastAP Thermosensitive Alkaline Phosphatase (Fermentas) to each 25 μl reaction, and incubated at 37 °C for 30 minutes, then heated to 80 °C for 15 minutes. Methods We propose the neutral term OBU (Operational Barcode Unit), as descriptor of presumed species level terminals found in an analysis of coi using multiple sequences, or a single unidentified specimen. Ideally, an OBU will be found eventually to represent a distinct species, and its sequence serve as a DNA barcode. We designate as ghost OBU a distinct standard DNA barcode sequence, or group of standard DNA barcode sequences, that cannot be identified as pertaining to any known species. Ghost OBUs may represent unidentified species, new species, NUMTs, within-species haplogroups, or bad sequence reads. Ethics statement. Specimens were already available in museum collections, purchased from fishermen or at markets; or collected in the wild using a beach seine or hand net and euthanized through immersion in buffered tricaine-methanesulphonate (MS 222) until cessation of opercular movements plus an additional 30 minutes, following the protocol in permits from the Swedish Environmental Protection Agency (dnr 412-7233-08 Nv) and the Stockholm Ethical Committee of the Swedish Board of Agriculture (dnr N 85/15). Collecting in Bangladesh was conducted under a permit to the University of Dhaka, and approval of the Ethical Review Committee, Faculty of Biological Sciences, University of Dhaka. Data Availability y All sequence and associated voucher data are available from BOLD and GenBank. Voucher metadata are available in Supplementary Information. 8 Scientific Reports | (2019) 9:9382 | https://doi.org/10.1038/s41598-019-45379-6 www.nature.com/scientificreports/ References 1. Department of Fisheries. 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New algorithms and methods to estimate maximum-likelihood phylogenies: Assessing the performance of PhyML 3 0 Syst Biol 59 307–321 https://doi org/10 1093/sysbio/syq010 (2010) 56. Guindon, S. et al. New algorithms and methods to estimate maximum-likelihood phylogenies: Assessing the performance of PhyML 3.0. Syst. Biol 59, 307–321, https://doi.org/10.1093/sysbio/syq010 (2010).hiii y p g y yq ( ) 7. The Zebrafish Information Network, https://wiki.zfin.org/display/general/ZFIN+Zebrafish+Nomenclature+Conventions (2018) Acknowledgements g Research on Bangladeshi freshwater fishes was supported by the project “Genetic characterization of freshwater fishes in Bangladesh using DNA barcodes”, funded by the Swedish Research Council, contract D0674001 to Sven Kullander and Abdur Rob Mollah. We thank several colleagues for helpful comments on this manuscript, and for support in identification of specimens: Ralf Britz, Kevin Conway, Ng Heok Hee, Maurice Kottelat; and the Chairman of the Department of Zoology, University of Dhaka, for logistic support. Author Contributions S.O.K. initiated the project, acquired funding, managed project administration, specimen curation and internal reports, performed the taxonomic analyses, and wrote the main part of the manuscript. M.N. developed methods of analysis, generated and analyzed molecular sequences, curated tissue samples, and participated in the writing of the final manuscript. M.M.R. contributed specimens, organized fieldwork, participated in generation of sequences, taxonomic determinations, curation of specimens, and made substantial contributions to the manuscript. A.R.M. Participated in funding acquisition, fieldwork, and logistics. All authors reviewed the manuscript. Scientific Reports | (2019) 9:9382 | https://doi.org/10.1038/s41598-019-45379-6 Additional Information Supplementary information accompanies this paper at https://doi.org/10.1038/s41598-019-45379-6 Competing Interests: The authors declare no competing interests. Publisher’s note: Springer Nature remains neutral with regard to jurisdictional claims in published maps and institutional affiliations. Open Access This article is licensed under a Creative Commons Attribution 4.0 International License, which permits use, sharing, adaptation, distribution and reproduction in any medium or format, as long as you give appropriate credit to the original author(s) and the source, provide a link to the Cre- ative Commons license, and indicate if changes were made. The images or other third party material in this article are included in the article’s Creative Commons license, unless indicated otherwise in a credit line to the material. 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Traveltime Analysis of VSP Seismograms in a Horizontal Transversely Isotropic Medium: A Physical Modeling Result
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TAO, Vol. 12, No. 4, 685-693, December 2001 TAO, Vol. 12, No. 4, 685-693, December 2001 1 Institute of Applied Geophysics, Institute of Seismology, National Chung Cheng University, Min-hsiung Chia-yi, Taiwan, ROG 2Department of Applied Physics, National Chiayi University, Min-hsiung, Chia-yi, Taiwan, ROG *Corresponding author address: Prof. Young-Fa Chang, Institute of Applied Geophysics, Institute of Seismology, National Chung Cheng University, Min-hs'1ung, Chia-yi 621, Taiwan, ROG E-mail: seichyo@eq.ccu.edu.tw NOTES AND CORRESPONDENCE Traveltime Analysis of VSP Seismograms in a Horizontal Transversely Isotropic Medium: A Physical Modeling Result Young-Fa Chang 1 • * and Chih-Hsiung Chang2 (Manuscript received 7 November 2000, in final form 30 August 2001) (Manuscript received 7 November 2000, in final form 30 August 2001) ABSTRACT In order to investigate the effects of anisotropy in a VSP survey, the multi-component VSP data for observing the qP-, qSH- and qSV-waves were collected with two different source-to-well offsets in an elastic trans­ versely isotropic medium (TIM) with a horizontal symmetry axis (HTI). The traveltimes of the events were then computed using two different algorithms: first with elastic constants of the medium, and second with "iso­ tropic velocities". Comparing the traveltimes of computations with the re­ corded data, a very clear and important consequence can be derived. For strongly anisotropic media (the anisotropy for the qP- and qS-waves are 25% and 29%, respectively, in our laboratory work), the effects of anisot­ ropy increase with increasing source-to-well offset. As the source-to-well offset is small compared with the depth of the receiver ( offseUdepth $ 0.2 in our work), the effects of anisotropy can be ignored. However, in process­ ing a large source-to-well offset VSP survey ( offseUdepth :2: 0.35 in this study), special attention must be paid on the effects of anisotropy. 1. INTRODUCTION Much of the earth's crust appears to be more or less anisotropic (Crarnpin 1981; Crampin and Lovell 1991; Helbig 1993), making seismic wave propagation more complicated than in isotropic media. Increasing demand for fossil fuels and minerals has encouraged geophysicists to explore deep and small reservoirs that could be possibly missed by traditional techniques. 685 TAO, Vol. 12, No. 4, December 2001 686 Therefore, seismic exploration techniques are being extended from simple isotropic models to complex anisotropic models in order to construct the geological structures in more detail p p g g Among seismic exploration techniques, the vertical seismic profile (VSP) is the most powerful one for investigation of anisotropy and the lithology around the well can be clearly studied. Shear-wave splitting in the near-surface late Tertiary shale is clear in both crosshole and VSP data (Winterstein and Paulsson 1990). Recorded as the components in the principal anisotropic axis, the shear-waves of different polarities reveal different velocities and reflec­ tion coefficients (Lynn and Thomsen 1990). The effects of singularities in sedimentary basins were observed in examining the shear-wave splitting in multi-offset VSPs at a borehole site in the Paris Basin (Bush and Crampin 1991). Shear-wave splitting was observed on 3-component seismograms from earthquakes of 5 to 18 km depths in the crystalline basement beneath the Los Angeles basin (Li et al. 1994). Wide-aperture walkaway VSP data acquired through trans­ versely isotropic horizontal layers can be used to determine the qP-wave phase-slowness sur­ face (Sayers 1997). Multi-component VSPs may reveal a strong vertical birefringence near the surface (MacBeth et al. 1997). However, due to geological complexity and the limitation of sources and receivers, the anisotropic characteristics of the earth can not be easily identified or quantified from field data, but they can be observed in physical modeling. Ebrom et al. (1990) used a wave propaga­ tion experiment in a fractured medium to show that the moveout curves for a survey line oriented parallel to the fractures are hyperbolic, but those for a survey line oriented perpen­ dicular to the fractures are non-hyperbolic. Cheadle et al. (1991) used industrial phenolite to study the propagations of seismic waves in an orthorhombic anisotropic medium. Brown et al. (1991) showed that the effect of the shear-wave window and the variation of the hyperbolic NMO parameter with offset are clearly observed in ultrasonic waves propagation through an orthorhombic medium. Chang et al. 1. INTRODUCTION (1994) clearly demonstrated the phenomena of shear­ wave splitting in a transversely isotropic medium (TIM). Chang and Gardner (1997) investi­ gated the effects of subsurface fractures in a three-layer medium and observed that the hori­ zontal moveout velocity decreases from the strike direction toward the transverse direction of the fractures. In this paper, we study the effects of anisotropy on VSP data using physical models. The observed anisotropic traveltimes in a strongly TIM (phenolite) with a horizontal symmetry axis (HTI) are compared with its theoretical anisotropic traveltimes and the isotropic traveltimes for two different source-to-well geometries. 2. EXPERIMENTS A pair of small transducers of 0.25 inch diameter was used as source and receiver (Panarnetrics A133S for detecting in-line particle motion, and Ultran SWC18-5 for detecting off-line and perpen­ dicular particle motion). The source-transducer emits an ultrasonic pulse into the medium and the echo is detected by the receiver-transducer. The oscilloscope (Tektronix TDS 420) re­ ceives the data and converts the analogic data into digital data. A personal computer is used to record and process the digital data. The bulk density of phenolite is 1.4 g/cm3• The elastic constants of the phenolite normal­ ized by the density are A11=15.8, A13=5.0, A33=8.7, A44=2.2, and A66=4.5 (106 m'/s') (Chang and Chang 2001a). The experimental error of velocity in the experiment is within 1 %. The velocity-anisotropies ((Vmax-Vmin)/Vmax) of the qP- and qS-waves for the phenolite are 25% and 29%, respectively. 2. EXPERIMENTS Vertical fractures caused by the horizontal pressure or tension force of the earth are com­ monly seen in the geological strata, giving the strata HTI for the seismic waves. In this study, a strongly anisotropic phenolite, composed of layers of paper bonded with phenolic resin, was used to simulate a HTI stratum. This phenolite is transversely isotropic because the paper is made up of no preferred direction. The configuration of the model, apparatus and arrangement of the measurements are shown in Fig. 1. The pulser/receiver (Panarnetrics 5058PR) emits high pulse voltage to excite the transducer, 687 Young-Fa Chang & Chih-Hsiung Chang z . (qP) Unit: cm ""k: Source \J : Receiver PC (qSH) 4 WELL Fig. 1. Configuration of the physical modeling, arrangement of the sources and receiver, apparatus and directions used in this study. and then receives and amplifies the weak signals. An orthogonal coordinate was adopted to describe the polarizations of the waves (Fig. 1). The Z-axis is vertical. The X- and Y-axes are horizontal and are perpendicular and parallel to the vertical fractures, respectively. A pair of small transducers of 0.25 inch diameter was used as source and receiver (Panarnetrics A133S for detecting in-line particle motion, and Ultran SWC18-5 for detecting off-line and perpen­ dicular particle motion). The source-transducer emits an ultrasonic pulse into the medium and the echo is detected by the receiver-transducer. The oscilloscope (Tektronix TDS 420) re­ ceives the data and converts the analogic data into digital data. A personal computer is used to record and process the digital data. z . (qP) Unit: cm ""k: Source \J : Receiver PC (qSH) 4 WELL Fig. 1. Configuration of the physical modeling, arrangement of the sources and receiver, apparatus and directions used in this study. z . (qP) Unit: cm ""k: Source \J : Receiver PC (qSH) 4 WELL : Source : Receiver Fig. 1. Configuration of the physical modeling, arrangement of the sources and receiver, apparatus and directions used in this study. Fig. 1. Configuration of the physical modeling, arrangement of the sources and receiver, apparatus and directions used in this study. and then receives and amplifies the weak signals. An orthogonal coordinate was adopted to describe the polarizations of the waves (Fig. 1). The Z-axis is vertical. The X- and Y-axes are horizontal and are perpendicular and parallel to the vertical fractures, respectively. 3.RESULTS The sources were located on the top surface and the receivers were located in the well. The length and time have been rescaled to simulate field seismic data, and the length and time scale factors are 10000:1. Two source-to-well offsets, 100 and 500 m, were used. The receiv­ ers were located between 100 and 1450 m depth with an interval of 50 m. Since the transducers are small relative to the propagation distance of the wave, the transducers can be considered as points. As the dominant polarizations are in the Z direction, the dominant events that are the 688 TAO, Vol. 12, No. 4, December 2001 qP-waves in this profile can be observed. When the dominant polarizations are in the Y and X directions, the dominant events that are the qSH- and qSV-waves, respectively, can be observed. The VSPs with 100 m source-to-well offset for qP-, qSH- and qSV-waves are shown in Figs. 2, 3 and 4, respectively. And the VSPs with 500 m source-to-well offset for qP-, qSH- and qSV-waves are shown in Figs. 5, 6 and 7, respectively. The exact phase velocities of the body waves in a homogeneous TIM can be derived from the equations of motion. However, the group velocity can not be derived for a wave radiated from a point source. But it can be obtained from the phase velocity using a differential opera­ tor (Thomsen 1986). The solid lines in Figs. 2-7 are the theoretical traveltimes for the waves propagation in the phenolite with group velocities. To view the effects of the anisotropy on the propagation of elastic body waves in a HTI, the traveltimes of the elastic body waves propaga­ tion with isotropic velocities were computed for comparison. The isotropic velocities are the qP-, qSH-, and qSV-waves velocities in the Z-axis direction; they are 4000, 2100 and 1500 ml s, respectively. The dashed lines in Figs. 2-7 show the traveltimes of the waves computed with the isotropic velocities. The phenomenon of velocity cusps for qSV-waves in a HTI can be expected to show up in Fig.7 as the receivers are 400-500 m in depth by the solid line. 4. DISCUSSION AND CONCLUSIONS The P-waves are easier to generate and detect than the S-waves in both field and laboratory. Therefore, the qP-waves can be easily and clearly observed in the experimental data (Figs. 2 Depth (m) 100 ' 300 500 700 900 1100 1300 F { '· 90 90 ' - " 3. á::,. 9P,.,, qi--es 180 180 ¿À ÁÂ Â 270 270 ,o- "';, HI 360 360 G 'o ms ms Depth (m) 100 ' 300 500 700 900 1100 1300 F { '· 90 90 ' - " 3. á::,. 9P,.,, qi--es 180 180 ¿À ÁÂ Â 270 270 ,o- "';, HI 360 360 G 'o ms ms Fig. 2. The Z-Z component VSP with 100 m source-to-well offset. The domi­ nant events in the profile are the qP-waves. The solid line is the theoreti­ cal traveltimes of the qP-waves and the dashed line is the traveltimes for the P-waves propagation using an isotropic velocity. Fig. 2. The Z-Z component VSP with 100 m source-to-well offset. The domi­ nant events in the profile are the qP-waves. The solid line is the theoreti­ cal traveltimes of the qP-waves and the dashed line is the traveltimes for the P-waves propagation using an isotropic velocity. Fig. 2. The Z-Z component VSP with 100 m source-to-well offset. The domi­ nant events in the profile are the qP-waves. The solid line is the theoreti­ cal traveltimes of the qP-waves and the dashed line is the traveltimes for the P-waves propagation using an isotropic velocity. Young-Fo Chang & Chih-Hsiung Chang 689 ms Depth (m) 100 300 500 700 900 1100 1300 200 400 600 ms Fig. 3. The Y-Y component VSP with 100 m source-to-well offset. The domi­ nant events in the profile are the qSH-waves. The solid line is the theo­ retical traveltimes of the qSH-waves and the dashed line is the traveltimes for the SH-waves propagation using an isotropic velocity. ms Depth (m) 100 300 500 700 900 1100 1300 200 400 600 ms Fig. 3. The Y-Y component VSP with 100 m source-to-well offset. The domi­ nant events in the profile are the qSH-waves. The solid line is the theo­ retical traveltimes of the qSH-waves and the dashed line is the traveltimes for the SH-waves propagation using an isotropic velocity. 200 400 600 800 100 ms Depth (m) 100 300 500 - ,_ à' E ;>-, " - , ___ D ... "_ --. 4. DISCUSSION AND CONCLUSIONS - -.. 9Si; :-.., ,,,, " qye>s 700 900 1 100 1300 I l)J1 --â...,- ""'- ,,_ reflected qP-waves ' "e- , ·- - .. ---¾ . ' > r )> '-;-... > ,,> "'- > 200 400 600 800 100 ms 200 400 600 800 100 ms Depth (m) 100 300 500 - ,_ à' E ;>-, " - , ___ D ... "_ --. - -.. 9Si; :-.., ,,,, " qye>s 700 900 1 100 1300 I l)J1 --â...,- ""'- ,,_ reflected qP-waves ' "e- , ·- - .. ---¾ . ' > r )> '-;-... > ,,> "'- > 200 400 600 800 100 ms Fig. 4. The X-X component VSP with 100 m source-to-well offset. The qSV­ waves are weak and can only be observed in the shallow traces. The reflected qP-waves are the qP-waves reflected from the face opposite to that the receivers located. The solid line is the theoretical traveltimes of the qSV-waves and the dashed line is the traveltimes for the SV-waves propagation using an isotropic velocity. Fig. 4. The X-X component VSP with 100 m source-to-well offset. The qSV­ waves are weak and can only be observed in the shallow traces. The reflected qP-waves are the qP-waves reflected from the face opposite to that the receivers located. The solid line is the theoretical traveltimes of the qSV-waves and the dashed line is the traveltimes for the SV-waves propagation using an isotropic velocity. 690 TAO, Vol. 12, No. 4, December 2001 140 240 340 440 540 ms Depth (m) 100 300 500 - B qPß '"" Wq"Yes ' C- -- '. - 700 c - ; , - ,c,, '--;:-c 900 1100 1300 c 140 240 ;:-.... ""'» c' JC )< 340 .. " • - ........ . , •' ¼ "'>c ) c ..... 440 > '• > tf:i:_ a'les 540 ms Fig. 5. The Z-Z component VSP with 500 m source-to-well offset. The domi­ nant events in the profile are the qP-waves. The reflected qP-waves are the qP-waves reflected from the bottom of the model. The solid line is the theoretical traveltimes of the qP-waves and the dashed line is the traveltimes for the P-waves propagation using an isotropic velocity. 140 240 340 440 540 ms Depth (m) 100 300 500 - B qPß '"" Wq"Yes ' C- -- '. 4. DISCUSSION AND CONCLUSIONS - 700 c - ; , - ,c,, '--;:-c 900 1100 1300 c 140 240 ;:-.... ""'» c' JC )< 340 .. " • - ........ . , •' ¼ "'>c ) c ..... 440 > '• > tf:i:_ a'les 540 ms Fig. 5. The Z-Z component VSP with 500 m source-to-well offset. The domi­ nant events in the profile are the qP-waves. The reflected qP-waves are the qP-waves reflected from the bottom of the model. The solid line is the theoretical traveltimes of the qP-waves and the dashed line is the traveltimes for the P-waves propagation using an isotropic velocity. Depth (m) 100 300 ms 500 700 I I I qP½waves 900 1100 1300 340 540 740 940 ms Fig. 6. The Y-Y component VSP with 500 m source-to-well offset. The domi­ nant events in the profile are the qSH-waves. The solid line is the theo­ retical traveltimes of the qSH-waves and the dashed line is the traveltimes for the SH-waves propagation using an isotropic velocity. Depth (m) 100 300 ms 500 700 I I I qP½waves 900 1100 1300 340 540 740 940 ms Fig. 6. The Y-Y component VSP with 500 m source-to-well offset. The domi­ nant events in the profile are the qSH-waves. The solid line is the theo­ retical traveltimes of the qSH-waves and the dashed line is the traveltimes for the SH-waves propagation using an isotropic velocity. Young-Fa Chang & Chih-Hsiung Chang 691 340 540 740 940 ms Depth (m) 100 300 500 700 900 1100 1300 -. - . t·- , ' ¹ q-L.I, I t , es " <; ,_.....__,.. reflected qP 1 1 "'-... ... ºwaves  ....... ¸ A·--. ' ''- ."- '-"' - "" ' .lI -rt ::-- .---. ''- • r- . . ' '- :-, 340 540 740 940 ms Fig. 7. The X-X component YSP with 500 m source-to-well offset. The qSY­ waves are too obscure to be observed. The reflected qP-waves are the qP-waves reflected from the face opposite to that the receivers located. The solid line is the theoretical traveltimes of the qSY-waves and the dashed line is the traveltimes for the SY-waves propagation using an isotropic velocity. 340 540 740 940 ms Depth (m) 100 300 500 700 900 1100 1300 -. - . t·- , ' ¹ q-L.I, I t , es " <; ,_.....__,.. reflected qP 1 1 "'-... ... 4. DISCUSSION AND CONCLUSIONS ºwaves  ....... ¸ A·--. ' ''- ."- '-"' - "" ' .lI -rt ::-- .---. ''- • r- . . ' '- :-, 340 540 740 940 ms Fig. 7. The X-X component YSP with 500 m source-to-well offset. The qSY­ waves are too obscure to be observed. The reflected qP-waves are the qP-waves reflected from the face opposite to that the receivers located. The solid line is the theoretical traveltimes of the qSY-waves and the dashed line is the traveltimes for the SY-waves propagation using an isotropic velocity. and 5). The qSH-waves are irreverent to qP- and qSY-waves. Hence, qSH-waves can be seen in Figs. 3 and 6; however, they are blurrier than qP-wave YSPs, and the small amplitude and high frequency converted qP-waves arrive before the qSH-waves. Although the same trans­ ducers are used for measuring the qSH- and qSY-waves, with a 90 degree difference in polarization, the qSY-waves show an obstacle in the recorded data (Figs. 4 and 7). The qSY­ waves are generally noisier than the qSH-waves in the ultrasonic transmission experiments. This is because the attenuation of medium for the qSY-wave is stronger than the qSH-wave, and the qSY-wave is somewhat like to convert to qP-wave. Although the qSY-waves are obscure and are contaminated by the direct qP-waves and converted qP-waves, they still can be recognized in the shallow traces of Fig. 4; but in Fig. 7 they are too weak to be observed. In Figs. 2-6, the good agreement between the theoretical (solid lines) and observed traveltimes confirms that the events we observed are the quasi body waves propagating with the group velocity. The isotropic arrivals advanced those of the anisotropic arrivals (in Figs. 2-3 and 5-6) is because that the isotropic velocities (P- and S-waves) used in this study are the maximum velocities of the qP- and qSH-waves. But the isotropic velocity of the SY-waves is not the maximum velocity of the qSY-wave (Figs. 4 and 7). and 5). The qSH-waves are irreverent to qP- and qSY-waves. Hence, qSH-waves can be seen in Figs. 3 and 6; however, they are blurrier than qP-wave YSPs, and the small amplitude and high frequency converted qP-waves arrive before the qSH-waves. Although the same trans­ ducers are used for measuring the qSH- and qSY-waves, with a 90 degree difference in polarization, the qSY-waves show an obstacle in the recorded data (Figs. 4 and 7). 4. DISCUSSION AND CONCLUSIONS The qSY­ waves are generally noisier than the qSH-waves in the ultrasonic transmission experiments. This is because the attenuation of medium for the qSY-wave is stronger than the qSH-wave, and the qSY-wave is somewhat like to convert to qP-wave. Although the qSY-waves are obscure and are contaminated by the direct qP-waves and converted qP-waves, they still can be recognized in the shallow traces of Fig. 4; but in Fig. 7 they are too weak to be observed. In Figs. 2-6, the good agreement between the theoretical (solid lines) and observed traveltimes confirms that the events we observed are the quasi body waves propagating with the group velocity. The isotropic arrivals advanced those of the anisotropic arrivals (in Figs. 2-3 and 5-6) is because that the isotropic velocities (P- and S-waves) used in this study are the maximum velocities of the qP- and qSH-waves. But the isotropic velocity of the SY-waves is not the maximum velocity of the qSY-wave (Figs. 4 and 7). 0: When the source,to-well offset is small (offset/depth :0:: 0.2 in our work), it can be seen that the curves of the predicted arrivals of the quasi body waves in our modeling HTI fit the ones that were computed using the isotropic velocities (Figs. 2-4), especially for depths greater 692 TAO, Vol. 12, No. 4, December 2001 than 500 m. The departure between these curves becomes smaller as the receiver runs deeper. As the source-to-well offset is increased (offset/depthö 0.35 in this study) (Figs. 5-7) or the depth of the small offset VSPs is reduced (Figs. 2-4 ), deviation between the curves of arrivals that are respectively computed from the elastic constant and isotropic velocities is also increased. The conclusion thus can be drawn that the idea of isotropic velocity can be confidently applied to the processing of VSP data .when the offset of the source to the well is small relative to the depth of the receiver. For a relatively large offset, the effects of anisotropy must be taken into account in the processing of VSP data. Otherwise, the VSP data can be erroneously processed and interpreted. Acknowledgment This research was financially supported by the National Science Council under the contract no. NSC 89-2116-M-194-004. Acknowledgment This research was financially supported by the National Science Council under the contract no. NSC 89-2116-M-194-004. B., and L.A. Thomsen, 1990: Reflection shear-wave data collected near the princi- REFERENCES Brown, R. J., D. C. Lawton, and S. P. Cheadle, 1991: Scaled physical modelling of anisotro­ pic wave propagation: Multioffset profiles over an orthorhombic medium. Geophys. J. Int., 107, 693-702. Bush, I., and S. Crampin, 1991: Paris Basin VSPs: case history establishing combinations of fine-layer (or lithologic) anisotropy and crack anisotropy from modelling shear wavefields near point singularities. Geophys. J. Int., 107, 433-477. Chang, C. H., G. H.F. Gardner, and J. A. McDonald, 1994: A physical model of shear-wave propagation in a transversely isotropic solid. Geophysics, 59, 484-487. Chang, C. H., and G. H.F. Gardner, 1997: Effects of vertically aligned subsurface fractures on seismic reflections: A physical model study. Geophysics, 62, 245-252. Chang, Y. F., and C. H. Chang, 2001a: Laboratory results for the features of body wave propagation in a transversely isotropic medium. Geophysics, in press. Cheadle, S. P., R. J. Brown, and D. C. Lawton, 1991: Orthorhombic anisotropy: A physical seismic modeling study. Geophysics, 56, 1603-1613. Crampin, S., 1981: A review of wave motion in anisotropic and cracked elastic-media. Wave motion, 3, 343-391. Crampin, S., and J. H. Lovell, 1991: A decade of shear-wave splitting in the earth's crust: what does it mean? what use can we make of it? and what should we do next ? Geophys. J. Int., 107, 387-407. Ebrom, D. A., R.H. Tatham, K. K. Sekharan, J. A. McDonald, and G. H.F. Gardner, 1990: Hyperbolic traveltime analysis of first arrivals in an azimuthally anisotropic medium: A physical modeling study. Geophysics, 55, 185-191. Helbig, K., 1993: Simultaneous observation of seismic waves of different polarization indi­ cates subsurface anisotropy and might help to unravel its cause. J. Applied Geophysics, 30, 1-24. Li, Y. G., T. L. Teng, and T. L. Henyey, 1994: Shear-wave splitting observations in the north­ ern Los Angles basin, southern California. Bull. Seism. Soc. Am., 84, 307-323. Lynn, H.B., and L.A. Thomsen, 1990: Reflection shear-wave data collected near the princi- Young-Fa Chang & Chih-Hsiung Chang pal axes of azimuthal anisotropy. Geophysics, 55, 147-156. MacBeth, C., X. Y. Li, X. Zeng, D. Cox, and J. Queen, 1997: Processing of a nine-component near-offset VSP for seismic anisotropy. Geophysics, 62, 676-689. Young-Fa Chang & Chih-Hsiung Chang 693 MacBeth, C., X. Y. Li, X. Zeng, D. Cox, and J. Queen, 1997: Processing of a nine-component near-offset VSP for seismic anisotropy. Geophysics, 62, 676-689. MacBeth, C., X. Y. Li, X. Zeng, D. Cox, and J. Queen, 1997: Processing of a nine-component near-offset VSP for seismic anisotropy. Geophysics, 62, 676-689. MacBeth, C., X. Y. Li, X. Zeng, D. Cox, and J. Queen, 1997: Processing of a nine-component near-offset VSP for seismic anisotropy. Geophysics, 62, 676-689. Sayers, C. M., 1997: Determination of anisotropic velocity models from walkaway VSP data acquired in the presence of dip. Geophysics, 62, 723-729. Sayers, C. M., 1997: Determination of anisotropic velocity models from walkaway VSP data acquired in the presence of dip. Geophysics, 62, 723-729. Sayers, C. M., 1997: Determination of anisotropic velocity models from walkaway VSP data acquired in the presence of dip. Geophysics, 62, 723-729. Thomsen, L., 1986: Weak elastic anisotropy. Geophysics, 51, 1954-1966. Thomsen, L., 1986: Weak elastic anisotropy. Geophysics, 51, 1954-1966. Winterstein, D. F., and B. N. P. Paulsson, 1990: Velocity anisotropy in shale determined from crosshole seismic and vertical seismic profile data. Geophysics, 35, 470-479. Winterstein, D. F., and B. N. P. Paulsson, 1990: Velocity anisotropy in shale determined from crosshole seismic and vertical seismic profile data. Geophysics, 35, 470-479.
https://openalex.org/W2070752776
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English
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On the Existence of Rudimentary Antlers in the Okapi
Proceedings of the Zoological Society of London
1,907
public-domain
3,134
* For explanation of the Plates, see p. 134. 26 PROF E RAY LA 26 PROF E RAY LA eb. 5, 126 RAY LANKESTER ON 3. On the Hxistence of Rudimentary Antlers in the Okapi. By E. Ray Lanxester, M.A., D.Sc., LL.D., F.B.S., F.Z.8., Director of the British Museum (Natural His- tory). | | (Plates VI. & VII.* and Text-figures 49-55.) We know a great deal more as to the horns of the Okapi than was the case when I communicated my description of that animal to the Society in 1901, and founded the genus Okapia. The two skulls sent home by Sir Harry Johnston—the first seen in Europe—were hornless, and it was at first a matter of doubt as to whether the Okapi wasa hornless Giraffid, or whether the male possessed horns whilst these two skulls were the one immature and the other that of a hornless female. During the printing of my memoir additional specimens were received in Brussels, and were transmitted to Dr. Forsyth Major in London for study and description. I saw in Dr. Forsyth Major’s possession a fine adult Okapi skull which had a pair of well-developed bony cones rising each by a broad base from the frontals, of which they appeared to form part. No suture was visible. An outline of this skull was published in my memoir by kind permission of Dr. Forsyth Major. I also was able to examine and to mention the existence of a curious structure discovered by that gentleman in regard to these ossicusps ; and I described it in the following terms :—‘“‘ The fine bony cones three inches long, which have made their appearance in the Text-fig. 49. Text-fig. 49. Drawing of a fore and aft section through the tip of the ossicone of an adult Okapi in the collection of the Museum of the Congo Independent State. The section and drawing were made by Dr. Forsyth Major. Th ti h th t ti f t fi f th f i t th The section shows the penetration of transverse fissures from the surface into the interior of the horn-tip. p a, dense ivory-like bone; 6, posteriorly-placed transverse fissure; c, more anterior transverse fissure (marking off and presumably about to cut off and detach an anterior seement or plate of bone as a rudimentary “ antler ”) Brussels skull, show no suture at their base, nor any indication of origin as separate cap-like structures. For all that one can see they may be direct outgrowths of the frontal bone itself. Curiously ici LON eae anvils Left. | Right. Left. Right. H.Grénvold dei London Stereoscopic Co PLOT N= ene S Ol Oi i Left. Right. Right. Right. H.Grénvold 4 Co imp- 4 Co imp- ‘0 the uncertainty which exists as to the origin of skin and skull which »s sent home from Africa as belonging to one individual, whereas they in certain cases belong to distinct individuals, it is still doubtful as to wuetner tne female Okapi has or has not in the adult condition a small knob-like protuberance of the integument, separable from the subjacent bone and representing the horn of the male. PLOT N= ene S Ol Oi i eas LOO, see valle G.M Woodward del. London Stereoscopic Co imp. G.M Woodwa 1907. RUDIMENTARY ANTLERS IN THE 127 enough, the point and posterior margin of the bony cone are polished as though it had protruded through the skin like a cervine antler. The point is separated by a suture from the rest of the ‘ ossicone,’ forming a small terminal cap of bone a third of an inch in depth. This curious structure, as well as a possible second suture a little lower down the ossicone, was pointed out to me by Dr. Forsyth Major. These appearances will be figured in that gentleman’s memoir on the Brussels’ specimens.” This is the first and so far the only published notice of the antler-like tips of the Okapi’s horns. The figure prepared by Dr. Major of the section made by him through the end of this Okapi’s ossicone is reproduced in the text-figure here appended (text-fig. 49). Dr. Major does not himself propose to publish anything further at present on the Okapi, and the little drawing has been placed in my hands by him. A tracing of it was also kindly sent to me by M. Fraipont, of Liége. The further history of our knowledge of the horns of the Okapi has been complicated by the arrival in Europe of various specimens, concerning the sex of which either erroneous informa- tion or none at all has been given by the natives from whom the specimens were obtained. Thus Dr. Forsyth Major was led to suppose that the female Okapi has a small unattached ossicone, some two inches in length, when adult, but he subsequently came to the conclusion that this supposed female was in reality a young male. In ‘ La Belgique Coloniale,’ No. 21, May 1902, Dr. Forsyth Major wrote :—“ L’Okapi posséde deux cornes frontales, recouvertes dune peau velue, plus petites, de forme conique et presque verti- cales chez la femelle; plus grandes, dirigées obliquement en arriére et en peu triangulaires chez le male.” subsequent date Dr. Forsyth Major came to the conclu- t the specimen supposed to be a female possessing small sicones, was in reality a young male (Proc. Zool. Soe. ». * One of these broad-skulled specimens has, however, been found to possess a pair of completely detached bony ossicones of minute size embedded in the integument. The specimen is a little older (as indicated by the dentition) than Sir Harry John- ston’s larger individual (that mounted in the British Museum), but is not quite adult. It belongs to Messrs. Rowland Ward. I give here figures of the minute ossicone (text-figs. 50 & 51). PLOT N= ene S Ol Oi i 339), and that the female Okapi is hornless, whilst the ie possesses “horns” which make their appearance as conical structures, ossifying independently of the sub- nes (as in the Giraffe) and becoming firmly ankylosed mtal bone in the adult—a boss-like upgrowth of which 0 the structure of the complete horn. s little room for doubt that this is the true account of r, though we still are in want of full information as to icters of the adult female Okapi*. In a subsequent vation I shall be able to give more precisely the characters » types of skull, supposed to be that of the horn-bearing the hornless female, respectively. The skulls carrying sed or merely loosely-attached bony cone on the frontals At a subsequent date Dr. Forsyth Major came to the conclu- on that the specimen supposed to be a female possessing small * ossicones, was in reality a young male (Proc. Zool. Soe. ». 339), and that the female Okapi is hornless, whilst the ie possesses “horns” which make their appearance as conical structures, ossifying independently of the sub- nes (as in the Giraffe) and becoming firmly ankylosed mtal bone in the adult—a boss-like upgrowth of which 0 the structure of the complete horn. s little room for doubt that this is the true account of r, though we still are in want of full information as to icters of the adult female Okapi*. In a subsequent vation I shall be able to give more precisely the characters » types of skull, supposed to be that of the horn-bearing the hornless female, respectively. The skulls carrying sed or merely loosely-attached bony cone on the frontals 128 b. 5, RAY LANKESTER ON (supposed to be those of males) are longer and narrower than the equally large or larger skulls devoid of any bony cones in connec- tion with the frontals (supposed to be those of females) *. What- ever opinion is held, or whatever decision may be ultimately arrived at in regard to these two types of skull, it is the fact that they are very distinct from one another and that all the Okapi skulls which I have examined can be definitely assigned to one or the Text-fig. 50. a. Text-fig. 50. PLOT N= ene S Ol Oi i Rudimentary free ossicone of hemispherical shape from the skin overlying the frontal bossed region of the skull of an Okapi of the broad-skulled type—sub-adult (deciduous molars very much worn, premolars not yet visible ; third lower molar in use on both sides, fifth cusp shows slight wear). a, natural size; 6, enlarged. Text-fig. 51. Section of the ossicusp drawn in text-fig. 50, to show the incomplete ossification. Section of the ossicusp drawn in text-fig. 50, to show the incomplete ossification. other of these two types. There is no third form known. The two types may perhaps be best distinguished as O. johrestoni (the name I gave to the broad hornless sub-adult skull accompanying 1907. RUDIMENTARY ANTLERS IN THE 129 the skin sent home by Sir Harry Johnston) and 0. liebrechtsi, the name given by Dr. Forsyth Major to the more elongate and narrow type of skull, which is that usually provided with bony cones attached to or ankylosed with the frontal bones. It is important to note that Dr. Major figures a skull (Proc. Zool. Soe. loc. cit. p. 423) which is hornless and is regarded by him as that of a female of the elongate type, O. liebrechtsi. I hope shortly to publish some measurements and outlines of these two types of skull. I have examined three of the UO. johnstoni-type, and five of the O. liebrechtsi-type. Though there is considerable variation in the number and breadth of the white stripes on the fore and hind limbs of the skins of Okapi received in this country (including the excellent specimens obtained independently by Major Powell Cotton and by Captain Boyd Alexander from widely separated localities, the former from the Ituri Forest, the latter from the Welle River), I have seen no evidence that a different striping of the skin is associated with the difference of skull-form. On the contrary, there is positive evidence that the striping of the skin is very nearly identical (though no two specimens are exactly alike) in animals which possessed the liebrechtsi form of skull with that exhibited by the mounted specimen (0. johnstoni) with hornless skull, sent home by Sir Harry Johnston, figured by me, and now in the British Museum. Nevertheless, it is true that direct and convincing evidence is as yet wanting for the conclusion that O. liebrechtsi is merely the male of O. johnstoni. y j When I had an opportunity (in 1904) of examining the fine skin of the adult (supposed) male Okapi, presented by the Congo State to the Museum of Paris, which is set up in the publie gallery there, I was especially anxious to note the state of the horn-tips. Proc. Zoou. Soc.—1907, * Since reading this paper, I have been kindly permitted by the authorities of the Royal Scottish Museum to examine the horns of one of the specimens above referred to by making a section of the tip of the horn. The piece cut out has been drawn and then carefully replaced and cemented in position, so that no injury is done to the specimen. The skull lent to me by the Royal Scottish Museum is that of which I had already drawn the horn-tips in figs. 1 to 8, Plate VI., before it had passed from the possession of Messrs. Rowland Ward & Co. The sections drawn on an enlarged scale in text-figs. 52 & 53 explain themselves. It is seen that the grooves or fissures visible on the surface do not extend very deeply, but that there is evidence of resorptive activity in the form of certain branching canal-like structures lying deeply within the bony matter, which have probably been excavated by resorptive ingrowths from the soft surface tissues. Text-fig. 51. I found that they were represented in the mounted specimen and were seen projecting through the skin which clothed the ‘‘ossicone” up to a limit of about half-an-inch from the tip. From this level the dense bony matter was naked. It showed in each horn two fine transverse grooves, as 10 the ossicone examined and sliced by Dr. Forsyth Major. This went far to prove that the condition noted by him was not exceptional or morbid, and accordingly I have examined the ossicones of other specimens of adult male Okapis, as opportunity occurred. Several skins and imperfect skeletons have been received in London by dealers in zoological specimens, and I am especially indebted to Messrs. Rowland Ward & Co. for the opportunity of examining the ossicones of four adult Okapis. Of two of these individuals I have had the ossicones drawn (PI. VI.) so as to show the free termination from different points of view. The two other specimens examined by me presented the same remarkable appearances as those figured, and as shown by the Paris Okapi, but I was unable to procure carefully drawn figures of them. Thus, including the Brussels skull examined by Dr. Forsyth Major, I have ascertained the existence of these transverse grooves or fissures in six adult male Okapis. I have 9 Zoou. Soc.—1907, No. IX. 9 b. 5, 130 RAY LANKESTER ON also evidence of their existence in a plaster cast of another specimen which passed through the hands of Messrs. Rowland Ward & Co. An examination of the figures given in Plate VI. shows that in all four ossicones (the right and left of two adult male Okapis) the free terminal region is smooth and polished, forming a cap of about half an inch in length, whilst this region is followed by a rougher substance, furrowed on the surface. The polished region projects beyond the skin, the rougher region is clothed by the living integument. In all there are very deep horizontal fissures in the polished material of the “cap.” These fissures are some- what irregular in form, and it is impossible without making a section (which I had not permission to do)* through the solid material to ascertain their depth. They are of the same nature as those shown in the text-figure in section (text-figure 49, p. 126). Text-fig. 51. I think there can be little doubt that these transverse fissures are caused by the ingrowth of the living tissue after the pro- trusion of the dense polished cap, so as to cut off the protruding portion and provide for its breaking off—Just as an antler is cut off and prepared for disruption in the Cervidee. A small conical piece is thus thrown off from the end of the horn or ossicusp, and may be regarded as a rudimentary or minute “antler.” But the process of discarding these minute points or antlers in the Okapi appears to differ from what occurs in the Cervide, not only in the minute size of the discarded segments, but in the fact that the preparation for the breaking off a second (and even a third) segment takes place before the first piece has been got rid of. The living tissue having absorbed the bony matter by a horizontal ingrowth and having created a transverse break in the continuity of the osseous substance (see text-figure), recedes for a distance of a sixth of an inch or less, and then again penetrates inwards, forming a new horizon of disruption; and from the appearance of the specimens figured in Plate VI., especially figs. 4 and 5, it seems that this process of the recession of the living investment of the horn-tip and the subsequent ingrowth of the living tissue, may be again repeated before the most anterior piece is broken off, so that these horizontal fissures are visible on the surface of the horn-tip, following one another somewhat irregularly. 1907. 131 RUDIMENTARY ANTLERS IN THE Text-fig. 52. Post. A. B. Text-fig. 52. Text-fig. 52. Text-fig. 52. Post B. A. Lankester, E. Ray. 1907. "On the Existence of Rudimentary Antlers in the Okapi." Proceedings of the Zoological Society of London 1907, 126–135. https://doi.org/10.1111/j.1096-3642.1907.tb01804.x. View This Item Online: https://www.biodiversitylibrary.org/item/97670 DOI: https://doi.org/10.1111/j.1096-3642.1907.tb01804.x Permalink: https://www.biodiversitylibrary.org/partpdf/72470 A. The diagram C shows the direction im which certain cuts have been made in the left osseous horn (ossicone or ossicusp) of the Edinburgh Okapi (also illustrated in P). VI. figs. 1 to 8). The diagram C shows the direction im which certain cuts have been made in the left osseous horn (ossicone or ossicusp) of the Edinburgh Okapi (also illustrated in P). VI. figs. 1 to 8). ) g ) a, anterior border; b, posterior border.’ L, left side; R, right side. d i h i h l i f h f f h bi ) g ) a, anterior border; b, posterior border.’ L, left side; R, right side. A i d i h i h l i f h f f h bi p g A isa drawing three times the natural size of the cut surface of the bisected horn, the bisection being effected in a plane erected on the line ab of the diagram C. It shows the eating in of the transverse fissures into the dense bony substance, and a number of irregular spaces and fissures which are probably cavities due to re-sorption of the bone. B is a drawing of the other half of the same bisected horn-tip. A isa drawing three times the natural size of the cut surface of the bisected horn, the bisection being effected in a plane erected on the line ab of the diagram C. It shows the eating in of the transverse fissures into the dense bony substance, and a number of irregular spaces and fissures which are probably cavities due to re-sorption of the bone. B is a drawing of the other half of the same bisected horn-tip. Text-fig. 53. Drawing of three times the natural size of the surface of a section obtained by cutting half of the same bisected horn-tip through a plane erected on the line R of the diagram, separating the shaded from the unshaded area. The penetration of the transverse fissures is shown. The cavities m, ”, 0 correspond to the transverse fissures labelled m, n, 0 in fig. 5 of Plate VI., representing the same specimen before it was divided. A9? A9? This file was generated 8 August 2024 at 17:38 UTC Holding Institution Smithsonian Libraries and Archives Copyright & Reuse Copyright & Reuse Copyright Status: Public domain. The BHL considers that this work is no longer under copyright protection. py g Copyright Status: Public domain. The BHL considers that this work is no longer under copyright protection. This document was created from content at the Biodiversity Heritage Library, the world's largest open access digital library for biodiversity literature and archives. 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Erratum in Jayapalan &amp; Natarajan, The role of CDK5 and GSK3B kinases in hyperphosphorylation of microtubule associated protein tau (MAPT) in Alzheimer’s disease
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open access www.bioinformation.net Erratum Volume 10(8) Erratum: A numerical error in methodology section under Sequence retrieval and modeling side heading Bioinformation - 2013 Dec 9(20): 1023-1030 The role of CDK5 and GSK3B kinases in hyperphosphorylation of microtubule associated protein tau (MAPT) in Alzheimer’s disease Saranya Jayapalan & Jeyakumar Natarajan* Data Mining and Text Mining Laboratory, Department of Bioinformatics, Bharathiar University, Coimbatore-641046, India Erratum in: Bioinformation - 2014 Aug 30 10(8): 555 [Numerical error corrected - On page 1024 (Methodology section: Sequence retrieval and modeling), in second column, in line3 and line18 the entry for length of MAPT sequence currently read as “358” It should read “352”] ISSN 0973-2063 (online) 0973-8894 (print) Bioinformation 10(8): 555 (2014) 555 © 2014 Biomedical Informatics
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Unitarity of the box diagram
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Published for SISSA by Springer Received: September 26, 2018 Accepted: November 8, 2018 Published: November 14, 2018 Roji Piusa and Ashoke Senb a Perimeter Institute for Theoretical Physics, Waterloo, ON N2L 2Y5, Canada b Harish-Chandra Research Institute, HBNI, Chhatnag Road, Jhusi, Allahabad 211019, India E-mail: rpius@perimeterinstitute.ca, sen@hri.res.in Abstract: The complete proof of cutting rules needed for proving perturbative unitarity of quantum field theories usually employs the largest time equation or old fashioned perturbation theory. None of these can be generalized to string field theory that has non-local vertices. In arXiv:1604.01783 we gave a proof of cutting rules in string field theory, which also provides an alternative proof of cutting rules in ordinary quantum field theories. In this note we illustrate how this works for the box diagram of φ4 field theory, avoiding the contributions from anomalous thresholds. Keywords: Scattering Amplitudes, String Field Theory ArXiv ePrint: 1805.00984 Open Access, c The Authors. Article funded by SCOAP3 . https://doi.org/10.1007/JHEP11(2018)094 JHEP11(2018)094 Unitarity of the box diagram Contents 1 Introduction and summary 1 2 The issues 2 3 Unitarity of the box diagram 3 1 14 Introduction and summary Cutkosky’s diagrammatic analysis shows that the discontinuities of a Feynman diagram across the ‘normal threshold’ singularities produce the result needed for unitarity of the S-matrix [1, 2]. However typically a Feynman diagram possesses many other Landau singularities e.g. anomalous thresholds at one loop [3, 4] and more complicated singularities at higher loop, and there are discontinuities associated with these singularities as well. A recent discussion on these may be found in [5, 6]. For this reason the standard approach to proving the cutting rules needed for unitarity makes use of indirect methods e.g. the largest time equation [7, 8] or old fashioned perturbation theory [9] based on time ordered diagrams. A recent analysis along similar line, suitable for dealing with vertices with finite number of time derivatives, can be found in [10]. Unfortunately these approaches are not suitable for proving the cutting rules for the Feynman diagrams arising in string field theory, since the vertices are non-local, not only in space but also in time, involving exponentials of quadratic functions of momenta. For this reason in [11] we developed a different approach to proving the cutting rules in such theories based on direct analysis of Feynman diagrams.1 Although originally developed for string field theory, this approach also gives an alternative, diagrammatic proof of cutting rules in ordinary quantum field theories. Another approach to proving unitarity in local theories by directly dealing with momentum space Feynman diagrams was suggested in [13]. We suspect that this approach is closely related to the one described in [11], but the precise relation is not clear at present. Since [11] gave an iterative proof of the cutting rules to all orders in perturbation theory, the analysis was necessarily somewhat abstract. In this paper we complement the analysis of [11] by showing how the method works for establishing the cutting rules for the box diagram that appears in the computation of one loop eight point amplitude in φ4 field theory. We emphasize however that the purpose of this note is not to prove unitarity of the 1 There have been two recent papers [6, 12] on unitarity of non-local field theories of the kind that arise in string field theory, but both seem to only focus on the analysis of discontinuity across normal thresholds. –1– JHEP11(2018)094 4 Unitarity of tree diagrams 2 The issues In this section we shall briefly discuss the issues that plague the proof of unitarity directly in momentum space. The singularities of a Feynman diagram are associated with Landau singularities where the integrand has poles due to certain number of internal propagators going on-shell and furthermore the integration contour over loop momenta are pinched, i.e. it is not possible to move away from the poles by deforming the integration contour in the complex loop momentum plane. At such singularities, the integral typically has a branch cut, leading to a discontinuity of the amplitude across the branch cut. In [1] Cutkosky gave a general formula for computing the discontinuity across a given threshold singularity. If a certain number of propagators go on-shell at a singularity, then the discontinuity in the amplitude from the corresponding branch cut is computed by replacing the i/(−k 2 −m2 +i) factor in each of these propagators by 2πδ(−k 2 − m2 ). Let us consider a singularity where the on-shell internal propagators are such that together they can be interpreted as an intermediate state in the original amplitude, e.g. if the set consists of the propagators carrying momenta ` and pA + pB − ` in figure 1. (We are assuming that the incoming particles come from the left and the outgoing particles move to the right.) Branch points associated with such singularities are known as normal thresholds. In this case the discontinuity computed from Cutkosky’s formula can be regarded as a product of two on-shell amplitudes, integrated over the phase space of the intermediate states. Therefore this looks similar to a contribution to −i T † T = −i T † |nihn|T that is needed for unitarity of the T-matrix — related to the S-matrix via S = 1 − i T . Such contributions are usually represented as cut diagrams, where the cut is a single line that divides the diagram into a left half and a right half, with the cut propagators representing the on-shell propagators. While this goes a long way towards proving unitarity of the theory, there are some missing ingradients: 1. A given Feynman diagram may have singularities other than normal threshold, e.g. if the propagators carrying momenta `, ` + pC and pA − ` in figure 1 were on-shell at the singularity. Such singularities are known as anomalous thresholds. Cutkosky’s –2– JHEP11(2018)094 box diagram for which there are many derivations. The goal is to illustrate how the iterative all order diagrammatic proof of the cutting rules given in [11] works for the box diagram. The rest of the paper is organized as follows. In section 2 we discuss some general issues that arise while trying to prove unitarity of amplitudes written as momentum space integrals. In section 3, which is the main body of the paper, we show how the method developed in [11] is used to prove cutting rules for the box diagram of φ4 field theory. This proof assumes the validity of cutting rules for connected and disconnected tree diagrams. For completeness, in section 4 we give a proof of cutting rules for tree diagrams, again by making use of the general method described in [11]. We shall use mostly plus signature for the space-time metric and denote a D dimensional momentum as (`0 , ~`) where `0 denote the time component and ~` denotes the spatial components. formula can still be used to compute the discontinuity across such branch points; however in this case the on-shell states cannot collectively be regarded as an intermediate state in the sum −i T † |nihn|T . 2. In computing −i T † T we need to reverse the signs of i in the propagators of the amplitude to the right of the cut so that it represents a matrix element of T † . This does not follow from Cutkosky’s formula for discontinuity. 3 Unitarity of the box diagram R Usually in φ4 theory with interaction term −(λ/4!) dD x φ4 one takes the propagator of momentum k to be −i/(k 2 + m2 − i) and the vertex to be −iλ/4!. Furthermore in the computation of the T-matrix we have an overall factor of i. However, as in [11], we shall use a slightly different but equivalent convention where for computation of the T-matrix we use the following rules: 1. The propagator of momentum k is given by 1/(−k 2 − m2 + i). 2. The vertex is given by λ/4!. 3. For each loop integral we have a factor of i. 4. If the diagram has nc disconnected components then we have a factor of i1−nc . P We shall drop the overall momentum conserving delta function (2π)D δ (D) ( i pi ), associated with each connected component, from the expressions for the amplitudes. The –3– JHEP11(2018)094 While for any specific graph one can do a more detailed analysis taking into account all these effects, the general proof of unitarity based on this approach becomes cumbersome. These problems were overcome in [7, 8] where a different proof of unitarity was given based on the ‘largest time equation’. An alternative proof was given in [9] based on the old fashioned perturbation theory. However unlike Cutkosky’s original analysis, which did not depend on the detailed structure of the vertices as long as they do not introduce additional singularities at finite momentum, the analysis of [7–9] requires working in coordinate space where at least for the time coordinate the propagator and vertices are expressed in the position space instead of the momentum space. Unfortunately for string field theory, for which the vertices are exponentials of quadratic functions of momenta, there is no convenient representation of the vertices in the coordinate space. Therefore the analysis of [7–9] do not apply. This difficulty was overcome in [11] that expressed T − T † as a sum over cuts diagrams by working directly in momentum space. Furthermore the part of the contribution to the right of the cut was shown to be hermitian conjugated, representing a contribution to T † . This method is well-suited for string field theory and other non-local theories, but also for ordinary quantum field theories with local vertices, giving an alternative proof of unitarity. In the next two sections we shall illustrate how this works for the box diagram and tree diagrams in φ4 theory. pB & pD . ↑ (pA − `) ↑ (` + pC ) `→ pC Figure 1. The box diagram of the eight point amplitude in φ4 theory. The external momenta pA , pB , pC , pD entering at the four vertices are taken to be positive if ingoing. space-time dimension D = d + 1 will be chosen such that the box diagram of figure 1 has no ulltra-violet divergence. This requires D ≤ 7. With this convention the contribution of the box diagram shown in figure 1 to the T-matrix is given by, Z i 4 dD ` I(pA , pB , pC , pD ) = λ {−`2 − m2 + i}−1 {−(` + pC )2 − m2 + i}−1 2 (2π)D {−(pA − `)2 − m2 + i}−1 {−(pA + pB − `)2 − m2 + i}−1 . (3.1) pA , pB , pC and pD denote net external momenta entering the vertices. In this convention an outgoing particle will have negative p0 . Since each of pA , pB , pC and pD receives contribution from two incoming or outgoing external states, they can be space-like or timelike and arbitrarily large in magnitude. Therefore all the singularities that can appear in the box diagram can be present here. In particular by taking pA to be a large time-like momentum we can mimick the case of a massive external particle above the threshold of production of a pair of φ particles — this is the situation in which the anomalous threshold is commonly discussed.2 Our goal will be to compute the quantity D ≡ I(pA , pB , pC , pD ) − I(−pA , −pB , −pC , −pD )∗ , (3.2) that represents a contribution to T − T † . We write down the expression for I(−pA , −pB , −pC , −pD )∗ by taking the complex conjugate of (3.1) and changing the signs of all the external momenta. Making a change of variables ` → −` in the resulting expression, 2 In fact we do not even need to assume in our analysis that the external momenta are on-shell. –4– JHEP11(2018)094 % pA (pA + pB − `) → we have Z i dD ` I(−pA , −pB , −pC , −pD )∗ = − λ4 {−`2 − m2 − i}−1 {−(` + pC )2 − m2 − i}−1 2 (2π)D {−(pA − `)2 − m2 − i}−1 {−(pA + pB − `)2 − m2 − i}−1 . (3.3) This gives i 4 λ 2 Z dd ` (2π)d Z  d`0 {−`2 − m2 + i}−1 {−(` + pC )2 − m2 + i}−1 2π {−(pA − `)2 − m2 + i}−1 {−(pA + pB − `)2 − m2 + i}−1 +{−`2 − m2 − i}−1 {−(` + pC )2 − m2 − i}−1 2 2 {−(pA − `) − m − i} −1 2 2 −1 {−(pA + pB − `) − m − i}  . (3.4) Unitarity of the S-matrix demands that T −T † must be equal to −i T † T . This translates to the cutting rules which tell us that D is given by the sum over all cuts of the box diagram, with the following rules for evaluating a cut diagram: 1. A cut must divide the diagram into the left half and the right half, with the convention that the incoming particles come from the left and the outgoing particles travel to the right. 2. A cut propagator corresponds to the replacement: P (k) ≡ −k 2 1 − m2 + i ⇒ Pc (k) ≡ −2 π i δ(−k 2 − m2 ) θ(k 0 ) , (3.5) where k denotes the momentum flowing along the propagator from the left side of the cut to the right side. The −i factor in the expression for Pc may seem unfamiliar, but in our convention this combines with the factor of i from loop integral to give the correct integration measure over the phase space. 3. The part of the amplitude to the right of the cut is replaced by its hermitian conjugate — involving complex conjugation and reversal of the signs of all external momenta. 4. Cut on an external line has no effect. 5. If a cut diagram has nL disconnected components on the left of the cut and nR disconnected components on the right of the cut, then it should be multiplied by an additional factor of (−1)nR −1 . This factor is needed to ensure that cutting rules lead to the unitarity relation T − T † = −i T † T [11]. We shall first prove that for fixed ~` in (3.4), the contribution to D from the `0 integral vanishes unless the integration contour is pinched between two singularities. 3 For this we 3 As in [11], we shall only allow deformations of loop energy integration contour into the complex plane, but keep the integration contours for spatial components of the loop momenta always along the real axes. –5– JHEP11(2018)094 D= D R = A − A∗ , (3.6) Z Z i 4 dd ` d`0 A= λ {−`2 − m2 + i}−1 {−(` + pC )2 − m2 + i}−1 d 2 2π (2π) R {−(pA − `)2 − m2 + i}−1 {−(pA + pB − `)2 − m2 + i}−1 , (3.7) where and A∗ is obtained from A by 1. replacing R by R0 , 2. reversing the signs of all the external momenta, and 3. complex conjugation. We shall prove that DR is given by the sum over cuts of the contributions to A. The full cutting rule is then obtained by adding the contributions from each small region R of this type. In this section we shall prove the cutting rule for DR assuming that it holds for all tree diagrams — including disconnected ones. This analysis will follow closely the one given in section 5.2.3 of [11] for one vertex irreducible diagrams. In the next section we shall describe the proof of cutting rules for connected and disconnected tree diagrams. Now for ~` ∈ R a certain number of propagators become nearly on-shell when `0 takes the value where its integration contour is nearly pinched. Since for small R, `0 lies within 4 For string field theory the `0 integration contour for both terms have their ends fixed at ±i∞ [11]. However a similar cancellation occurs for these contours as well. In this case bad behaviour in some directions at ∞ prevents us from deforming the contours to ∞, but the relevant contours can be deformed to each other. –6– JHEP11(2018)094 deform the `0 integral to ∞ in the lower half plane for the first term inside the square bracket in (3.4) and to ∞ in the upper half plane for the second term in the square bracket in (3.4), picking up residues from the poles that the contour passes through during the deformation. Since the poles of the first term are complex conjugates of the poles in the second term, we pick residues from exactly the same set of poles with i replaced by −i. Furthermore in the first term the poles are traversed in the clockwise direction while in the second term the poles are traversed in the anti-clockwise direction. As long as there are no nearby poles, we can set  = 0 while evaluating these residues. In this case their contributions exactly cancel. This argument breaks down if the contours are pinched since the residues diverge as  → 0, and we have to carefully take the limit to see if there is any left-over contribution after we combine the results of the two terms. 4 The pinch singularities occur when a pair of poles in the `0 plane approach the integration contour from the opposite sides. Therefore in the ~` space they occur on a subspace of codimension 1 or higher (if more than two poles approach the same point). We shall call this the pinched subspace. We shall focus on the integration over a small neighborhood R of the pinched subspace. R is a (D − 1) dimensional space, i.e. of codimension zero. We denote by R0 the image of R under ~` → −~`, and consider the quantity 3 pA − ` 4 ` 1 2 pA + pB − ` ` + pC a small range at the pinch, we can assign definite signs to the energies carried by each internal propagator at the pinch. We shall now associate with the region R a reduced diagram that is obtained from the original diagram by shrinking to points all propagators that are not nearly on-shell near the pinch. Furthermore we shall draw the nearly on-shell propagators such that energy flows from left to right near the pinch. For definiteness, and to consider a situation of maximal complexity, we shall consider a region R for which all four internal propagators are nearly on-shell at the pinch, and5 `0 > 0, `0 + p0C > 0, p0A − `0 > 0, p0A + p0B − `0 > 0 , (3.8) at the pinch. The corresponding reduced diagram is shown in figure 2. We have dropped the external legs from this diagram to avoid cluttering. We also number the propagators carrying momenta `, ` + pC , pA − ` and pA + pB − ` by 1, 2, 3 and 4 respectively. It is easy to see that the corresponding integral for A∗ , after making a change of integration variable `0 → −`0 , will be pinched at the same value of `0 . Let us denote by P (k) the propagator with momentum k: P (k) ≡ −k 2 1 1 1 p p = , 2 − m + i k 0 − ~k 2 + m2 + iε k 0 + ~k 2 + m2 − iε where ε is positive for positive . Therefore we can express (3.7) as Z Z i 4 dd ` d`0 A= λ P (`) P (` + pC ) P (pA − `) P (pA + pB − `) . d 2 2π R (2π) (3.9) (3.10) Since the pinch is assumed to occur at the positive values of `0 , `0 + p0C , p0A − `0 and p0A + p0B − `0 , the relevant poles of the propagators that take part in pinching the contour, are at q q 0 ` = ~`2 + m2 − iε, `0 = −p0C + (~` + p~C )2 + m2 − iε, q q `0 = p0A − (~ pA − ~`)2 + m2 + iε, `0 = p0A + p0B − (~ pA + p~B − ~`)2 + m2 + iε . (3.11) 5 A similar analysis can be carried out for all other reduced diagrams. –7– JHEP11(2018)094 Figure 2. The reduced diagram of figure 1 associated with R when at the pinch all propagators are nearly on-shell and the energies carried by the propagators lie in the range (3.8). The internal lines are drawn in a way so that at the pinch energy flows from the left to the right along each of the propagators. Since four propagators become on-shell at the pinch, the pinched subspace has comension 4, and R is a small neighborhood of this codimension 4 subspace. Note that at the pinch the poles from the propagators 1 and 2 are in the lower half `0 plane while the poles from the other propagators are in the upper half `0 plane. Therefore while deforming the `0 contour to the lower half plane, we shall pick up residues from the poles of the propagators 1 and 2 at the pinch. For this reason the set {1, 2} will play a special role in our analysis. We now define P 0 (k) ≡ 1 1 p p = P (k) − Pc (k) , k 0 − ~k 2 + m2 − iε k 0 + ~k 2 + m2 − iε (3.12) Pc (k) ≡ −2 π i δ(−k 2 − m2 ) θ(k 0 ) , (3.13) is the cut propagator. It follows from (3.12) that P (`)P (`+pC ) = P 0 (`)P 0 (`+pC )+Pc (`)P (`+pC )+P (`)Pc (`+pC )−Pc (`)Pc (`+pC ) . (3.14) Using this in (3.10) we can express A as b + A(1) + A(2) − A(12) , A=A (3.15) where b = i λ4 A 2 Z i 4 λ 2 Z i = λ4 2 Z i = λ4 2 Z A(1) = A (2) (12) A Z R dd ` (2π)d Z R dd ` (2π)d Z R dd ` (2π)d Z R dd ` (2π)d d`0 0 P (`) P 0 (` + pC ) P (pA − `) P (pA + pB − `) , 2π (3.16) d`0 Pc (`) P (` + pC ) P (pA − `) P (pA + pB − `) , 2π (3.17) d`0 P (`) Pc (` + pC ) P (pA − `) P (pA + pB − `) , 2π (3.18) d`0 Pc (`) Pc (` + pC ) P (pA − `) P (pA + pB − `) . 2π (3.19) In writing (3.17)–(3.19) we have used the notation of [11] in which A(i1 ···in ) is obtained from A by replacing the i1 , · · · in -th propagators by cut propagators. The diagrammatic representations of A(1) , A(2) and A(12) have been shown in figures 3–5 with the thick vertical line across a propagator representing a cut propagator Pc . We emphasize that these are not yet cut diagrams as the cut does not divide the graph into a left half and a right half, and we neither reverse the momenta not complex conjugate any part of the graph. Instead these should be regarded as tree amplitudes since a cut propagator can be regarded as a pair of incoming and outgoing lines with identical momentum. By a similar manipulation we can express A∗ as b∗ + A(1)∗ + A(2)∗ − A(12)∗ . A∗ = A (3.20) b the relevant poles in the `0 plane, We now note that in the expression (3.16) for A responsible for the pinch in the original amplitude A, are all in the upper half plane, since –8– JHEP11(2018)094 where 3 3 4 4 = 1 1 2 2 1 Figure 3. Representation of A(1) . On the left hand side the vertical line through propagator 1 represents that it is a cut propagator. On the right hand side this is made explicit by replacing the cut propagator by a pair of external lines — one outgoing and one incoming. 4 3 JHEP11(2018)094 3 4 = 1 2 2 1 2 Figure 4. Representation of A(2) . 3 4 3 4 1 2 = 1 2 1 2 Figure 5. Representation of A(12) . in the locations of the poles of the integrand, the signs of the iε in the first line of (3.11) are reversed. Therefore the `0 contour is not pinched, and by our previous argument, b−A b∗ = 0 . A (3.21) On the other hand, since A(1) , A(2) and A(12) are tree amplitudes, and since we are assuming that cutting rules hold for tree amplitudes, A(1) − A(1)∗ , A(2) − A(2)∗ and A(12) − A(12)∗ are given by sum over cuts of the tree diagrams. This gives, in the notation of [11]: (1) (1) (1) A(1) − A(1)∗ = A1 + A2 + A12 , (12) A(12) − A(12)∗ = A1 (12) + A2 (2) (2) (2) A(2) − A(2)∗ = A1 + A2 + A21 , (12) (12) + A12 + A21 . (3.22) (i ···i ) Here Aj11···jmn for 1 ≤ ik ≤ 2, 1 ≤ jk ≤ 2 represents sum over all cuts of A(i1 ···in ) satisfying the following properties: 1. The cut can be viewed as a cut of the original graph contributing to the amplitude A. –9– 2. The cut crosses the j1 , · · · jm ’th propagators in the set {1, 2} and possibly other propagators outside the set {1, 2}. (i ···i ) (1) (12) A2 = A2 , (2) (12) A 1 = A1 , (1) (12) A12 = A12 , (2) (12) A21 = A21 . (3.23) These identities can be verified by explicitly examining the equalities of figures 7 and 13, 9 and 12, 8 and 14, and figures 11 and 15. Using (3.15), (3.20), (3.21), (3.22) and (3.23) we now get (1) (1) (1) (2) (1) (2) (2) (2) (12) A − A∗ = A1 + A2 + A12 + A1 + A2 + A21 − A1 = A1 + A2 . (12) − A2 (12) (12) − A12 − A21 (3.24) The diagrammatic representation of the right hand side, given by the sum of the left hand sides of figures 6 and figure 10, is shown in figure 16. We now see that this is precisely given by the sum of all possible cuts of the reduced diagram shown in figure 2. In particular possible contributions from anomalous thresholds, represented by figure 7 and figure 9, cancel with the contributions from figures 13 and 12 and do not appear in the final expression. These cancellations are special cases of the general results described in eqs. (5.26)–(5.33) of [11]. We end this section with a few remarks: 1. Our analysis automatically includes other reduced diagrams that are obtained by contracting one or more propagators in figure 2. We simply have to set to zero all terms where the corresponding propagator is replaced by a cut propagator. For example if we take the triangle diagram obtained by contracting the propagator 3 in figure 2, the final answer for A − A∗ will include sum over only the second and third diagrams in figure 16. 2. This does not cover all the cases however. An example is shown in figure 17 where at the pinch poles on the same side come from non-adjacent propagators. We can analyze this by repeating the analysis, with the role of the momenta ` and ` + pC in (3.14) now played by ` and ` − pA − pB . The rest of the analysis proceeds as before, – 10 – JHEP11(2018)094 On the other hand Ai11j n describes sum over cuts of A(i1 ···in ) which pass through the j-th and i1 -th propagators in the set {1, 2} and possibly other propagators outside the set {1, 2}, but which are not regular cuts of the original amplitude A since, viewed in the context of the original graph, the i1 -th propagator carries energy across the cut in the wrong direction. Explicit diagrammatic representation of all the terms on the right hand side of (3.22) has been given in figures 6–15. In particular figures 8, 11, 14 and 15 describe (1) (2) (12) (12) contributions to A12 , A21 , A12 and A21 respectively. As is clear from the right hand sides of these figures, these are perfectly good cuts of the tree amplitude A(i1 ···in ) , even though the left hand sides of these figures show that they are not valid cuts of A. We shall now write down a few identities following from the simple observation that a propagator cut twice has the same expression as the propagator cut once, since the cut passing through an external line has no effect. Therefore we have = + + (1) = + + (1) Figure 7. Contributions to A2 . Unless cancelled, this would represent contributions from anomalous threshold. with the role of the set {1, 2} played by the set {1, 4}. The final result for A − A∗ , (1) (4) (14) according to the general result of [11], will be given by A1 + A4 + A14 , which is (12) simply the sum over all cuts of figure 17. (The corresponding contribution A12 was not present in the previous example since figure 2 has no cut that passes through both propagators 1 and 2.) 3. In our analysis we have assumed that for a given R, the `0 contour has a single pinch point to which two or more poles approach. We can also have more than one pinch on the `0 contour, with two or more poles approaching each pinch point. Since after we factorize each propagator as in (3.9), each denominator factor is linear in `0 and has a single zero, different denominator factors must be responsible for different pinches. Therefore we can divide the denominators into different sets, with the first set S1 responsible for the first pinch, the second set S2 responsible for the second pinch and so on. Different pinch points will have different reduced diagrams associated with them, since the list of singular propagators and the direction of energy flow through these propagators will depend on the pinch. – 11 – JHEP11(2018)094 Figure 6. Contributions to A1 . The figure on the left hand side expresses it as a sum of cut diagrams of the original graph, with the propagator 1 replaced by the cut propagator. The right hand side shows this as a sum of cuts of a tree diagram in which the propagator 1 is replaced by a pair of incoming and outgoing lines. We have not shown the external states of the original amplitude A in any of the diagrams. = (1) + = + (2) Figure 9. Contributions to A1 . + = + (2) Figure 10. Contributions to A2 . = (2) Figure 11. Contributions to A21 . – 12 – JHEP11(2018)094 Figure 8. Contribution to A12 . In the left hand side the cut seems to cross the propagator 1 of the original diagram in the reverse direction so that it appears as if the energy of the propagator 1 flows from the right of the cut to the left of the cut. However since the propagator 1 is already on-shell, the correct representation of the diagram is on the right hand side where it is represented as the cut of a tree diagram. In this representation there is nothing unusual. + = + + . = + (12) Figure 13. Contributions to A2 . = (12) Figure 14. Contributions to A12 . = (12) Figure 15. Contributions to A21 . – 13 – JHEP11(2018)094 (12) Figure 12. Contributions to A1 + + + Figure 16. Complete contribution to A − A∗ . ` −pC − ` 2 4 pA − ` ` − pA − pB Figure 17. A reduced diagram in which non-adjacent propagators 1 and 4 have poles on the same side at the pinch. At the pinch the energy flows from left to right in each of the propagators. In the figure the momenta are labelled so that they flow from left to right. We can now carry out the analysis by first treating the product of denominators in b is still the set S1 as in (3.14). The main difference will be that now the `0 contour in A pinched due to the other set of denominators belonging to S2 , S3 , · · · etc. The other terms contain delta functions that force `0 to be at the first pinch and therefore the denominators in the other sets remain finite. These terms can be analyzed as before. b by decomposing the second set of denominators, belonging to We can now analyze A the set S2 , as in (3.9) and repeat the analysis. We continue this till we reach a stage where we have a sum of terms where in one term the contour is not pinched (the b and in the other terms the delta-function fixes `0 at a pinch. The final analog of A) result will then be given by the sum of cuts of all the reduced diagrams corresponding to all the pinches. 4 Unitarity of tree diagrams Since the proof of cutting rules for the box diagram assumed the validity of cutting rules for connected and disconnected tree diagrams, we shall prove the cutting rules for tree diagrams in this section. The analysis is a straightforward application of sections 5.2.2 and 5.3 of [11]. We begin with the simple diagram shown in figure 18(a) with p0 > 0. Its expression is given by A = λ2 (−p2 − m2 + i)−1 . – 14 – (4.1) JHEP11(2018)094 3 1 p→ p→ (a) (b) U U P P D D (b) (a) Figure 19. (a) Schematic representation of a generic connected tree diagram in which the blobs U and D themselves are connected tree diagrams. (b) Example of such a generic connected tree diagram. Therefore A − A∗ = λ2 (−p2 − m2 + i)−1 − λ2 (−p2 − m2 − i)−1 = λ2 (−2π i) δ(−p2 − m2 ) . (4.2) On the other hand the cut diagram shown in figure 18(b) has the same expression. (We can drop the θ(p0 ) term from the cut propagator since p0 has been chosen to be positive.) This proves the cutting rule for figure 18(a). Next we shall prove the cutting rules for any connected tree amplitude assuming that it holds for all connected tree amplitudes with at least one less vertex. For this we follow closely the analysis of section 5.2.2 of [11] of ‘one vertex reducible’ diagrams. Let P be any vertex of the amplitude to which at least two internal lines are connected. Then the general form of the diagram can be represented as in figure 19(a), with each of the blobs U and D describing some connected tree diagram. If AU and AD are the amplitudes associated – 15 – JHEP11(2018)094 Figure 18. (a) A tree diagram in φ4 theory with six external lines and one internal propagator. In drawing this we have made an exception to our conventions and have drawn the external lines. The internal line has energy p0 flowing from the left to the right. (b) Cut diagram of (a). with the tree diagrams U and D respectively, then the full amplitude A is given by6 A = λ−1 AU AD , (4.3) where the λ−1 factor accounts for the fact that both AU and AD includes a factor of λ from the vertex P whereas in A we have only one factor of λ from the vertex. Therefore A − A∗ = λ−1 {AU AD − A∗U A∗D } . (4.4) AU − A∗U = ∆U L + ∆U R , AD − A∗D = ∆DL + ∆DR . (4.5) Using (4.5) and some trivial rearrangement of terms we can express (4.4) as A − A∗ = λ−1 {A∗U ∆DL + ∆U L ∆DL + ∆U L A∗D − ∆U R ∆DR + AU ∆DR + ∆U R AD } . (4.6) This can be verified e.g. by expressing both sides in terms of A∗U , A∗D , ∆U R , ∆U L , ∆DR and ∆DL . The diagrammatic representations of the six terms in (4.6) have been shown in figure 20. Special attention should be paid to the minus sign of the fourth term on the right hand side of (4.6). This is compatible with the fourth term in figure 20 due to the (−1)nR −1 factor that multiplies each cut diagram, nR being the number of disconnected components on the right of the cut. If we denote by nU R and nDR the number of disconnected components on the right of the cut in U and in D, then the product of the cut diagrams of U and D carries a factor of (−1)nU R −1+nDR −1 . On the other hand the fourth cut diagram of figure 20 carries a factor of (−1)nU R +nDR −1 . The two differ by a sign showing that the fourth diagram of figure 20 is indeed given by −λ−1 ∆U R ∆DR . We now note that the six cut diagrams of figure 20 exhaust all possible cuts of the diagram 19(a). This shows that A − A∗ is indeed given by the sum over all cut diagrams of A in accordance with the cutting rules. Finally we turn to the proof of cutting rules for disconnected tree diagrams following the analysis of section 5.3 of [11]. Again the proof will proceed via induction, namely we shall prove the result assuming that it holds for diagrams with less number of vertices. For this let us suppose that the graph contains two pieces B and C that are disconnected from each other. This has been shown in figure 21. B and C themselves may be disconnected graphs, but each will contain less number of vertices and therefore satisfy cutting rules. 6 Note that some of the external lines of U at P are internal lines of D and some of the external lines of D at P are internal lines of U . Therefore they are generically off-shell. This does not have any effect on our analysis since the validity of cutting rules does not require the external lines to be on-shell. – 16 – JHEP11(2018)094 Now since AU and AD are themselves connected tree amplitudes with less number of vertices than A, AU − A∗U and AD − A∗D are given by sum over cut diagrams of U and D. We divide each of these cut diagrams into two classes: ∆U R and ∆U L will denote the sum over cut diagrams of U for which the cut passes on the left and right of P respectively, and similarly ∆DR and ∆DL will denote the sum over cut diagrams of D for which the cut passes on the left and right of P respectively. Therefore we have '$ '$ '$ '$ '$ '$ U U U U U U &% &% &% &% &% &% '$ '$ '$ '$ '$ '$ D D D D D D &% &% &% &% &% &% # B "! # C "! Figure 21. Schematic representation of a tree amplitude with at least two disconnected components. The blobs B and C represent tree amplitudes which themselves may have additional disconnected components. # # B B # # # # # B B B B B "! "! "! "! "! "! "! # # # # # # # C C C C C C C "! "! "! "! "! "! "! (a) (b) (c) (d) (e) (f) (g) Figure 22. Diagrammatic representation of the seven terms in (4.8). These can also be interpreted as cuts of figure 21. Denoting by B and C the expressions for the amplitudes associated with the graphs B and C, we have the full amplitude A given by A = −i B C , (4.7) where the factor of −i arises due to the fact that the total number of disconnected components of A is equal to the sum of the number of disconnected components of B and of C, and – 17 – JHEP11(2018)094 Figure 20. Diagrammatic representation of the six terms in (4.6). These can also be interpreted as cuts of figure 19(a). therefore due to the (i)1−nc factor in the expression for the amplitude with nc disconnected components, the product B C has one more factor of i compared to A. This gives A − A∗ = −i (B C + B ∗ C ∗ ) = −iB ∗ C − iB C ∗ + i (B − B ∗ ) (C − C ∗ ) + i (B − B ∗ ) C ∗ − i(B − B ∗ ) C +i B ∗ (C − C ∗ ) − i B (C − C ∗ ) . (4.8) Acknowledgments We would like to thank Alok Laddha and Terry Tomboulis for useful discussions. The research of R.P. was supported in part by Perimeter Institute for Theoretical Physics. Research at Perimeter Institute is supported by the Government of Canada through Industry Canada and by the Province of Ontario through the Ministry of Research and Innovation. This research of A.S. was supported in part by the J. C. Bose fellowship of the Department of Science and Technology, India. Open Access. This article is distributed under the terms of the Creative Commons Attribution License (CC-BY 4.0), which permits any use, distribution and reproduction in any medium, provided the original author(s) and source are credited. References [1] R.E. Cutkosky, Singularities and discontinuities of Feynman amplitudes, J. Math. Phys. 1 (1960) 429 [INSPIRE]. [2] M. Fowler, Introduction to Momentum Space Integration Techniques in Perturbation Theory, J. Math. Phys. 3 (1962) 936. [3] S. Mandelstam, Unitarity Condition Below Physical Thresholds in the Normal and Anomalous Cases, Phys. Rev. Lett. 4 (1960) 84 [INSPIRE]. [4] R.E. Cutkosky, Anomalous Thresholds, Rev. Mod. Phys. 33 (1961) 448. [5] R. Zwicky, A brief Introduction to Dispersion Relations and Analyticity, in Proceedings of Quantum Field Theory at the Limits: from Strong Fields to Heavy Quarks (HQ 2016), Dubna Russia (2016), pg. 93 [arXiv:1610.06090] [INSPIRE]. [6] P. Chin and E.T. Tomboulis, Nonlocal vertices and analyticity: Landau equations and general Cutkosky rule, JHEP 06 (2018) 014 [arXiv:1803.08899] [INSPIRE]. – 18 – JHEP11(2018)094 The second step is the result of trivial algebraic manipulation. The seven terms in (4.8) can be diagrammatically represented as the seven cut diagrams shown in figure 22. The additional minus signs in the contributions from figure 22(c), (d) and (f), given respectively by the third, fourth and sixth terms in (4.8), account for the fact that if nR denotes the number of disconnected components to the right of the diagram, then the sum of nR − 1 for the component diagrams differ from (nR − 1) of the full diagram by 1. We now note that the seven terms in figure 22 are in one to one correspondence with the cuts of A. This proves the validity of the cutting rule for the disconnected amplitude A. [7] M.J.G. Veltman, Unitarity and causality in a renormalizable field theory with unstable particles, Physica 29 (1963) 186 [INSPIRE]. [8] G. ’t Hooft and M.J.G. Veltman, Diagrammar, NATO Sci. Ser. B 4 (1974) 177. [9] G.F. Sterman, An Introduction to quantum field theory, Cambridge University Press, Cambridge U.K. (1993). [10] E.T. Tomboulis, Causality and Unitarity via the Tree-Loop Duality Relation, JHEP 05 (2017) 148 [arXiv:1701.07052] [INSPIRE]. [12] F. Briscese and L. Modesto, Cutkosky rules and perturbative unitarity in Euclidean nonlocal quantum field theories, arXiv:1803.08827 [INSPIRE]. [13] D. Anselmi, Algebraic cutting equations, Annals Phys. 394 (2018) 294 [arXiv:1612.07148] [INSPIRE]. – 19 – JHEP11(2018)094 [11] R. Pius and A. Sen, Cutkosky rules for superstring field theory, JHEP 10 (2016) 024 [Erratum ibid. 1809 (2018) 122] [arXiv:1604.01783] [INSPIRE].
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Chiral algebras in Landau-Ginzburg models
˜The œJournal of high energy physics/˜The œjournal of high energy physics
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Published for SISSA by Springer Published for SISSA by Springer Published for SISSA by Springer Received: January 22, 2018 Accepted: March 2, 2018 Published: March 13, 2018 Received: January 22, 2018 Accepted: March 2, 2018 Published: March 13, 2018 Received: January 22, 2018 Accepted: March 2, 2018 Published: March 13, 2018 JHEP03(2018)079 Open Access, c⃝The Authors. Article funded by SCOAP3. Mykola Dedushenko Mykola Dedushenko Joseph Henry Laboratories, Princeton University, Princeton NJ, 08540, U.S.A. 3 Landau-Ginzburg models 3.1 Quasihomogeneous case 3.1.1 The OPE in the cohomology 3.1.2 Classical and quantum chiral algebra 3.1.3 Non-abelian global symmetries 4 N = (2, 2) models 4 N = (2, 2) models 5 Examples 5.1 Degenerate examples 5.2 N = 2 minimal models 5.2.1 The Ak+1 series 5.2.2 D and E series of minimal models 5 Examples 5 Examples 5.1 Degenerate examples 5.2 N = 2 minimal models 5.2.1 The Ak+1 series 5.2 N = 2 minimal models 5.2.1 The Ak+1 series 5.2.2 D and E series of minimal models 6 Discussions and further directions 39 6 Discussions and further directions 1 Introduction Contents 1 Introduction 2 N = (0, 2) theories 3 2.1 Conventions and some generalities 3 2.2 Supercurrent multiplet and RG invariance 5 2.2.1 General case 5 2.2.2 Emergent conformal invariance in the cohomology 8 2.2.3 OPE in the cohomology 10 2.3 Chiral algebras of superconformal theories 10 2.4 The operator cohomology and the superspace 12 2.4.1 Classical and quantum observables 12 2.4.2 Classical observables and the cohomology 12 2.4.3 The cohomology of Q+ in F0 and of D+ in bF0 14 2 N = (0, 2) theories 2.1 Conventions and some generalities 2.2 Supercurrent multiplet and RG invariance 2.2.1 General case 2.2.2 Emergent conformal invariance in the cohomology 2.2.3 OPE in the cohomology JHEP03(2018)079 2.3 Chiral algebras of superconformal theories 2.4.2 Classical observables and the cohomology b 2.4.3 The cohomology of Q+ in F0 and of D+ in bF0 E-mail: dedushenko@gmail.com E-mail: dedushenko@gmail.com Abstract: Chiral algebras in the cohomology of the Q+ supercharge of two-dimensional N = (0, 2) theories on flat spacetime are discussed. Using the supercurrent multiplet, we show that the answer is renormalization group invariant for theories with an R-symmetry. For N = (0, 2) Landau-Ginzburg models, the chiral algebra is determined by the operator equations of motion, which preserve their classical form, and quantum renormalization of composite operators. We study these theories and then specialize to the N = (2, 2) models and consider some examples. Keywords: Conformal and W Symmetry, Conformal Field Theory, Extended Supersym- metry, Field Theories in Lower Dimensions ArXiv ePrint: 1511.04372 Open Access, c⃝The Authors. Article funded by SCOAP3. Open Access, c⃝The Authors. Article funded by SCOAP3. https://doi.org/10.1007/JHEP03(2018)079 https://doi.org/10.1007/JHEP03(2018)079 Contents 1 Introduction 1 2 N = (0, 2) theories 3 2.1 Conventions and some generalities 3 2.2 Supercurrent multiplet and RG invariance 5 2.2.1 General case 5 2.2.2 Emergent conformal invariance in the cohomology 8 2.2.3 OPE in the cohomology 10 2.3 Chiral algebras of superconformal theories 10 2.4 The operator cohomology and the superspace 12 2.4.1 Classical and quantum observables 12 2.4.2 Classical observables and the cohomology 12 2.4.3 The cohomology of Q+ in F0 and of D+ in bF0 14 3 Landau-Ginzburg models 14 3.1 Quasihomogeneous case 17 3.1.1 The OPE in the cohomology 20 3.1.2 Classical and quantum chiral algebra 22 3.1.3 Non-abelian global symmetries 24 4 N = (2, 2) models 25 5 Examples 29 5.1 Degenerate examples 29 5.2 N = 2 minimal models 30 5.2.1 The Ak+1 series 30 5.2.2 D and E series of minimal models 37 6 Discussions and further directions 39 1 Introduction Two-dimensional theories with N = (0, 2) supersymmetry have been attracting attention over the last couple of decades. A motivation largely came from their potential phenomeno- logical relevance for heterotic string compactifications, which require the internal theory to be an N = (0, 2) SCFT. But these theories are interesting and rich quantum field the- ories by themselves, which makes them a good object to study and apply various physical ideas. Thinking in that direction, gauge theories are of course of particular importance in – 1 – theoretical physics and deserve attention in various dimensions and with various amounts of supersymmetry. But besides that, N = (0, 2) gauged linear sigma models are known to be a useful tool to construct N = (0, 2) SCTFs, and hence heterotic string vacua, as infrared (IR) fixed points of the renormalization group (RG) flow (see [1, 2] or just [3] and references therein). Recently, the dynamics of two-dimensional N = (0, 2) supersymmetric gauge theories, both abelian and non-abelian, have seen an increasing interest, especially due to devel- opments in [4–6]. At the same time, more basic models of N = (0, 2) interacting matter without gauge fields, sometimes referred to as N = (0, 2) Landau-Ginzburg (LG) mod- els, have been studied, some references being [2, 7, 8]. One can think of starting from an N = (0, 2) LG model with global flavor symmetries and then gauging these global symmetries to obtain an N = (0, 2) gauge theory. Therefore, it might be beneficial for certain questions to first study properties of LG models and then ask what happens to these properties after gauging. JHEP03(2018)079 The property we want to study in this paper is the chiral algebra in the cohomology of one of the supercharges. The supercharges Q+ and Q+ of the (0, 2) theory satisfy: {Q+, Q+} = 2P++, (1.1) (1.1) where P++ is the right-moving translation generator. Since Q 2 + = 0, one can study its cohomology, and the above equation implies that it is holomorphic, or, in terms of light- cone coordinates x±± (in Lorenzian signature), the cohomology depends non-trivially only on x−−, while differentiation with respect to x++ annihilates cohomology classes. This observation was first made in [9] and then in [10] used to elucidate some properties of N = (2, 2) LG models and their IR fixed points. 1 Introduction Then, part of the analysis from [10] was extended to N = (0, 2) gauge theories in [11]. Chiral algebras of N = (0, 2) half-twisted sigma models were studied to some extent in the literature due to their connection with the theory of chiral differential operators. In particular, the perturbative approach was developed in [12] and [13], and some non- perturbative aspects were studied in [14] and [15]. There was also a number of papers on topological rings (which are finite sectors of chiral algebras in N = (0, 2) theories), some examples being [16–19]. However, it seems that systematic analysis of chiral algebras in N = (0, 2) LG models and gauge theories has not been performed yet. Our goal is to make a small step in this direction. In this paper we first study some general properties of N = (0, 2) theories on R1,1. Then we restrict to a certain class of models, namely LG models, and later consider LG models with N = (2, 2) supersymmetry and give some examples. An important question about chiral algebras of N = (0, 2) theories is how they behave under the RG flow. It turns out that in theories with the R-symmetry chiral algebra is an RG invariant, while it is not completely clear whether this is the case for more general theories without R-symmetry. The RG invariance underlies all approaches to extract some useful information about the CFT in the IR, such as, for example, in [10] and [11]. It seems that this has never been proved in the literature though. – 2 – We study RG invariance of the chiral algebra on general grounds using the N = (0, 2) supercurrent multiplet described in [20]. If the theory has an R-symmetry, the supercurrent multiplet becomes what is called the R-multiplet. In such a situation, only using manipulations with the R-multiplet, we show that there is a stress-energy tensor in the cohomology. This fact underlies the finding of [11] that the stress-energy tensor in the cohomology is not spoiled by anomaly if and only if the R-symmetry is non-anomalous. So we obtain conformal symmetry in the chiral algebra. It is interesting to note that this stress-energy tensor is the one of the half-twisted N = (0, 2) theory. 2 N = (0, 2) theories In this section we discuss some general aspects of two-dimensional (0, 2)-supersymmetric theories and their chiral algebras. 1 Introduction Since the stress-energy tensor in cohomology is identified with the left-moving stress-energy tensor of the CFT in the IR, we can say the following: in N = (0, 2) theories with the R-symmetry, the RG flow from the UV to the IR performs a half-twist. JHEP03(2018)079 Having conformal symmetry in the cohomology is a very strong restriction. It turns out that because of it, the chiral algebra cannot depend on any dimensionful constants. This fact allows one to argue that for the LG models, the chiral algebra is in fact tree level exact, and the OPE of the cohomology classes can be computed using the free field correlators. This makes chiral algebra a potentially powerful tool for obtaining exact results. We should note, however, that in this discussion we assume that there are no non-perturbative effects. This seems a reasonable assumption for LG models on a topologically trivial space with a topologically trivial target only. In section 2 of this paper we discuss general aspects of N = (0, 2) theories, namely the supercurrent multiplet and its ambiguities, conformal invariance in the cohomology and its implications for the OPE. We also discuss what is the chiral algebra of SCFT’s and relate it to the notion of a chiral ring of [21]. We then discuss the superspace technique to describe the Q+ cohomology. In section 3 we discuss general properties of N = (0, 2) LG models and specify to the quasi-homogeneous superpotentials. We then review the statement that the OPE of the cohomology classes can be computed using the free theory and argue that the chiral algebra is tree level exact (but still different from the classical algebra due to the singularities one encounters in defining composite operators). In section 4 we specialize to N = (2, 2) supersymmetry, and in section 5 we discuss a few examples, focusing on the Ak+1 series of N = 2 minimal models. We then conclude in section 6 and mention some further directions. 2 N = (0, 2) theories 2 N = (0, 2) theories 2.1 Conventions and some generalities The two-dimensional theories with (0, 2) supersymmetry are characterized by the existence of two conserved supercharges Q+ and Q+ of positive (or right-handed) chirality acting on the Hilbert space of the theory. They satisfy: Q2 + = Q 2 + = 0, {Q+, Q+} = 2P++, (2.1) + + {Q+, Q+} = 2P++, (2.1) (2.1) – 3 – – 3 – where 2P++ = P0 + P1 is a light-cone momentum. The standard geometric realization of supersymmetry is to consider the superspace R2|2 with bosonic coordinates x0, x1 and fermionic coordinates θ+ and θ +. Superfields are distributions on this superspace taking values in operators acting on the Hilbert space. The supercharges Q+ and Q+ act on operators (and therefore on superfields) by commutators, and the geometric realization of this action is through the differential operators: Q+ = ∂ ∂θ+ + iθ + ∂ ∂x++ , Q+ = −∂ ∂θ + −iθ+ ∂ ∂x++ , (2.2) (2.2) JHEP03(2018)079 so that for an arbitrary superfield F, we have [Q+, F]± = Q+F, where [. . . ]± denotes a graded commutator. These operators obviously satisfy the required relation [Q+, Q+] = −2i ∂ ∂x+ . We also have another pair of differential operators on R2|2, D+ and D+, given by: so that for an arbitrary superfield F, we have [Q+, F]± = Q+F, where [. . . ]± denotes a graded commutator. These operators obviously satisfy the required relation [Q+, Q+] = −2i ∂ ∂x+ . We also have another pair of differential operators on R2|2, D+ and D+, given by: D+ = ∂ ∂θ+ −iθ + ∂ ∂x++ , D+ = −∂ ∂θ + + iθ+ ∂ ∂x++ , (2.3) (2.3) for which the key property is that they anticommute with Q+ and Q+ and hence can be used in constructing supersymmetric Lagrangians. for which the key property is that they anticommute with Q+ and Q+ and hence can be used in constructing supersymmetric Lagrangians. We also adopt the convention in which hermitian conjugation reverses the order of fermions, that is (θ1θ2)† = θ2θ1. The basic superfields are The basic superfields are The basic superfields are 1) Chiral superfields satisfying D+Φ = 0. The component expansion contains a complex scalar φ and a left spinor ψ+: 1) Chiral superfields satisfying D+Φ = 0. 3) Real superfield. 2.1 Conventions and some generalities The component expansion contains a complex scalar φ and a left spinor ψ+: Φ = φ + iθ+ψ+ −iθ+θ +∂++φ (2.4) (2.4) The antichiral superfield satisfies D+Φ = 0 and is given by: The antichiral superfield satisfies D+Φ = 0 and is given by: Φ = φ + iθ +ψ+ + iθ+θ +∂++φ. (2.5) (2.5) 2) Fermi superfields satisfying D+Λ = E(Φ), where E(Φ) is a chiral superfield con- structed as a holomorphic function of basic chiral superfields. The component ex- pansion contains a right-handed spinor λ and an auxiliary field G: 2) Fermi superfields satisfying D+Λ = E(Φ), where E(Φ) is a chiral superfield con- structed as a holomorphic function of basic chiral superfields. The component ex- pansion contains a right-handed spinor λ and an auxiliary field G: 2) Fermi superfields satisfying D+Λ = E(Φ), where E(Φ) is a chiral superfield con- structed as a holomorphic function of basic chiral superfields. The component ex- pansion contains a right-handed spinor λ and an auxiliary field G: Λ = λ + θ+G −iθ+θ +∂++λ −θ +E(Φ), (2.6) (2.6) where E itself has to be expanded in components. The opposite chirality Fermi superfield satisfies D+Λ = −E(Φ) and is given by: where E itself has to be expanded in components. The opposite chirality Fermi superfield satisfies D+Λ = −E(Φ) and is given by: Λ = λ + θ +G + iθ+θ +∂++λ −θ+E(Φ). (2.7) (2.7) – 4 – If U is a real superfield, it can always be thought of as a real part of some chiral superfield (not necessarily a local one; also we will allow for superfields which are chiral only on-shell). We will denote the imaginary part of this chiral superfield by eU. Then U + ieU is chiral on-shell and U −ieU is antichiral. The relation between U and eU is: D+ eU = iD+U, D+ eU = −iD+U, (2.8) + + D+ eU = −iD+U, (2.8) (2.8) up to equations of motion. This eU is defined up to a term which is constant on-shell. 1Our conventions are different from [20]. 2.1 Conventions and some generalities If the component expansion of U is JHEP03(2018)079 U = u + iθ+χ+ + iθ +χ+ + θ+θ +∂++v, (2.9) (2.9) where we wrote the highest component as a derivative of some function v, then the com- ponent expansion of eU is: where we wrote the highest component as a derivative of some function v, then the com- ponent expansion of eU is: eU = v + θ+χ+ −θ +χ+ −θ+θ +∂++u, (2.10) (2.10) again up to terms which vanish on equations of motion. e again up to terms which vanish on equations of motion. e Note that if we want components of U and eU to be local operators, then U cannot be an arbitrary local real superfield. Its highest component, written as ∂++v above, should be a derivative of a local field. Only in such a case v above is also local and hence eU is also the local superfield. 2.2 Supercurrent multiplet and RG invariance 2.2.1 General case The general N = (0, 2) multiplet containing the stress-energy tensor and the supersymme- try current was described in [20]. It is referred to as the supercurrent multiplet. It consists of real superfields S++, T−−−−and a complex superfield W−satisfying:1 ∂−−S++ = D+W−−D+W−, D+T−−−−= ∂−−W−, D+T−−−−= ∂−−W−, D+W−= C, (2.11) (2.11) D+W−= C, (2.11) where C is a complex constant (a space-filling brane current). The component expansions which solve these constraints are: S++ = j++ −2iθ+S+++ −2iθ +S+++ −2θ+θ +T++++, W−= −S+−−−iθ+  T++−−+ i 2∂−−j++  −θ +C + iθ+θ +∂++S+−−, W−= −S+−−+ iθ +  T++−−−i 2∂−−j++  −θ+C −iθ+θ +∂++S+−−, T−−−−= T−−−−−θ+∂−−S+−−+ θ +∂−−S+−−+ 1 2θ+θ +∂2 −−j++. ( (2.12) 1Our conventions are different from [20]. – 5 – – 5 – Applying constraints (2.11) to these expansions implies conservation of S+ (the supersym- metry current), conservation of T (the stress-energy tensor) and symmetry of T: Applying constraints (2.11) to these expansions implies conservation of S+ (the supersym- metry current), conservation of T (the stress-energy tensor) and symmetry of T: ∂++S+−−+ ∂−−S+++ = 0, ∂++T±±−−+ ∂−−T±±++ = 0, T++−−−T−−++ = 0. (2.13) (2.13) Quite naturally, constraints (2.11) do not determine the supercurrent multiplet uniquely. There are two types of ambiguities which preserve both the conservation laws and the form of equations (2.11). One ambiguity corresponds to improvement transformations: Quite naturally, constraints (2.11) do not determine the supercurrent multiplet uniquely. There are two types of ambiguities which preserve both the conservation laws and the form of equations (2.11). One ambiguity corresponds to improvement transformations: JHEP03(2018)079 S++ →S++ + [D+, D+]U, W−→W−+ ∂−−D+U, W−→W−+ ∂−−D+U, T−−−−→T−−−−+ ∂2 −−U, (2.14) (2.14) where U is an arbitrary real scalar superfield. These transformations do not change con- served charges. Another ambiguity corresponds to the possibility of modifying the supercurrent multi- plet by another conserved current (say, corresponding to some flavor symmetry), satisfying an additional requirement of locality which will be explained in a moment. If we have another conserved superspace current I±±, that is a pair of real superfields satisfying: ∂−−I++ + ∂++I−−= 0, (2.15) (2.15) then we can use it to shift the supercurrent multiplet, i.e. define a new multiplet: S++ →eS++ = S++ + I++, W−→f W−= W−+ i 2D+I−−, W−→f W−= W−−i 2D+I−−, T−−−−→eT−−−−= T−−−−+ 1 2∂−−eI−−. (2.16) (2.16) Note that in the last equation we use eI−−, a real superfield related to I−−as in (2.8). 2.2.1 General case That is, eI−−is such that I−−+ ieI−−is chiral. The new superfields eS++, f W−and eT−−−− will also satisfy the constraints (2.11). However, most conserved charges will be shifted by this transformation. Note that for the above transformation to make sense in a local QFT, both I±± and eI−−have to be local, so there is an extra requirement on I±± that not only it has to be a conserved local superspace current, but also eI−−has to be local. In the cases of interest for us, this will actually be the case. Note that in the last equation we use eI−−, a real superfield related to I−−as in (2.8). That is, eI−−is such that I−−+ ieI−−is chiral. The new superfields eS++, f W−and eT−−−− will also satisfy the constraints (2.11). However, most conserved charges will be shifted by this transformation. Note that for the above transformation to make sense in a local QFT, both I±± and eI−−have to be local, so there is an extra requirement on I±± that not only it has to be a conserved local superspace current, but also eI−−has to be local. In the cases of interest for us, this will actually be the case. – 6 – One can easily read offthe action of Q+ on various components of the supercurrent multiplet, and we are interested in the following: One can easily read offthe action of Q+ on various components of the supercurrent multiplet, and we are interested in the following: {Q+, S+++} = −i  T++++ + i 2∂++j++  , {Q+, S+−−} = i  T++−−−i 2∂−−j++  , {Q+, S+++} = −i  T++++ + i 2∂++j++  , {Q+, S+−−} = i  T++−−−i 2∂−−j++  , {Q+, T++++} = ∂++S+++, {Q+, T++−−} = −∂++S+−−, {Q+, T−−−−} = −∂−−S+−−. (2.17) {Q+, T++++} = ∂++S+++, JHEP03(2018)079 {Q+, T−−−−} = −∂−−S+−−. (2.17) (2.17) We see that neither component of the stress-energy tensor is annihilated by Q+, so com- ponents of T by itself do not represent any Q+-cohomology classes. However, certain relations hold in the cohomology, in particular T++−−−i 2∂−−j++ is Q+-exact. If we define the “virial current” Vµ as: We see that neither component of the stress-energy tensor is annihilated by Q+, so com- ponents of T by itself do not represent any Q+-cohomology classes. However, certain relations hold in the cohomology, in particular T++−−−i 2∂−−j++ is Q+-exact. 2.2.2 Emergent conformal invariance in the cohomology In the previous subsection we considered a general N = (0, 2) theory in 2d, which a priori did not have any R-symmetries. The lowest component j++ of the superfield S++ did not satisfy any conservation laws and, moreover, was not even accompanied by j−−. As was noted in [20], if we restrict to the case C = 0 and W−= i 2D+R−−, where R−−is another real superfield (and also relabel S++ by R++), we get what is called an R-multiplet. The equation relating S++ and W−becomes simply ∂−−R++ + ∂++R−−= 0, so the lowest component j−−of R−−together with j++ form a conserved R-current. So we have: R−−= j−−−2iθ+S+−−−2iθ +S+−−−2θ+θ +T++−−, (2.23) (2.23) JHEP03(2018)079 with ∂++j−−+ ∂−−j++ = 0. In this situation, it becomes possible to define a new stress- energy tensor: with ∂++j−−+ ∂−−j++ = 0. In this situation, it becomes possible to define a new stress- energy tensor: eT++++ = T++++ + i 2∂++j++, eT++−−= T++−−−i 2∂−−j++, eT−−−−= T−−−−−i 2∂−−j−−, (2.24) (2.24) which is also symmetric and conserved (by virtue of the conservation of j), but also it satisfies: which is also symmetric and conserved (by virtue of the conservation of j), but also it satisfies: eT++−−= {Q+, . . . }, {Q+, eT−−−−} = 0. (2.25) eT++++ = {Q+, . . . }, eT++−−= {Q+, . . . }, eT−−−−̸= {Q+, . . . }, {Q+, eT−−−−} = 0. (2.25) eT++++ = {Q+, . . . }, eT−−−−̸= {Q+, . . . }, (2.25) This procedure for N = (0, 2) theories is known as a half-twisting. The above relations demonstrate that when it can be performed, one has the full 2d conformal invariance in the cohomology of Q+: the cohomology class represented by eT−−−−plays the role of the holomorphic2 stress-energy tensor. It also ensures that the Q+-cohomology is invariant under the RG flow. The RG invariance of the chiral algebra implies that it carries a useful information about the IR fixed point. Let us also take a closer look at the ambiguities of the supercurrent multiplet in the presence of R-symmetry. The improvement transformations are determined by a real superfield U though (2.14), which tells us how R++, W−and T−−−−are improved. 2.2.1 General case If we define the “virial current” Vµ as: V−−= 0, V++ = ij++, (2.18) (2.18) then we have: T µ µ = ∂µVµ −{Q+, 4iS+−−}, (2.19) (2.19) which looks like condition for an effective scale-invariance [22], with the effective current for constant dilatations given by dµ = xνTνµ −Vµ. This current is “almost conserved”: ∂µdµ = {Q+, . . . }. (2.20) (2.20) The current dµ itself is not Q+-closed. Even though dµ is not precisely conserved, only up to Q+-exact terms, we still can try to define a “charge” D corresponding to this current. If we have a local operator O(0) inserted at the origin, we define the action of D on this operator as follows. Pick a contour C enclosing O(0) and define: [D, O(0)] = I C ⋆d(x)O(0) = I C dxµϵµνdν(x)O(0). (2.21) (2.21) This definition is clearly contour-dependent, since dµ(x) is not conserved. As we deform the contour a bit, [D, O(0)] changes by [∂µdµ(x), O] integrated over the area swept by the deformation of the contour. But ∂µdµ(x) is Q+-exact, so if O(0) is Q+-closed, the change in [D, O(0)] under the contour deformation is Q+-exact. This means that [D, O(0)] is well-defined up to a Q+-exact piece when it acts on Q+-closed operators. Moreover, one can check that: [D, Q+] = Q+, (2.22) (2.22) which shows that D maps Q+-closed operators into Q+-closed operators. So we conclude that D is a well-defined operator in the cohomology. It generates scale-transformations there. Since D is not Q+-closed itself, we can say that scale transformations act as outer automorphisms in the cohomology. – 7 – 2To be more precise, we should Wick rotate to the Euclidean signature in order to have holomorphy. 2.2.2 Emergent conformal invariance in the cohomology On R−−it acts by: R−−→R−−−2∂−−eU, (2.26) (2.26) where the relation between U and eU is as in (2.8), that is where the relation between U and eU is as in (2.8), that is U = u + iθ+χ+ + iθ +χ+ + θ+θ +∂++v, eU = v + θ+χ+ −θ +χ+ −θ+θ +∂++u. (2.27) (2.27) For an improvement transformation of the R-multiplet to make sense, we have to assume that both U and eU are local superfields. In view of the comment we made before, this For an improvement transformation of the R-multiplet to make sense, we have to assume that both U and eU are local superfields. In view of the comment we made before, this 2To be more precise, we should Wick rotate to the Euclidean signature in order to have holomorphy. 2To be more precise, we should Wick rotate to the Euclidean signature in order to have holomorphy. – 8 – restricts the class of allowed U. While for a general supercurrent multiplet the improvem ent transformations were parametrized by an arbitrary local real superfield U, for the R- multiplet they are parametrized by such a local real superfields U that eU is also local. Thus the R-multiplet allows a smaller class of improvements then a general supercurrent multiplet. This is not surprising after all. For the general supercurrent multiplet, only the stress-energy tensor and the supersymmetry currents are conserved, so improvements should only preserve their conservation. In the R-multiplet, on the other hand, we also have the conserved R-current, so preserving its conservation (and the R-charge value) restricts the class of allowed improvements. In terms of component currents, the improvement transformation is: JHEP03(2018)079 j−−→j−−−2∂−−v, T++−−→T++−−−∂++∂−−u, j++ →j++ + 2∂++v, j−−→j−−−2∂−−v, T++++ →T++++ + ∂2 ++u, T++−−→T++−−−∂++∂−−u, T−−−−→T−−−−+ ∂2 −−u, S+++ →S+++ + i∂++χ+, S+−−→S+−−−i∂−−χ+, S+++ →S+++ −i∂++χ+, S+−−→S+−−+ i∂−−χ+. (2.28) S+−−→S+−−−i∂−−χ+, S+−−→S+−−+ i∂−−χ+. (2.28) (2.28) As expected, this transformation does not spoil conservation of any of these currents. It does not shift values of any conserved charges either. Also, it is easy to check that components eT++++ and eT++−−of the half-twisted stress-energy tensor are shifted by Q+- exact terms. On the other hand, eT−−−−is shifted by ∂2 −−(u + iv), which is, being the lowest component of chiral superfield U + ieU, is Q+-closed but generally is not Q+-exact. Therefore, there is a family of possible holomorphic stress tensors in the Q+-cohomology, corresponding to different improvements. 3Even if the operator representing the cohomology class in the full theory is not left-moving, the class it represents is left-moving. Since Lorenz-invariance of the full theory induces Lorenz-invariance in the cohomology, one indeed can use the argument made in the text. 2.2.3 OPE in the cohomology This is possible only for p = 0, that is µ should not be there. All dependence on dimensionful coupling constants of the original supersymmetric theory will therefore be hidden in the Q+-exact term. This simple observation will be helpful later. It will imply that one can turn offall dimensionful couplings for the OPE computation. In the models we are going to study this will mean that it is enough to compute OPE in the free theory. 2.2.3 OPE in the cohomology If we have two operators O1 and O2 representing nontrivial Q+-cohomology classes, we can consider their OPE. On very general grounds we have: O1(x++, x−−)O2(0, 0) = X n,m (x++)n(x−−)mOn,m(0, 0). (2.33) (2.33) Now recall that the operator ∂++ acts trivially in the cohomology, that is if O1 is Q+- closed, then ∂++O1 is Q+-exact, and thus so is ∂++O1(x++, x−−)O2(0, 0). Acting with ∂++ on the right-hand side then gives a Q+-exact answer, that is: Now recall that the operator ∂++ acts trivially in the cohomology, that is if O1 is Q+- closed, then ∂++O1 is Q+-exact, and thus so is ∂++O1(x++, x−−)O2(0, 0). Acting with ∂++ on the right-hand side then gives a Q+-exact answer, that is: JHEP03(2018)079 X n,m n(x++)n−1(x−−)mOn,m(0, 0) = [Q+, . . . ]. (2.34) (2.34) This implies that all terms except those with n = 0 are Q+-exact. If the cohomology classes represented by Oi have scaling dimensions hi, we can then write: O1(x++, x−−)O2(0, 0) = X k 1 (x−−)h1+h2−hk Ok(0, 0) + [Q+, . . . ]. (2.35) (2.35) Note also that, since in the cohomology we have left-movers only, scaling dimensions and spins coincide.3 This, in particular, implies an obvious conclusion that no dimensionful constants can appear in the OPE of the cohomology classes. Any dimensionful constant will have non-trivial dimension but trivial spin, and therefore its appearance will either break scaling or Lorentz-invariance of the OPE. Indeed, if we have some dimensionful parameter µ, then in the expression: Note also that, since in the cohomology we have left-movers only, scaling dimensions and spins coincide.3 This, in particular, implies an obvious conclusion that no dimensionful constants can appear in the OPE of the cohomology classes. Any dimensionful constant will have non-trivial dimension but trivial spin, and therefore its appearance will either break scaling or Lorentz-invariance of the OPE. Indeed, if we have some dimensionful parameter µ, then in the expression: O1(x++, x−−)O2(0, 0) = X k µp (x−−)∆Ok(0, 0) + [Q+, . . . ], (2.36) (2.36) scaling invariance implies ∆= h1 + h2 −hk −h(µ)p, where h(µ) is the dimension of while Lorentz invariance implies ∆= h1 + h2 −hk. This is possible only for p = 0, that µ should not be there. scaling invariance implies ∆= h1 + h2 −hk −h(µ)p, where h(µ) is the dimension of µ, while Lorentz invariance implies ∆= h1 + h2 −hk. 2.2.2 Emergent conformal invariance in the cohomology Another ambiguity, namely shifting by the superspace current I±±, works in a straight- forward way: R++ →R++ + I++, R−−→R−−+ I−−, T−−−−→T−−−−+ 1 2∂−−eI−−. (2.29) (2.29) If we denote the components of I±± by: If we denote the components of I±± by: I±± = i±± −2iθ+I+±± −2iθ +I+±± −2θ+θ +H++±±, (2.30) (2.30) and introduce a local operator h−−such that and introduce a local operator h−−such that ∂++h−−= H++−−, (2.31) ∂++h−−= H++−−, (2.31) then the shifting transformation in components works as: then the shifting transformation in components works as: j±± →j±± + i±±, S+±± →S+±± + I+±±, S+±± →S+±± + I+±±, T++±± →T++±± + H++±±, T−−−−→T−−−−−∂−−h−−. (2.32) (2.32) This ambiguity will naturally arise in a later discussion. This ambiguity will naturally arise in a later discussion. – 9 – 2.3 Chiral algebras of superconformal theories For superconformal theories, the N = (0, 2), d = 2 super-Poincare algebra of symmetries is enhanced to Vir ⊕g SVir, where Vir denotes the left-handed Virasoro algebra (generated by the holomorphic stress-energy tensor) and the g SVir denotes the right-handed N = 2 – 10 – super-Virasoro algebra (generated by the corresponding anti-holomorphic currents). The left-handed algebra might be enlarged to the super-Virasoro as well (or even some larger W-algebra) if we have more symmetries on the left, but it graded-commutes with the N = 2 Virasoro on the right in any case. Let us restrict to the NS sector of the g SVir. The operators Q+ and Q+ can be identified as eG+ −1/2 and eG− −1/2 respectively — two of the fermionic generators of g SVir (we put tildes on g SVir and on its generators to emphasize that this is an anti-holomorphic algebra). In a conformal case, we have the radial quantization Hilbert space H, and we assume that it has an inner product, such that eG− 1/2 =  eG+ −1/2 † is a special supersymmetry generator. Part of the super-Virasoro algebra relations are: JHEP03(2018)079 { eG− −1/2, eG+ −1/2} = 2eL−1, { eG+ −1/2, ( eG+ −1/2)†} ≡{ eG+ −1/2, eG− 1/2} = 2eL0 −eJ0. (2.37) (2.37) Recall that in conformal case we have a state-operator correspondence. Therefore, instead of computing the operator cohomology, we can equivalently ask for the cohomology of eG+ −1/2 acting on the Hilbert space H. The second equation in (2.37) shows that, by the standard Hodge theory argument, this cohomology can be identified with the kernel of 2eL0 −eJ0. Also, in a unitary theory, it shows that 2eL0 −eJ0 ≥0. Now, every state in the Hilbert space is built by acting with eL−n, eJ−n, eG+ −α, eG− −α, n, α > 0 on a superconformal primary state. It is easy to see that all these operators except eG+ −1/2 increase the eigenvalue of 2eL0−eJ0, while eG+ −1/2 does not change it. Therefore, if the primary state has 2eL0 −eJ0 > 0, then all states in its superconformal family have 2eL0 −eJ0 > 0 and thus do not contribute to the cohomology. On the other hand, if some primary state |∆⟩has zero eigenvalue of 2eL0 −eJ0, then so does eG+ −1/2|∆⟩, while other states in the same conformal family have 2eL0 −eJ0 > 0. 2.3 Chiral algebras of superconformal theories But (2eL0 −eJ0)|∆⟩= 0 and the second equation of (2.37) imply that eG+ −1/2|∆⟩= 0. Therefore, in such a case there is just one non-trivial state in the superconformal family which contributes to the cohomology — the primary state itself. This way we prove that in the NS sector of a unitary N = (0, 2) superconformal theory there is an isomorphism: H(H, eG+ −1/2) ≃{Primaries of ] SVir with 2eL0 −eJ0 = 0} = {|ψ⟩∈H : eLn|ψ⟩= eJn|ψ⟩= eG+ α−1|ψ⟩= eG− α |ψ⟩= (2eL0 −eJ0)|ψ⟩= 0, n, α > 0}. H(H, eG+ −1/2) ≃{Primaries of ] SVir with 2eL0 −eJ0 = 0} = {|ψ⟩∈H : eLn|ψ⟩= eJn|ψ⟩= eG+ α−1|ψ⟩= eG− α |ψ⟩= (2eL0 −eJ0)|ψ⟩= 0, n, α > 0}. (2.38) Notice that these are what is usually called the chiral primaries with respect to ] SVir. In fact, this is essentially the construction of [21] applied to N = (0, 2) theories. In the N = (2, 2) case, [21] describe the chiral ring of the N = (2, 2) model by studying the set of (anti)chiral primaries both with respect to the left- and the right-moving super-Virasoro algebras. For the N = (0, 2) theories, we have in (2.38) only the chiral primary condition with respect to the right-moving super-Virasoro algebra. For that reason, the object we – 11 – get is not just the chiral ring: it involves holomorphic OPEs as part of its structure and is usually referred to as the W-algebra, or also chiral algebra. Another remark is that for N = (2, 2) theories, the chiral algebra that we study encodes the (c, c) and (a, c) rings of [21] as a part of its structure. Indeed, by considering the subspace of H(H, eG+ −1/2) annihilated by 2L0 −J0, where L0 and J0 are from the left- moving SVir algebra, we get the space {|ψ⟩∈H : (2L0 −J0)|ψ⟩= (2eL0 −eJ0)|ψ⟩= 0}, which is the space of chiral primaries with respect to both SVir and ] SVir, and therefore gives rise to the (c, c) ring under the OPE. Analogously, picking the subspace annihilated by L0 + J0, we get the (a, c) ring. 2.3 Chiral algebras of superconformal theories JHEP03(2018)079 One consequence of this is that in N = (2, 2) theories, the (anti)chiral primaries, which form the (c, c) or (a, c) rings of the theory, always show up in the chiral algebra as primaries of the left-moving ] SVir. In the simplest cases they will generate the whole chiral algebra, but as we will see later, there might be other primary operators in the algebra, which are not simply elements of the (c, c) or (a, c) ring. 2.4.1 Classical and quantum observables In the models we are going to study later in this paper, the chiral algebra will turn out to be tree-level exact. As we will argue, no loop corrections will contribute to the cohomology. However, despite our usual intuition that “tree level” means “classical”, it is important to understand that the quantum chiral algebra in the Q+-cohomology is not the same as the classical one. The distinction comes from the way we multiply operators. In classical field theory, to multiply fields we use the usual point-wise multiplication of functions on space-time. In quantum theory, even at the tree level, we should subtract singularities which appear when different operators collide, which for example gives the usual notion of normal ordering in CFT. It might happen (and it will happen in concrete examples) that the classical composite operator is Q+-closed, but the singular part we need to subtract to define the quantum operator is not Q+-closed. This subtlety should be taken into account when computing the chiral algebra of the theory. But still, as a step in this direction, it is useful to understand the structure of the classical chiral algebra first. 2.4.2 Classical observables and the cohomology Let us introduce the space of classical observables F and the space of classical superob- servables bF. We will sometimes refer to a generic field as φ and to a generic superfield as Φ. Both of these spaces classically carry the structures of supercommutative algebras. Definition 1. F is a supercommutative algebra of polynomials of fields φ and their deriva- tives ∂n −−∂m ++φ whose coefficients are analytic functions on a space-time, modulo classical equations of motion. In other words, F = Cω(M)[. . . , φ, ∂n −−∂m ++φ, . . . ]/I, where Cω(M) denotes analytic functions on M, and I denotes an ideal generated by the equations of motion and all their derivatives. – 12 – If the classical equations of motion do not depend on space-time coordinates explicitly (only through the coordinate-dependence of the generating fields), we can introduce: Definition 2. F0 is a subalgebra of F of observables which do not depend on a space-time point explicitly. In other words, it is generated by the same fields and their derivatives as F (and also modulo equations of motion), but the coefficients are taken to be just complex numbers rather than functions. There are straightforward superspace analogs of these: Definition 3. bF is a supercommutative algebra of polynomials of superfields Φ and their bosonic and super-derivatives ∂n −−∂m ++Dp +D q +Φ whose coefficients are analytic functions on superspace, modulo classical superspace equations of motion. JHEP03(2018)079 If the superspace equations of motion do not include any explicit dependence on a superspace point, i.e. if they have the form of a polynomial of generating fields ∂n −−∂m ++Dp +D q +Φ with complex coefficients, we again can define a subalgebra: Definition 4. bF0 is a subalgebra of bF of superobservables which do not depend on a space- time point explicitly. In other words, it is generated by the same superfields and their derivatives as bF (and also modulo superspace equations of motion), but the coefficients are taken to be just complex numbers rather then functions. Our goal is to compute the cohomology of Q+ acting on F in the situation when the equations of motion do not depend on the superspace point explicitly. The first observation is that the operator Q+ only acts on the generating fields of the algebra F, it does not act on the c-number functions which can possibly multiply these fields. Proprosition 2. For any a ∈F0 there exists a unique A ∈bF0 such that a = A . The problem which we are addressing is to find the cohomology of Q+ in F0. That is, the classes of fields a ∈F0 which satisfy [Q+, a] = 0, modulo those a for which a = [Q+, b], b ∈F0. Now from the Proposition 2, we know that there exist A, B ∈bF0, such that a = A| and b = B|. The equation [Q+, a] = 0 implies then Q+A| = 0. There is a small subtlety here which shows why it is correct to look for the cohomology of D+ rather than Q+: D+ acts on bF0 by definition, while Q+ = D+ +2iθ+∂+ does not, as it introduces an explicit dependence on θ (therefore Q+ acts from bF0 to a bigger space bF). However, we can write: D+A| = Q+A| = 0. But D+A ∈bF0, so we can apply Proposition 1 and conclude that D+A = 0. Analogously a = [Q+, b] implies A = D+B. This proves the Proprosition 3. The cohomology of Q+ in F0 (denoted H(F0)) is isomorphic to the cohomology of D+ in bF0 (denoted H( bF0)). The isomorphism H( bF0) →H(F0) is defined by taking the lowest component of the superfield. This proposition shows why in the rest of the paper we are going to study the coho- mology of D+. 2.4.3 The cohomology of Q+ in F0 and of D+ in b F0 2.4.3 The cohomology of Q+ in F0 and of D+ in b F0 Take an arbitrary A ∈bF0. A is some general superfield, and it can be expanded into components with respect to the Grassmann coordinates. The most basic property it satis- fies is that the supersymmetry transformations of its components are encoded in the way differential operators Q+ and Q+ act on it. This follows simply from the fact that this holds for the generating superfields from which A is constructed and the fact that we do not allow explicit dependence on the superspace coordinates in the algebra bF0. So we have: [Q+, A] = Q+A, (2.42) (2.42) JHEP03(2018)079 and the same for Q+. Supersymmetry relates all components of A and it is straightforward to see that: prosition 1. If the lowest component A of the superfield A ∈bF0 vanishes, then A = 0. Proprosition 1. If the lowest component A of the superfield A ∈bF0 vanishes, then A = 0. The algebras F0 and bF0 are related in an obvious way: any element of F0 can be found as a component of some superfield in bF0. In particular, we can always find a superfield A which contains a given element a ∈F0 as its lowest component. Moreover, supersymmetry defines this A uniquely, so: roprosition 2. For any a ∈F0 there exists a unique A ∈bF0 such that a = A . 2.4.2 Classical observables and the cohomology This means that it is enough to compute the cohomology of Q+ acting on F0. To be more rigorous, we can introduce operators of multiplication by xµ called m(xµ): ∀O ∈F, m(xµ)O = xµO, (2.39) (2.39) and notice that they commute with Q+. Then we can introduce a bigrading on F by saying that an explicit factor of (x0)n(x1)m has degree (n, m). After this it becomes obvious that H(F) ≃ M n,m≥0 Hn,m(F), (2.40) (2.40) where the bar over the right hand side means that we should actually consider a completion of this space with respect to some norm, because we have to allow infinte sums (series) to account for the possibility of having analytic functions as coefficients. As we mentioned, Q+ does not act in any way on xµ, and because of that: Hn,m(F) ≃H(F0). (2.41) (2.41) Therefore, from now on we will only study the cohomology in F0, which of course only makes sense when the equations of motion do not depend on the superspace point explicitly. Therefore, from now on we will only study the cohomology in F0, which of course only makes sense when the equations of motion do not depend on the superspace point explicitly. – 13 – 3 Landau-Ginzburg models The N = (0, 2) Landau-Ginzburg (LG) model is described by a set of chiral superfields Φi, i = 1 . . . n and Fermi superfields Λa, a = 1 . . . m. The action is (we assume summation over repeated indices, even if they both appear upstairs or downstairs; sometimes we will write the sum sign explicitly to avoid possible confusion): S = 1 π Z d2xd2θ  −i 2Φ i∂−−Φi −1 2Λ aΛa  + 1 π Z d2xdθ+ΛaJa(Φ)|θ +=0 + h.c., (3.1) (3.1) – 14 – where in general D+Λa = Ea(Φ) and P a Ea(Φ)Ja(Φ) = 0. The classical superspace equations of motion are: D+∂−−Φ i = iΛ a ∂Ea ∂Φi −2iΛa ∂Ja ∂Φi , D+∂−−Φi = −iΛa ∂E a ∂Φ i + 2iΛ a ∂Ja ∂Φ i , D+Λ a = −2Ja(Φ), D+Λa = 2Ja(Φ). D+∂−−Φ i = iΛ a ∂Ea ∂Φi −2iΛa ∂Ja ∂Φi , D+∂−−Φi = −iΛa ∂E a ∂Φ i + 2iΛ a ∂Ja ∂Φ i , D+Λ a = −2Ja(Φ), D+Λa = 2Ja(Φ). (3.2) D+Λa = 2Ja(Φ). (3.2) (3.2) JHEP03(2018)079 The supersymmetry currents of this theory are: The supersymmetry currents of this theory are: The supersymmetry currents of this theory are: S+++ = i 2ψi +∂++φ i, S+++ = −i 2ψ i +∂++φi, S+−−= i 2λaE a(φ) −iλ aJa(φ), S+−−= iλaJa(φ) −i 2λ aEa(φ). (3.3) (3.3) It is not hard to find a superfield S++ such that S+++ = i 2D+S++| and S+++ = −i 2D+S++|: S++ = 1 2D+ΦiD+Φ i. (3.4) (3.4) If we also introduce: W−= i 2Λ aEa −iΛaJa, T−−−−= ∂−−Φi∂−−Φ i + i 2Λa∂−−Λ a −i 2∂−−ΛaΛ a, (3.5) (3.5) then we find that: then we find that: ∂−−S++ = D+W−−D+W−, D+T−−−−= ∂−−W−, D+W−= 0. (3.6) + D+W−= 0. (3.6) (3.6) We see that these are precisely the relations (2.11) of the N = (0, 2) d = 2 supercurrent, and moreover, the component expansions of S++, W−and T−−−−, written as in (2.12), include the supersymmetry currents (3.3). Therefore, we have described the supercurrent multiplet of the theory (3.1). In a generic situation, it is not an R-multiplet, because there are no R-symmetries. We see that these are precisely the relations (2.11) of the N = (0, 2) d = 2 supercurrent, and moreover, the component expansions of S++, W−and T−−−−, written as in (2.12), include the supersymmetry currents (3.3). 3 Landau-Ginzburg models Therefore, we have described the supercurrent multiplet of the theory (3.1). In a generic situation, it is not an R-multiplet, because there are no R-symmetries. The algebra bF0 is a supercommutative algebra freely generated by superfields Φi, Φ i, Λa, Λ a and their derivatives (with respect to ∂−−, ∂++, D+ and D+ applied arbi- trary number of times) modulo the relations. The relations are: the ones that follow from {D+, D+} = 2i∂++ and D2 + = D 2 + = 0, the chirality conditions D+Φi = 0, D+Λa = Ea(Φ) and the superspace equations of motion as written above. All differential corollaries of the relations should also be included as relations of course. – 15 – It is not too hard to find a set of independent generators G, so that all the relations will be taken into account and we will have simply bF0 ≃C[G], a polynomial algebra generated by those generators. It is not too hard to find a set of independent generators G, so that all the relations will be taken into account and we will have simply bF0 ≃C[G], a polynomial algebra generated by those generators. We will now find this G. First of all, due to the chirality conditions, Φi can only appear with the D+ derivative (moreover, with at most one, because D2 + = 0), and Φ i — with D+. The chirality condition for Λa allows to replace D+Λa by Ea(Φ), while the equation of motion D+Λa = −2Ja(Φ) allows to replace D+Λa by an expression without derivatives. Therefore it is enough to consider only bosonic derivatives acting on Λa. However the simple relation: JHEP03(2018)079 2i∂++Λa = {D+, D+}Λa = D+D+Λa + D+D+Λa = D+Ea(Φ) + 2D+Ja(Φ) (3.7) (3.7) shows that ∂++ derivatives acting on Fermi superfields can also be removed. Therefore, in the generating set G, it is enough to include only ∂n −−Λa and ∂n −−Λ a, with n ≥0, and the appropriate derivatives of bosonic chiral superfields. By appropriate derivatives of bosonic chiral superfields we mean the following. First, we need to include ∂n −−Φi and ∂n −−Φ i with n ≥0. D+Φi and D+Φ i should also be included, but there is no need to include expressions like D+∂n −−Φi, because, as equations of motion for Φi show, D+∂−−Φi and D+∂−−Φ i can be replaced by expressions without derivatives. 3 Landau-Ginzburg models Expressions like ∂n ++Φi, D+∂n ++Φi and their complex conjugates have to be included, they cannot be reduced to expressions without derivatives. Finally, there is no need to include both ∂++ and ∂−− derivatives because of: 2i∂++∂−−Φ i = D+D+∂−−Φ i = D+  iΛ a ∂Ea ∂Φi −2iΛa ∂Ja ∂Φi  . (3.8) (3.8) So, to summarize, we write the generating set explicitly: G =  ∂n −−Φi, ∂n −−Φ i, ∂n ++Φi, D+∂n ++Φi, ∂n ++Φ i, D+∂n ++Φ i, ∂n −−Λa, ∂n −−Λ a, n ≥0 . (3.9) G =  ∂n −−Φi, ∂n −−Φ i, ∂n ++Φi, D+∂n ++Φi, ∂n ++Φ i, D+∂n ++Φ i, ∂n −−Λa, ∂n −−Λ a, n ≥0 . (3.9) To emphasize once again, we claim that: G =  ∂n −−Φi, ∂n −−Φ i, ∂n ++Φi, D+∂n ++Φi, ∂n ++Φ i, D+∂n ++Φ i, ∂n −−Λa, ∂n −−Λ a, n ≥0 . (3.9) G =  ∂n −−Φi, ∂n −−Φ i, ∂n ++Φi, D+∂n ++Φi, ∂n ++Φ i, D+∂n ++Φ i, ∂n −−Λa, ∂n −−Λ a, n ≥0 . (3.9) To emphasize once again, we claim that: To emphasize once again, we claim that: To emphasize once again, we claim that: To emphasize once again, we claim that: bF0 ≃C[G]. (3.10) (3.10) Using the relations satisfied by the fields, it is not hard to describe the action of D+ in terms of the generators in G. We have: Using the relations satisfied by the fields, it is not hard to describe the action of D+ in terms of the generators in G. We have: D+(∂n −−Φi) = 0, D+(∂n −−Φ i) = ∂n−1 −−  iΛ a ∂Ea ∂Φi −2iΛa ∂Ja ∂Φi  , D+(∂n ++Φi) = 0, D+(D+∂n ++Φi) = 2i∂n+1 ++ Φi, D+(∂n ++Φ i) = D+∂n ++Φ i, D+(D+∂n ++Φ i) = 0, D+(∂n −−Λa) = ∂n −−Ea(Φ), D+(∂n −−Λ a) = −2∂n −−Ja(Φ). (3.11) D+(∂n −−Φi) = 0, D+(∂n −−Φ i) = ∂n−1 −−  iΛ a ∂Ea ∂Φi −2iΛa ∂Ja ∂Φi  , (3.11) D+(∂n −−Λ a) = −2∂n −−Ja(Φ). (3.11) D+(∂n −−Λ a) = −2∂n −−Ja(Φ). (3.11) From these formulas we can guess that polynomials of ∂n −−Φi should be in the cohomology. However, we need some extra assumptions about Ea and Ja in order to move further. – 16 – 3.1 Quasihomogeneous case As we have already learned, it is interesting to consider the case when the theory has an R-symmetry. In such a case, we expect to have a stress-energy tensor in the cohomology. It is not hard to check that the following transformation: θ+ →e−iϵθ+, Φi →e−iϵαiΦi, Λa →e−iϵeαaΛa (3.12) , Λa →e−iϵeαaΛa (3.12) (3.12) is a symmetry of the classical action if and only if the following quasihomogeneity conditions are satisfied: JHEP03(2018)079 is a symmetry of the classical action if and only if the following quasihomogeneity conditions are satisfied: eαaJa + X i αiΦi ∂Ja ∂Φi = Ja, −eαaEa + X i αiΦi ∂Ea ∂Φi = Ea, (3.13) i −eαaEa + X i αiΦi ∂Ea ∂Φi = Ea, (3.13) (3.13) where αi and eαa are real numbers. It is a matter of a standard calculation to find the real conserved current j±± for this R-symmetry. It is then straightforward to write a superfield which has it as the lowest component. The answer is: R++ = −i 2 X i αi  Φi∂++Φ i −Φ i∂++Φi + 1 2 X i (1 −αi)D+ΦiD+Φ i R−−= −i 2 X i αi  Φi∂−−Φ i −Φ i∂−−Φi − X a eαaΛaΛ a. R−−= −i 2 X i αi  Φi∂−−Φ i −Φ i∂−−Φi − X a eαaΛaΛ a. (3.14) (3.14) It is not a coincidence that we called it R. In fact, one can check that the equations of motion imply ∂−−R++ + ∂++R−−= 0. Therefore, higher components of R are also conserved currents. This is the supercurrent multiplet discussed before provided we can find another real superfield Y−−−−(which has possibly improved stress-energy tensor T−−−− as its lowest component) satisfying the required constraints. As one can check from (2.11), the condition4 on Y−−−−is: D+Y−−−−= i 2D+∂−−R−−. (3.15) (3.15) Note that D+Y−−−−= −i 2D+∂−−R−−is then satisfied automatically. This defines Y−−−− uniquely up to an arbitrary function of x−, as Y−−−−→Y−−−−+ f(x−) preserves the above constraints. A simple computation allows to find a real superfield such that it satisfies these constraints: Y−−−−= X i h ∂−−Φi∂−−Φ i −αi 4 ∂2 −−(Φ iΦi) i + X a  i 2Λa∂−−Λ a −i 2∂−−ΛaΛ a  . (3.16) 4The fact that this Y−−−−together with Y++++ = i 4[D+, D+]J++ and Y++−−= i 4[D+, D+]J−−form a conserved superspace current then follows automatically. 3.1 Quasihomogeneous case 4The fact that this Y−−−−together with Y++++ = i 4[D+, D+]J++ and Y++−−= i 4[D+, D+]J−−form a conserved superspace current then follows automatically. – 17 – Therefore, we actually have a supercurrent multiplet described by R++, R−−and Y−−−−, which is, moreover, an R-multiplet in the terminology of [20], as reviewed in section 2.2. The Q+-cohomology class represented by the twisted stress-energy tensor eT−−−−from (2.24) promotes to the D+-cohomology class represented by the superfield: eY = Y−−−−−i 2∂−−J−− = X i h ∂−−Φi∂−−Φ i −αi 2 ∂−−(Φi∂−−Φ i) i + X a  i 2Λa∂−−Λ a −i 2∂−−ΛaΛ a + ieαa 2 ∂−−(ΛaΛ a)  . (3.17) (3.17) JHEP03(2018)079 This is precisely the stress-energy tensor in the cohomology as found in [11]. At first sight, one could think that this is the end of the story. However, there are some subtleties here, which we will now discuss. First of all, how is this R-multiplet related to the more general supercurrent multiplet which we found in (3.6)? The answer is simple. If we also define V−= i 2D+R−−, (3.18) (3.18) then R++, V−and Y−−−−form a supercurrent multiplet related to S++, W−and T−−−− by the improvement transformation: then R++, V−and Y−−−−form a supercurrent multiplet related to S++, W−and T−−−− by the improvement transformation: R++ = S++ + [D+, D+]U, V−= W−+ ∂−−D+U, Y−−−−= T−−−−+ ∂2 −−U, U = − X i αi 4 ΦiΦ i. (3.19) U = − X i αi 4 ΦiΦ i. (3.19) (3.19) Note that the superfield U cannot be represented as a real part of some local chiral su- perfield. Therefore this is an example of the improvement transformation allowed for the general supercurrent multiplet but not allowed for the R-multiplet. As we will see mo- mentarily, there might exist several R-multiplets which are not equivalent to each other as R-mulitplets (cannot be related to each other by the R-multiplet improvements), but they all are related to the same supercurrent multiplet S++, W−, T−−−−by the more general improvement described above. So now we will discuss the possibility of having several inequivalent R-multiplets. Note that the quasihomogeneity conditions (3.13) might have more than one solution. 3.1 Quasihomogeneous case (3.27) (3.27) – 19 – For the D+-closed element eY−−−−= Y−−−−−i 2J−−, we have: For the D+-closed element eY−−−−= Y−−−−−i 2J−−, we have: r the D+-closed element eY−−−−= Y−−−−−i 2J−−, we have: eY−−−−→eY−−−−−i 2∂−−(I−−+ ieI−−) = eY−−−−−i 2∂−−F−−. (3.28) (3.28) So the cohomology class [ eY−−−−] gets shifted by −i 2[∂−−F−−]. Let us summarize. We have the family of R-current multiplets generated by shifts using the superspace current I±±. In the cohomology this corresponds to having an extra left-moving U(1) current [F−−] generating an ambiguity of the stress-energy tensor in the cohomology, as we can do shifts of the cohomology class [ eY−−−−] by [∂−−F−−]. JHEP03(2018)079 But the conformal theory to which our LG model flows in the IR supposedly should have a unique stress-energy tensor, which thus gives a preferred stress-energy tensor for the chiral algebra in the Q+-cohomology. One can ask a natural question: which of the R-current multiplets above corresponds to the true stress-energy tensor of the theory in the IR? The answer is simple: the correct stress-energy tensor is the one, for which the U(1) current [F−−] is a primary operator in the cohomology, at least when it is possible to make it primary (we will discuss this point later). It is clear that this corresponds to extrem- izing the central charge of the corresponding Virasiro algebra (see the next subsection). To turn this statement into a criteria for picking the unique solution (αi, eαa) of (3.13), we need to understand first how to compute the operator product expansions (OPE) in the cohomology. 3.1 Quasihomogeneous case This corresponds to having an extra flavor U(1) symmetry, which can then mix with the R- symmetry to give another solution of (3.13) (in terms of current, this means to replace the R-symmetry current jR by jR + jF , where jF is a Flavor symmetry current). The flavor symmetry does not rotate the thetas, so it acts just as: Φi →e−iϵqiΦi, Λa →e−iϵeqaΛa. (3.20) Φi →e−iϵqiΦi, Λa →e−iϵeqaΛa. (3.20) – 18 – The condition that this is a symmetry of the classical action is: The condition that this is a symmetry of the classical action is: eqaJa + X i qiΦi ∂Ja ∂Φi = 0, −eqaEa + X i qiΦi ∂Ea ∂Φi = 0. (3.21) (3.21) We can see now that if {αi, eαa} is some solution of (3.13) and {qi, eqa} is some solution of (3.21), then {αi+qi, eαa+eqa} is another solution of (3.13). This is actually the ambiguity of the supercurrent multiplet which we were discussing before. In case we have extra superspace currents, the basic supercurrent mutiplet S++, W−, T−−−−can be shifted. Let us belabor this point somewhat further. JHEP03(2018)079 One can compute the current corresponding to the flavor symmetry (3.20) and find the real superfield which contains it as the lowest component: I−−= − X a eqaΛaΛ a −i 2 X i qi  Φi∂−−Φ i −Φ i∂−−Φi , I++ = −1 2 X i qiD+ΦiD+Φ i + i 2 X i qi  Φ i∂++Φi −Φi∂++Φ i . (3.22) (3.22) On shell these are conserved at the level of superfields: On shell these are conserved at the level of superfields: ∂++I−−+ ∂−−I++ = 0. (3.23) (3.23) One can do a small computation to check that the following superfield: F−−= − X a eqaΛaΛ a −i X i qiΦi∂−−Φ i (3.24) (3.24) is chiral on-shell, i.e. it satisfies D+F−−= 0 provided the equations of motion hold. In par- ticular, it means that this F−−gives rise to the left-moving U(1) current in the cohomology. But it is also true that: Re F−−= I−−. (3.25) (3.25) Therefore, there exists a local expression for the superfield eI−−: eI−−= Im F−−= −1 2 X i qi∂−−(ΦiΦ i). (3.26) (3.26) So, according to the general discussion from the section 2.2, we can shift the R-multiplet using this I±±. Recall that the shift is: R++ →R++ + I++, R−−→R−−+ I−−, Y−−−−→Y−−−−+ 1 2∂−−eI−−. 3.1.1 The OPE in the cohomology The component action of the model that we study is: (3.29) S = SD + SF , (3.29) where the D-term action is: where the D-term action is: SD = 1 π Z d2x  −∂−−φ i∂++φi −i 2ψ i +∂−−ψi + −iλ a∂++λa −1 2GaG a + i 2∂iEa(φ)λ aψi + −i 2∂iE a(φ)ψ i +λa + 1 2Ea(φ)E a(φ)  , (3.30) (3.30) and the F-term is: and the F-term is: SF = 1 π Z d2x  GaJa(φ) + G aJa(φ) −iλaψi +∂iJa(φ) −iλ aψ i +∂iJa(φ)  . (3.31) (3.31) All couplings come from the E and J-type superpotentials. Note that φ is dimensionless in 2d, (and fermions are of dimension 1/2), therefore both Ea and Ja should have dimension 1. We will include an explicit coupling µ of dimension 1 in the theory, and replace Ea →µEa and Ja →µJa in the above action, thinking of Ea(φ) and Ja(φ) as dimensionless functions of dimensionless fields φi now. In section 2.2.3 we saw that in order to compute the OPE of the cohomology classes we can turn offall dimensionful couplings in the theory. In particular, we can tune µ to – 20 – zero. This will remove all interactions from the above action. Thus to compute the OPE of the cohomology classes, it is enough to consider the free theory: zero. This will remove all interactions from the above action. Thus to compute the OPE of the cohomology classes, it is enough to consider the free theory: S0 = 1 π Z d2x  −∂−−φ i∂++φi −i 2ψ i +∂−−ψi + −iλ a∂++λa −1 2GaG a  . (3.32) (3.32) Its correlators can be conveniently combined into superfield correlators: Its correlators can be conveniently combined into superfield correlators: D Φ i(x, θ′)Φj(y, θ) E = δij log r−−r++ , D Λ a(x, θ′)Λb(y, θ) E = δab i r−−, (3.33) (3.33) JHEP03(2018)079 where r−−= x−−−y−−, r++ = x++ −y++ + iθ+θ + + iθ′+θ ′+ + 2iθ ′+θ+. (3.34) (3.34) Now we want to compute the OPE of eY from (3.17) with itself. eY represented a candidate stress-energy tensor in the cohomology and was given by: Now we want to compute the OPE of eY from (3.17) with itself. 3.1.1 The OPE in the cohomology (3.40) (3.40) In a generic situation, this equation allows to pick a unique solution (αi, eαa) and write a correct stress-energy tensor. If the action admits f independent U(1) flavor symmetries described by charges (qn i , eqn a), n = 1 . . . f, we should write the equation (3.40) for each of them. Again, generically, one can expect this to give a condition to pick the unique stress-energy tensor in the cohomology. In a generic situation, this equation allows to pick a unique solution (αi, eαa) and write a correct stress-energy tensor. If the action admits f independent U(1) flavor symmetries described by charges (qn i , eqn a), n = 1 . . . f, we should write the equation (3.40) for each of them. Again, generically, one can expect this to give a condition to pick the unique stress-energy tensor in the cohomology. JHEP03(2018)079 However, non-generic situations are possible, when this equation might either not fix the stress-energy tensor completely, or might have no solutions at all. We will explore such examples for the N = (2, 2) theories later: in such cases, indices a and i take the same set of values, we have eαa = αi=a, and flavor symmetries (which should differ from the N = (2, 2) R-symmetries) have eqa = qi=a. Therefore the equation (3.40) reduces to just P i qi = 0, which either holds identically and therefore imposes no constraints on αi, or does not hold at all. In a former situation, the ambiguity of choosing the unique stress- energy tensor is not removed and is just present in the IR. In a latter situation, there is no solution to (3.40), which means that [F−−] cannot be made primary by choosing the proper stress-energy tensor. There is an unwanted central term in the eYF−−OPE, which cannot be removed and signals that there is an obstruction for the IR compact CFT to exist. We will see in examples that there is a flat direction in the potential. This is usually interpreted as a lack of normalizable vacuum. One can also note that if we decide to study the gauge theory obtained by gauging the flavor symmetry with charges (qi, eqa), then the above equations become related to anomalies. 3.1.1 The OPE in the cohomology Namely, the central term in the F−−F−−OPE becomes just the gauge anomaly (so it is the t’Hooft anomaly in the LG model context): we need P i q2 i −P a eq2 a = 0 for the gauge theory to exist [5]. Then equation (3.40) becomes the condition for the R-symmetry defined by charges (αi, eαa) to be non-anomalous [11]. 3.1.1 The OPE in the cohomology eY represented a candidate stress-energy tensor in the cohomology and was given by: eY = X i h 1 −αi 2  ∂−−Φi∂−−Φ i −αi 2 Φi∂2 −−Φ ii + X a  i 2(1 + eαa)Λa∂−−Λ a −i 2(1 −eαa)∂−−ΛaΛ a  . (3.35) eY = X i h 1 −αi 2  ∂−−Φi∂−−Φ i −αi 2 Φi∂2 −−Φ ii + X a  i 2(1 + eαa)Λa∂−−Λ a −i 2(1 −eαa)∂−−ΛaΛ a  . (3.35) (3.35) Using the OPE above, we find that: Using the OPE above, we find that: eY(x) eY(y) ∼ c/2 (x−−−y−−)4 + 2 eY(y) (x−−−y−−)2 + ∂−−eY(y) x−−−y−−+ {Q+, . . . }, (3.36) (3.36) where the notation {Q+, . . . } for the unimportant term is slightly inaccurate: what we actually mean is that the term that we drop becomes Q+-exact after we put θ+ = θ + = 0, but as a shorthand we will denote it as {Q+, . . . }. The central term is: c = X i (2 −6αi + 3α2 i ) + X a (1 −3eα2 a). (3.37) (3.37) This matches the result of [11] and shows that we indeed have the stress-energy tensor in the cohomology. Before we found that in case there is a U(1) flavor symmetry, there is another D+- closed superfield F−−, which gives rise to the left-moving U(1) current in the cohomology. Recall that: F−−= − X a eqaΛaΛ a −i X i qiΦi∂−−Φ i. (3.38) (3.38) We can similarly compute its OPE: We can similarly compute its OPE: F−−(x)F−−(y) ∼ P i q2 i −P a eq2 a (x−−−y−−)2 + {Q+, . . . }. (3.39) (3.39) This current creates ambiguity, as we explained before: we can replace eY by eY + λ∂−−F−−for any λ ∈R and get another stress-energy tensor in the cohomology. The – 21 – unique one is picked by requiring that the [F−−] cohomology class be primary with respect to the correct stress-energy tensor, whenever it is possible to impose such a condition. Equivalently, since shifting by the current shifts the central charge, one can ask that the value of the central charge (3.37) be extremal with respect to the shifts (αi, eαa) →(αi + λqi, eαa + λeqa). Any of these two criteria of course give the same equation:5 X i qi(1 −αi) + X a eqaeαa = 0. 5While (3.37) is actually a left-moving central charge cL, this is also equivalent to the cR- extremization [23] since cL −cR is fixed by the gravitational anomaly. 3.1.2 Classical and quantum chiral algebra When we were discussing the OPE in the cohomology, we argued that, as a consequence of conformal invariance, there should be no dimensionful couplings present in the OPE. We can generalize that further to say that the chiral algebra should not depend on any dimensionful couplings at all. Any algebraic relations that involve dimensionful coupling constants would violate the combination of scale and Lorentz invariance. One of the basic facts about theories we study is that they are free in the UV. In fact, this provides an alternative argument for why the singular part of the OPE is independent – 22 – of couplings. Short-distance singularities of operators are simply governed by the free theory, even before passing to the Q+ cohomology (however, we find the argument based on Lorentz and scale invariance in the cohomology to be more transparent in our case). Independence of chiral algebras on dimensionful couplings implies a useful property, which can be thought of as a sort of non-renormalization theorem. The exact quantum chiral algebra in our theories is “almost determined” by the classical chiral algebra (and might be called “almost tree level exact”, although this name could be misleading). All we need to do to find the quantum counterpart is renormalize composite operators. Composite operators can be thought of as several fundamental fields brought into one point, and in the process we should subtract short-distance singularities. It might well happen (and will happen in concrete examples later) that even though the classical operator is in Q+ cohomology, the infinite piece you have to subtract is not annihilated by Q+. In this way, renormalization of composite operators representing classical cohomology classes can remove part of the classical cohomology. The claim is that what you obtain using this procedure is the exact answer. JHEP03(2018)079 To understand why this is true, we will think of an exact quantum theory as a set of local operators, which satisfy OPE relations and operator equations of motion. As we said, short-distance singularities are governed by the free theory, so singular part of the OPE does not care about interactions and operator equations of motion. Non-singular part of the OPE can be thought of as a definition of composite operators, and this is the point where we should be careful, as already noted before. The remaining thing we need to care about are operator equations of motion. 6In fact, the right hand side of (3.2) is already proportional to µ, so additional terms would be multiplied by higher powers of µ. 3.1.2 Classical and quantum chiral algebra If we stare at classical equations (3.2), we can understand that they do not have any short-distance singularities and can be made into operator equations. The question one might ask is whether they receive any corrections at the quantum level. If there were such corrections, they would be a result of interactions and would depend on the dimensionful coupling6 µ. If this could change the answer for chiral algebra, it would mean that the algebra depends on a dimensionful constant µ. We know that this is impossible on general grounds, so we expect that quantum corrections to operator equations of motion are not important for the chiral algebra computation. In fact, thinking slightly more general, the situation might be even simpler. Suppose we have some renormalizable field theory, and we define it in the path integral approach. This means that we choose our favorite regularization to make path integral finite-dimensional, define the action and the measure in this regularization and add counterterms, if needed. Or, alternatively, think in terms of bare fields and couplings, without any counterterms. The standard way to derive equations of motion which hold under correlators, i.e., operator equations of motion, is through integration by parts. For renormalizable field theories defined in this way, these equations of motion hold exactly when written in terms bare fields. If we write them in terms of physical fields and counterterms, then counterterms of course contribute to equations of motion, but their role is to renormalize composite – 23 – operators that appear in equations of motion. This becomes very clear in the example of the λφ4 theory. The equation of motion of the λφ4 with counterterms is: (□+ m2)φ = λφ3 + δmφ + δφ□φ + δλφ3, (3.41) (3.41) and by some simple manipulations with diagrams, one can see that these three terms on the right are precisely what one needs to define a composite operator λφ3. The mass counterterm δmφ removes singularity coming from the self-contraction in φ3, while the other two remove singularities coming from contractions between φ3 and one insertion of the interaction vertex λφ4/4. It is quite obvious that this continues to higher orders of perturbation theory, simply because the theory is renormalizable and has only these three counterterms. JHEP03(2018)079 It is not completely clear how general this argument is and whether it holds for gauge theories, but it definitely works for our LG models. 3.1.2 Classical and quantum chiral algebra Moreover, it is possible to show that our models do not need any counterterms at all. So our conclusion is that equations (3.2) hold exactly once we properly define compos- ite operators appearing there. This supports our claim that to compute quantum chiral algebra, we need to find the classical one and then check which part of it survives after the renormalization of composite operators. All these statements are true in perturbation theory. They might not hold if non- perturbative corrections become relevant. For example, instantons might lift cohomology classes [15], and this has to be studied separately. In our case we assume that the worldsheet and the target are topologically trivial, so non-perturbative corrections are not expected. 3.1.3 Non-abelian global symmetries (3.44) (3.44) If we write [tα, tβ] = ifγ αβtγ, then the OPE of these currents is given by: If we write [tα, tβ] = ifγ αβtγ, then the OPE of these currents is given by: Jα(x)Jβ(y) ∼tr(tαtβ) −tr(τατβ) (x−−−y−−)2 + fγ αβJγ(y) x−−−y−−+ {Q+, . . . }. (3.45) (3.45) We have tr(tαtβ) = 2xΦδαβ and tr(τατβ) = 2xΛδαβ, where xΦ and xΛ are Dynkin indices of the representations RΦ and RΛ respectively. Therefore, in the cohomology we find a current algebra of G at the level r = 2(xΦ −xΛ). We have tr(tαtβ) = 2xΦδαβ and tr(τατβ) = 2xΛδαβ, where xΦ and xΛ are Dynkin indices of the representations RΦ and RΛ respectively. Therefore, in the cohomology we find a current algebra of G at the level r = 2(xΦ −xΛ). 3.1.3 Non-abelian global symmetries In addition to U(1) global symmetries, the action may also have non-abelian linearly realized global symmetries that commute with SUSY. They generally are of the form Φi →Ai jΦj, Λa →Ba b Λb. The kinetic part of the action implies that A ∈U(NΦ) and B ∈U(NΛ), where NΦ is the number of chiral superfields Φi, i = 1 . . . NΦ, and NΛ is the number of Fermi superfields Λa, a = 1 . . . NΛ. It is clear that by a unitary transformation Φi →U i jΦj, Λa →V a b Λb one can always bring A and B into the diagonal form, and in such a basis they will describe just the U(1) global symmetry. Therefore, in order to have something new compared to the previous discussion, we assume that the action has some U(1) global symmetries and, on top of that, also has some non-abelian symmetries. Altogether, they close to a subgroup G ⊂ U(NΦ) × U(NΛ). The free theory has the full U(NΦ) × U(NΛ) symmetry, which is then broken to the subgroup G by the E and J superpotentials. Embedding G ⊂U(NΦ) × U(NΛ) defines an (NΦ + NΛ)-dimensional representation of G on superfields of our model. This representation is reducible and can be decomposed as a direct sum of an NΦ-dimensional representation RΦ on chiral superfields and an NΛ- dimensional representation RΛ on Fermi superfields. Let the Hermitian generators of this subgroup in the representation RΦ be called tα, α = 1 . . . |G|, and in the representation RΛ – 24 – — τα, α = 1 . . . |G|. The infinitesimal transformation is: — τα, α = 1 . . . |G|. The infinitesimal transformation is: Φi →Φi + iϵα(tα)i jΦj, Λa →Λa + iϵα(τα)a bΛb. (3.42) (3.42) The condition on J and E for this to be a symmetry is: The condition on J and E for this to be a symmetry is: (tα)i jΦj∂iJa(Φ) + (τα)b aJb(Φ) = 0, (tα)i jΦj∂iEa(Φ) −(τα)a bEb(Φ) = 0. (3.43) (3.43) JHEP03(2018)079 It is straightforward to repeat what we had done for abelian symmetries and to find the corresponding element in the D+-cohomology: Jα = (τα)a bΛbΛ a + i(tα)i jΦj∂−−Φ i. 4 N = (2, 2) models If in a general N = (0, 2) LG model as described before we put Ea = 0, take a to be the same sort of index as i, i.e., just put NΛ = NΦ (recall that everything is topologically trivial in our discussion) and take Ja(Φ) = ∂W(Φ) ∂Φi=a for some holomorphic superpotential W(Φ), we get a general N = (2, 2) LG model. In such a case (0, 2) superfields are promoted to (2, 2) chiral superfields: Φi = Φi + i √ 2θ−Λi −iθ−θ −∂−−Φi. (4.1) (4.1) With N = (2, 2) supersymmetry, we can go further in the discussion of general prop- erties of the chiral algebra in the Q+-cohomology. First of all, let us get rid of the trivially reducible case. Suppose that we can organize superfields Φi into two nonempty sets: {Φ1, Φ2, . . . , Φs}, {Φs+1, Φs+2, . . . , ΦNΦ}, so that the superpotential can be written as a sum: With N = (2, 2) supersymmetry, we can go further in the discussion of general prop- erties of the chiral algebra in the Q+-cohomology. First of all, let us get rid of the trivially reducible case. Suppose that we can organize superfields Φi into two nonempty sets: {Φ1, Φ2, . . . , Φs}, {Φs+1, Φs+2, . . . , ΦNΦ}, so that the superpotential can be written as a sum: W(Φ) = W (1)(Φ1, Φ2, . . . , Φs) + W (2)(Φs+1, Φs+2, . . . , ΦNΦ), (4.2) (4.2) This superpotential just describes 2 separate LG models which do not interact with each other. The space of observables in such a model is just the graded-symmetric tensor product of the spaces for each of the two models, and the supercharge is the sum Q+ = Q (1) + +Q (2) + , where each term in the sum acts on the corresponding factor in the graded-symmetric tensor product. It is a simple algebraic exercise to prove that the cohomology of such a Q+ is just the graded-symmetric tensor product of the cohomologies of Q (1) + and Q (2) + . – 25 – Therefore, without any loss of generality, it is enough to study superpotentials which cannot be decomposed as in (4.2), and can never be brought into such a decomposable form by a holomorphic change of coordinates on the target. 4 N = (2, 2) models We will assume this from now on. Note that it was shown in [8] that with such an assumption, no accidents happen in the IR, which also simplifies life a lot. Since we put Ea = 0, quasihomogeneity conditions (3.13) now always have at least one solution, eαa = 1, ∀a, αi = 0, ∀i. Therefore, according to our previous discussion, there is always a stress-energy tensor in the cohomology. It is interesting, however, to study the case when W(Φ) is quasi-homogeneous itself: JHEP03(2018)079 X i βiΦi ∂W ∂Φi = W(Φ). (4.3) (4.3) After all, as was noted in [24], this is the case most relevant for studying the IR fixed point of the LG model. With this property, if we take αi = βi and eαa = βi=a, we get another solution of (3.13). In other words, there exists a U(1) flavor symmetry corresponding to the solution qi = βi, eqa = βi=a −1 of (3.21). If there is only one such flavor symmetry, we can see that the equation (3.40) picks αi = βi and eαa = βi=a as defining the correct stress-energy tensor. Indeed, these values satisfy (3.40), while another solution, eαa = 1, αi = 0, inserted in (3.40), gives P i qi + P a eqa = P i(2βi −1), which is generically non-zero. The last sum being zero corresponds to various degenerate cases, for example if superpotential is just a quadratic polynomial (which means that all fields are massive, the IR theory is trivial and the chiral algebra should be trivial too). We will not concentrate on such cases. On the other hand, there can be more flavor symmetries in the model: Φi →e−iγiϵΦi, (4.4) (4.4) if one can find such a system of charges γi that: if one can find such a system of charges γi that: if one can find such a system of charges γi that: X i γiΦi ∂W ∂Φi = 0. (4.5) (4.5) This gives a solution qi = γi, eqa = γi=a of (3.21). Note that both the solution qi = βi, eqa = βi=a −1 and the solution qi = γi, eqa = γi=a describe flavor symmetries from the N = (0, 2) point of view, since they just satisfy (3.21). 4 N = (2, 2) models However, from the N = (2, 2) point of view, only the latter one is a flavor symmetry, while the former one becomes the left-handed R-symmetry of the N = (2, 2) SUSY, which is seen from the fact that Φ’s and Λ’s charges differ by one. The action of the LG model in the (2, 2) superspace is: S = 1 4π Z d2xd4θ Φ iΦi + 1 4π Z d2xd2θ W(Φ) + 1 4π Z d2xd2θ W(Φ) (4.6) (4.6) The superspace equations of motion are simply: The superspace equations of motion are simply: D+D−Φ i = ∂W ∂Φi . (4.7) D+D−Φ i = ∂W ∂Φi . (4.7) – 26 – – 26 – As was first noted in [10], we can find an element in the D+-cohomology represented by the (2, 2) superfield: J = X i 1 −βi 2 D−ΦiD−Φ i −iβiΦi∂−−Φ i  , (4.8) (4.8) which can then be expanded in components with respect to θ−and θ −: the lowest com- ponent is the left-handed R-current, the top component is the stress-energy tensor (which, using our earlier N = (0, 2) terminology, corresponds to the solution αi = βi, eαa = βi=a of (3.13)), and the fermionic components are the two left-handed supersymmetries. There- fore, this J generates a left-moving N = 2 superconformal algebra in the D+-cohomology. If there exist additional U(1) flavor symmetries characterized by weights γi satisfy- ing (4.5), then there is another D+-cohomology class represented by: JHEP03(2018)079 If there exist additional U(1) flavor symmetries characterized by weights γi satisfy ing (4.5), then there is another D+-cohomology class represented by: Ψ = 1 2 X i γiΦiD−Φ i, (4.9) (4.9) 7The argument is as follows. Presence of the central term in (4.17) implies through the operator-state correspondence that there is a state |ψ⟩in the IR CFT such that G− +1/2|ψ⟩= κ|0⟩+ Q+|φ⟩, where G− +1/2 is one of the superconformal generators, |0⟩is the vacuum state and |φ⟩is some state. Taking the dimension- zero component of this equality, we can assume that ψ has dimensions (1/2, 0), so that G− +1/2|ψ⟩has dimension zero. Since in a unitary theory there are no operators of negative dimension, Q+|φ⟩should not be there: G− +1/2|ψ⟩= κ|0⟩. Invariance of the vacuum implies G+ −1/2G− +1/2|ψ⟩= 0. Since in a unitary theory (G− +1/2)† = G+ −1/2, by multiplying with ⟨ψ|, the last equality implies G− +1/2|ψ⟩= 0, which gives a contradiction unless κ = 0. so the derivative: (4.15) θ− 12 = θ− 1 −θ− 2 , θ − 12 = θ − 1 −θ − 2 , θ12 = θ1 −θ2 , θ12 = θ1 −θ2 , r12 = x−− 1 −x−− 2 + iθ − 1 θ− 2 −iθ − 2 θ− 1 , (4.14) r12 = x−− 1 −x−− 2 + iθ − 1 θ− 2 −iθ − 2 θ− 1 , (4.14) (4.14) and the central charge is: c = 3 X i (1 −2βi). (4.15) (4.15) Equation (4.13) encodes the N = 2 superconformal algebra with the central charge c (this equation, but in slightly different conventions, was present in [10]). Of course we could have obtained the same value of the central charge using the more general equation (3.37), which holds for more general N = (0, 2) LG models. One would have to put αi = βi, eαa = βi=a there. JHEP03(2018)079 Notice that the central charge (4.15) is linear in βi. This means that if we have U(1) flavor symmetries such that (4.5) holds, we can no longer get rid of the ambiguity βi →βi + λγi by simply asking the central charge to take the extremal value. This is related to the fact that the OPE of the cohomology class represented by (4.9) with itself is regular: Ψ(x1, θ1)Ψ(x2, θ2) ∼{Q+, . . . }. (4.16) (4.16) that the OPE of [Ψ] with [J ] is the same as with [J +λD−Ψ], ∀λ ∈R. The J Ψ OPE is: J (x1, θ1)Ψ(x2, θ2) ∼κ θ− 12 (r12)2 −θ− 12θ − 12 (r12)2 Ψ(x2, θ2) −iθ− 12 r12 D−Ψ(x2, θ2) −2θ− 12θ − 12 r12 ∂−−Ψ(x2, θ2) − i r12 Ψ(x2, θ2) + {Q+, . . . }, (4.17) (4.17) where κ = P i γi. Compare this with what one expects for the OPE of J with some superconformal primary superfield P: where κ = P i γi. Compare this with what one expects for the OPE of J with some superconformal primary superfield P: J (x1, θ1)P(x2, θ2) ∼−2θ− 12θ − 12 (r12)2 ∆P(x2, θ2) −iθ− 12 r12 D−P(x2, θ2) −iθ − 12 r12 D−P(x2, θ2) −2θ− 12θ − 12 r12 ∂−−P(x2, θ2) − i r12 qP(x2, θ2) + {Q+, . . . }, (4.18) (4.18) where ∆is the conformal dimension of P and q is its R-charge. so the derivative: so the derivative: so the derivative: D−Ψ = X i γi 1 2D−ΦiD−Φ i + iΦi∂−−Φ i  (4.10) (4.10) generates ambiguity, because we can replace J →J + λD−Ψ, ∀λ ∈R. Of course, this is still the same ambiguity of the N = (0, 2) stress-tensor multiplet related to U(1) flavor symmetries that we were discussing before. The only difference is that by now we have dealt with the U(1) global symmetry which is the left-handed R-symmetry from the N = (2, 2) point of view (it was described by the charges qi = βi, eqa = βi=a −1), and what we are left with in (4.9) corresponds to the actual N = (2, 2) flavor symmetry. Similar to what we had for a more general N = (0, 2) case, we could have analyzed this ambiguity using the N = (2, 2) supersurrent multiplet, especially since its structure is described in details in the appendix C of [20]. However, we chose not to do this, as it would not give us anything essentially new compared to what we have already understood. Previous discussion of the OPE in the cohomology being determined by the free prop- agators of course still holds. The free propagator of chiral superfields is: D Φ i(x1, θ1)Φj(x2, θ2) E = δij log R−− 12 R++ 12  , (4.11) (4.11) where where R−− 12 = x−− 1 −x−− 2 + iθ− 1 θ − 1 + iθ− 2 θ − 2 + 2iθ − 1 θ− 2 , R++ 12 = x++ 1 −x++ 2 + iθ+ 1 θ + 1 + iθ+ 2 θ + 2 + 2iθ + 1 θ+ 2 . (4.12) (4.12) We can compute the OPEs: We can compute the OPEs: J (x1, θ1)J (x2, θ2) ∼− c 3(r12)2 −2θ− 12θ − 12 (r12)2 J (x2, θ2) −iθ− 12 r12 D−J (x2, θ2) −iθ − 12 r12 D−J (x2, θ2) −2θ− 12θ − 12 r12 ∂−−J (x2, θ2) + {Q+, . . . }, (4.13) – 27 – – 27 – where θ− 12 = θ− 1 −θ− 2 , θ − 12 = θ − 1 −θ − 2 , r12 = x−− 1 −x−− 2 + iθ − 1 θ− 2 −iθ − 2 θ− 1 , (4.14) c = 3 X i (1 −2βi). so the derivative: What we see is that for non-zero values of κ, Ψ is non-primary, and moreover it is not a descendant of any primary, as can be seen from unitarity and global superconformal invariance of the vacuum of the IR theory.7 Therefore, the non-zero κ becomes an obstruction for the IR CFT to exist. where ∆is the conformal dimension of P and q is its R-charge. What we see is that for non-zero values of κ, Ψ is non-primary, and moreover it is not a descendant of any primary, as can be seen from unitarity and global superconformal invariance of the vacuum of the IR theory.7 Therefore, the non-zero κ becomes an obstruction for the IR CFT to exist. 7The argument is as follows. Presence of the central term in (4.17) implies through the operator-state correspondence that there is a state |ψ⟩in the IR CFT such that G− +1/2|ψ⟩= κ|0⟩+ Q+|φ⟩, where G− +1/2 is one of the superconformal generators, |0⟩is the vacuum state and |φ⟩is some state. Taking the dimension- zero component of this equality, we can assume that ψ has dimensions (1/2, 0), so that G− +1/2|ψ⟩has dimension zero. Since in a unitary theory there are no operators of negative dimension, Q+|φ⟩should not be there: G− +1/2|ψ⟩= κ|0⟩. Invariance of the vacuum implies G+ −1/2G− +1/2|ψ⟩= 0. Since in a unitary theory (G− +1/2)† = G+ −1/2, by multiplying with ⟨ψ|, the last equality implies G− +1/2|ψ⟩= 0, which gives a contradiction unless κ = 0. – 28 – This happens for example in a model with two superfields X and Y and superpotential W = XY 2. Notice that since the ΨΨ OPE is regular, so is the D−ΨD−Ψ OPE. The absence of central term in it means, as we have mentioned in section 3.1.1, that the corresponding flavor symmetry can be gauged without encountering gauge anomalies. Possible non-zero value of κ then becomes the anomaly for the right-handed R-symmetry. This would be relevant if we were studying gauge theories. The theory can also have non-abelian flavor symmetries, which lead, as we have argued before, to the current algebra in the cohomology. In our discussion of general (0, 2) theories, the level of this current algebra was given by the difference of Dynkin indices: r = 2xΦ−2xΛ. The first term here corresponded to the way flavor symmetry acted on Φ’s, and the second — on Λ’s. 5 Examples In this section we will consider a few examples of applications of our machinery to the N = (2, 2) LG models, where we can say something about the chiral algebra and therefore draw some conclusions about the theory to which the model flows in the IR. so the derivative: In the (2, 2)-supersymmetric case, the flavor symmetry acts in the same way on Φ’s and Λ’s, as they are just components of the (2, 2) chiral superfields Φi. So xΦ = xΛ. We conclude that the current algebra in the cohomology corresponding to some flavor symmetry of the N = (2, 2) supersymmetric LG model always has level zero. JHEP03(2018)079 5.1 Degenerate examples Consider the theory of two chiral superfields X and Y with superpotential W = XY 2. (5.1) (5.1) This theory has a non-trivial flavor symmetry. A possible charge assignment is: γX = 2, γY = −1, so that This theory has a non-trivial flavor symmetry. A possible charge assignment is: γX = 2, γY = −1, so that γY = −1, so that γXX ∂W ∂X + γY Y ∂W ∂Y = 0. (5.2) (5.2) As we know from the equation (4.9), there is an extra operator Ψ in the cohomology as a result of this flavor symmetry. Since γX + γY = 1 ̸= 0, the OPE (4.17) tells us that this operator is not primary. Moreover, as we explained in the Footnote 7, an operator satisfy- ing (4.17) cannot be made primary in a unitary CFT with invariant vacuum. Therefore, its existence indicates that the RG flow does not end at any compact CFT: the deep IR theory does not have a normalizable vacuum. In fact, the superpotential has a flat direction Y = 0, and we can conclude that the low-energy mode describing propagation along this flat direction renders vacuum non-normalizable. One can get a compact conformal fixed point if we add a perturbation ϵX2n+1 to the superpotential. This actually corresponds to having the D series of minimal models at the IR fixed point, with the exact choice of the model depending on n, even for small ϵ. By sending ϵ →0, the compact IR fixed point will most likely go to infinity, signaling that the degenerate theory W = XY 2 will – 29 – behave differently. It would be great to have a better understanding of such non-compact IR theories. This flavor symmetry could be gauged, however, as we have noted before, this would make right-handed R-symmetry anomalous because of γX + γY ̸= 0. By considering a slightly different superpotential, namely: By considering a slightly different superpotential, namely: W = X2Y 2, (5.3) (5.3) we get again a theory with flavor symmetry, but the charges now can be chosen as γX = 1, γY = −1, so that γX + γY = 0. Therefore, the bad central term does not appear in (4.17), and the theory should have a better IR limit, even though the superpotential still has flat directions. 5.2 N = 2 minimal models A series of N = (2, 2) LG models are known to flow in the IR to the N = (2, 2) minimal models. These superconformal theories are relatively simple. The central charge is given by [25–29]: 3k A series of N = (2, 2) LG models are known to flow in the IR to the N = (2, 2) minimal models. These superconformal theories are relatively simple. The central charge is given by [25–29]: c = 3k k + 2, k ≥1, (5.4) (5.4) and there is a known spectrum of possible superconformal primaries. The A-D-E classifi- cation of modular-invariant theories is known [30–33], and the corresponding LG superpo- tentials have been identified before. So, we can try to compute the chiral algebra both for the LG model and for the minimal model which is supposed to arise in the IR, therefore providing more evidence for this relation, as well as demonstrating the power of chiral algebras. 5.1 Degenerate examples For example, if we gauge this flavor symmetry, the flat directions are removed and we still get a theory with the right-handed R-symmetry. We are not going to study this example any further. JHEP03(2018)079 5.2.1 The Ak+1 series For a given k, the diagonal Ak+1 minimal model is the simplest one. Its set of primaries has a subset of k + 1 fields which are chiral primary with respect to ] SVir. Let us call them Os, s = 0, . . . k, where O0 = 1 is the identity operator and Os has left-right conformal dimensions (h, h) = ( s 2(k+2), s 2(k+2)) and left-right U(1) charges (q, q) = ( s k+2, s k+2). As we see, they all are chiral primaries with respect to both SVir and ] SVir. Therefore, together with the N = 2 currents, they generate the chiral algebra of the theory, as well as the anti-chiral algebra abtained analogously by taking the cohomology of Q−. We expect to get the same result from the LG model description. It is obtained by considering only one chiral superfield Φ with the superpotential: W(Φ) = Φk+2 k + 2. (5.5) (5.5) The equations of motion are D+D−Φ −Φk+1 = 0, D−D+Φ −Φ k+1 = 0. (5.6) (5.6) – 30 – – 30 – Differentiating these equations and multiplying them by arbitrary polynomials of Φ, Φ and their derivatives, we get a differential ideal I. The algebra bF0 consists of arbitrary poly- nomials of variables ∂n +∂m −Dk +Dp −Φ and ∂n +∂m −D k +D p −Φ for non-negative integers n, m, k, p, modulo the ideal I: Differentiating these equations and multiplying them by arbitrary polynomials of Φ, Φ and their derivatives, we get a differential ideal I. The algebra bF0 consists of arbitrary poly- nomials of variables ∂n +∂m −Dk +Dp −Φ and ∂n +∂m −D k +D p −Φ for non-negative integers n, m, k, p, modulo the ideal I: bF0 = C[. . . , ∂n +∂m −Dk +Dp −Φ, ∂n +∂m −D k +D p −Φ, . . . ]/I. (5.7) (5.7) It is not hard to find another set of generators, which will generate bF0 as a super- commutative polynomial algebra itself. We already explained it in the context of general N = (0, 2) LG models. Namely, we can take: It is not hard to find another set of generators, which will generate bF0 as a super- commutative polynomial algebra itself. We already explained it in the context of general N = (0, 2) LG models. 5.2.1 The Ak+1 series Namely, we can take: G = {∂n −−Φ, ∂n ++Φ, D−∂n −−Φ, D+∂n ++Φ, ∂n −−Φ, ∂n ++Φ, D−∂n −−Φ, D+∂n ++Φ, n ≥0}. (5.8) JHEP03(2018)079 All other derivatives of elementary superfields Φ and Φ can be expressed, using equations of motion, as polynomials of these generators, and moreover, there are no further algebraic relations between these generators. So we have: All other derivatives of elementary superfields Φ and Φ can be expressed, using equations of motion, as polynomials of these generators, and moreover, there are no further algebraic relations between these generators. So we have: bF0 ≃C[G]. (5.9) (5.9) We will first compute the classical cohomology of D+ acting on this space. After that we will check which part of it survives at the quantum level, when we take care to subtract singular parts from composite operators. It is clear that the cohomology classes can only be destroyed by this subtraction. Indeed, suppose we define: We will first compute the classical cohomology of D+ acting on this space. After that we will check which part of it survives at the quantum level, when we take care to subtract singular parts from composite operators. It is clear that the cohomology classes can only be destroyed by this subtraction. Indeed, suppose we define: : AB : (z) = lim ϵ→0 (A(z + ϵ)B(z) −(singular in ϵ)) . (5.10) (5.10) If AB was classically in the cohomology but the singular part is not D+-closed, the operator : AB : is no longer in the cohomology. If AB was not in the cohomology even classically, then neither is : AB :, which is quite obvious. Finally, if AB was classically D+-exact, then there is no need to consider : AB :. Even if the singular part represented some non- trivial quantum cohomology class, we would find it by starting with some other classical cohomology class anyways. So, we will look for the classical cohomology first, and then check which part of it survives subtraction of singularities.8 If AB was classically in the cohomology but the singular part is not D+-closed, the operator : AB : is no longer in the cohomology. If AB was not in the cohomology even classically, then neither is : AB :, which is quite obvious. Finally, if AB was classically D+-exact, then there is no need to consider : AB :. 5.2.1 The Ak+1 series Even if the singular part represented some non- trivial quantum cohomology class, we would find it by starting with some other classical cohomology class anyways. So, we will look for the classical cohomology first, and then check which part of it survives subtraction of singularities.8 To find how D+ acts on bF0 in terms of the generators, we act with D+ on the generators from the set G and, using the equations of motion, express the result in terms of these generators again. To explicitly describe D+, it is convenient to write it as a sum: D+ = d0 + d1, (5.11) (5.11) D+ = d0 + d1, where d0 acts as follows: d0 : ∂n −−Φ 7→0, ∂n ++Φ 7→0, D−∂n −−Φ 7→0, ∂n+1 −−Φ 7→0, D+∂n ++Φ 7→0, D−∂n −−Φ 7→0, D+∂n ++Φ 7→2i∂n+1 ++ Φ, ∂n ++Φ 7→D+∂n ++Φ, (5.12) d0 : ∂n −−Φ 7→0, ∂n ++Φ 7→0, D−∂n −−Φ 7→0, ∂n+1 −−Φ 7→0, D+∂n ++Φ 7→0, D−∂n −−Φ 7→0, D+∂n ++Φ 7→2i∂n+1 ++ Φ, ∂n ++Φ 7→D+∂n ++Φ, (5.12) (5.12) 8In fact, computation of the classical cohomology is a hard combinatorial problem, while we are really only interested in quantum cohomology. So we will not determine the classical cohomology completely, only partly. As we will see, there is an N = 2 super-Virasoro algebra in cohomology, so our approach will be to look for those classical cohomology classes which have a chance to be superconformal primaries at the quantum level. – 31 – and d1 acts as: and d1 acts as: d1 : ∂n −−Φ 7→0, ∂n ++Φ 7→0, D−∂n −−Φ 7→0, D+∂n ++Φ 7→0, ∂n ++Φ 7→0, D+∂n ++Φ 7→0, D−∂n −−Φ 7→∂n −−(Φk+1), ∂n+1 −−Φ 7→i 2D−∂n −−(Φk+1). (5.13 (5.13) This explicitly describes how D+ acts on the generators, and then extends to the full algebra bF0 by linearity and Leibniz rule. Notice that d0 is just the D+ in the theory with zero superpotential, while d1 includes corrections due to the superpotential. This splitting of D+ is motivated by a perturbative computation of the D+-cohommology, i.e., the spectral sequence, which we are about to perform. JHEP03(2018)079 Let us introduce a filtration degree on bF0 by saying that for generators: ∀x ∈G, fdeg(x) = 1, (5.14) (5.14) which then extends multiplicatively on the whole bF0. 5.2.1 The Ak+1 series We then define: bF(p) 0 = {S ∈bF0 : fdeg(S) ≥p}, (5.15) bF(p) 0 = {S ∈bF0 : fdeg(S) ≥p}, (5.15) which gives a filtration: bF0 ∼= bF(0) 0 ⊃bF(1) 0 ⊃bF(2) 0 ⊃. . . (5.16) (5.16) Our differential D+ obviously preserves this filtration. In particular, d0 does not change the filtration degree, while d1 increases it by k, if k > 0. This allows us to apply spectral sequences to compute the cohomology of D+. But before that we will mention a trivial technical lemma we will need later. Lemma 1. Let V be a Z2-graded vector space and S(V ) = ⊕k≥0Sk(V ) be the graded- symmetric algebra of V . If there is a degree-1 differential d : V →V , i.e., d2 = 0, then by the Leibniz rule it extends to a differential acting on the graded-symmetric algebra d : S(V ) →S(V ), and moreover, its cohomology is: H(S(V ), d) = S (H(V, d)) . (5.17) (5.17) Now, having this lemma, we will proceed to compute the cohomology of D+. Now, having this lemma, we will proceed to compute the cohomology of D+. First let us consider the trivial case k = 0. Then both d0 and d1 do not change the filtration degree. We can define a vector space spanned by the elements of G: V = Span(G). Since D+ = d0 + d1 does not change the filtration degree, it acts as a linear operator on this V . Next we notice that bF0 ≃S(V ), so by the lemma H( bF0, D+) = S H(V, D+)  . To compute the cohomology of D+ acting as a linear operator on V , we notice that all elements of G are either not D+-closed or are D+-exact as a consequence of the equation of motion D+D−Φ = Φ. So the cohomology is trivial for k = 0 (stress-energy supercurrent J becomes D+-exact for k = 0 ad well). This could be expected because the k = 0 model is massive, and therefore the IR theory it flows to is empty. – 32 – > 0. Then at the zeroth order of spectral sequence we have: Now, suppose k > 0. 5.2.1 The Ak+1 series Then at the zeroth order of spectral sequence we have: Ep 0 = bF(p) 0 / bF(p+1) 0 , E0 ≡Gr( bF0) ∼= M p≥0 Ep 0, (5.18) (5.18) where Gr( bF0) is the graded space associated with the filtered space bF0, and the differential acting on it is just d0, which preserves grading. We note that E1 0 ≃Span(G), the vector space spanned by the generators from G (which all have degree 1). Since d0 preserves grading and, as one can easily see, E0 ≃S(E1 0), we just apply lemma and get H(E0, d0) = S H(E1 0, d0)  . By inspecting equations (5.12), we easily find the cohomology of d0 acting on E1 0. The answer is H(E1 0, d0) = Span(S0), where the set S0 is: JHEP03(2018)079 S0 = {∂n −−Φ, D−∂n −−Φ, ∂n+1 −−Φ, D−∂n −−Φ, n ≥0}. (5.19) (5.19) Therefore, we find the first term of the spectral sequence: Therefore, we find the first term of the spectral sequence: E1 = H(E0, d0) ≃C[S0]. (5.20) (5.20) Now, if k = 1, then for the first step of spectral sequence, d1 becomes the differential acting on E1. If k > 1, then the differential acting on E1 is just zero, and E2 = H(E1, 0) ≃ E1. Next, if k > 2, we find that E3 ≃E1, and so on. This procedure goes on until we get to the k-th term of spectral sequence: Ek ≃E1. As we know from spectral sequences, the differential acting on Ek should be the degree-k part of D+, i.e., d1. So for the next term we have: Ek+1 ≃H(E1, d1). (5.21) (5.21) Since there are no components of D+ of degree higher than k, the spectral sequence collapses here and we conclude that: Since there are no components of D+ of degree higher than k, the spectral sequence collapses here and we conclude that: H( bF0, D+) ≃H(E1, d1) ≃H(C[S0], d1). (5.22) (5.22) So all we need to do now is compute the cohomology of d1 acting on C[S0]. The way d1 acts on the elements of S0 is: So all we need to do now is compute the cohomology of d1 acting on C[S0]. The way d1 acts on the elements of S0 is: ∂n −−Φ 7→0, D−∂n −−Φ 7→0, ∂n+1 −−Φ 7→i 2D−∂n −−(Φk+1), D−∂n −−Φ 7→∂n −−(Φk+1). 5.2.1 The Ak+1 series (5.23) d1 : (5.23) Even though we have considerably simplified the original problem, the direct computation of the d1 cohomology is still too nasty. We can simplify it more by recalling that we already have a stress-energy supercurrent in the cohomology, and therefore it is enough to look for its superconformal primaries only. Our superpotential is of a quasi-homogeneous class, with β = 1 k+2, so the stress-energy supercurrent is: J = k + 1 2(k + 2)D−ΦD−Φ − i k + 2Φ∂−−Φ (5.24) (5.24) and the corresponding central charge is c = 3k k+2. Now suppose we found some polyno- mial P ∈C[S0] which represents a D+-cohomology class. We have the following techni- cal lemma: – 33 – – 33 – Lemma 2. Every d1-cohomology class [P] which is a superconformal primary with respect to J , can be represented as a polynomial of Φ, D−Φ, D−Φ and ∂−−Φ, that is P ∈ C[Φ, D−Φ, D−Φ, ∂−−Φ]. Lemma 2. Every d1-cohomology class [P] which is a superconformal primary with respect to J , can be represented as a polynomial of Φ, D−Φ, D−Φ and ∂−−Φ, that is P ∈ C[Φ, D−Φ, D−Φ, ∂−−Φ]. Lemma 2. Every d1-cohomology class [P] which is a superconformal primary with respect to J , can be represented as a polynomial of Φ, D−Φ, D−Φ and ∂−−Φ, that is P ∈ C[Φ, D−Φ, D−Φ, ∂−−Φ]. The idea is that having higher derivatives of Φ and Φ in the expression for P will result in higher poles in the J (x, θ)P(0, 0) OPE, which should not be there if [P] is primary. Elegant proof of this statement is not available at the moment, but calculations seem to show that it is true, so we leave it as a conjecture. The operators Φ, Φ2, . . . Φk are all in the cohomology and are primaries — we will write their OPE’s with J later. Φk+1 is exact and so is not in the cohomology, so any polynomial of Φ is just a linear combination of 1, Φ, Φ2, . . . , Φk in the cohomology. Since D−(P(Φ)) = P ′(Φ)D−Φ and (D−Φ)2 = 0, any polynomial of Φ and D−Φ is A(Φ) + D−B(Φ), where the second term is a descendant. Let us figure out now if there are any other primaries in the cohomology. 5.2.1 The Ak+1 series We try to construct d1-closed (or equivalently, D+- closed) polynomials from Φ, D−Φ, D−Φ and ∂−−Φ, which are not just polynomials of Φ and D−Φ. A simple computation shows that the most general such combination with even statistics is: JHEP03(2018)079 E = ∞ X n=0 Pn(Φ)(∂−−Φ)n (n + 1)(k + 1) 2 D−ΦD−Φ −iΦ∂−−Φ  , (5.25) (5.25) where Pn are arbitrary polynomials, while the most general odd closed element is: O = ∞ X n=1 Cn(Φ)(∂−−Φ)nD−Φ, (5.26) (5.26) where again Cn are arbitrary polynomials. where again Cn are arbitrary polynomials. To slightly simplify computations, we notice that since the operator d1 increases the introduced above filtration degree fdeg by k, one can grade the cohomology by this degree, and it is enough to assume that E has a given fixed degree (i.e., it is a homogeneous polynomial). Next, we notice that we could introduce another grading — by the number of derivatives in the expression. If we assign the bosonic derivative ∂−−a “derivative degree” 1 and the fermionic derivatives D−and D−a “derivative degree” 1/2, we can see that the operator d1 actually lowers the “derivative degree” by 1/2. Therefore, again, we can grade the cohomology by this degree, and it is enough to study the cohomology within the sector with a given “derivative degree”. Fixing values of these two degrees — the filtration degree and the “derivative degree” — we see that it is enough, without loss of generality, to consider: Es,n = Φs(∂−−Φ)n (n + 1)(k + 1) 2 D−ΦD−Φ −iΦ∂−−Φ  , Os,n = Φs(∂−−Φ)nD−Φ. (5.27) (5.27) where s and n are non-negative integers. A simple calculation gives: D−Es,n = −i [s + 1 + (n + 1)(k + 1)] Os,n+1. (5.28) (5.28) – 34 – This suggests that any odd element of the above form Os,n+1 that we could have possibly found in the cohomology would always by a descendant of some even element. This is also true for Os,0 = ΦsD−Φ = 1 s+1D−Φs+1. Therefore, it is enough to study the expres- sion Es,n given above. Can it represent a nontrivial cohomology class, and can it be a superconformal primary? Observables Es,n and their lifting. Notice that for s ≥k: bservables Es,n and their lifting. Notice that for s ≥k: Observables Es,n and their lifting. 5.2.1 The Ak+1 series Careful computation of [Q+, e1], followed by taking the ϵ →0 limit, shows that: [Q+, e1] = −(k + 1)  (k + 1)φk∂−−ψ−−1 2ψ−∂−−φk  −i(k + 2)[Q+, ∂2 −−φ]. (5.37) (5.37) JHEP03(2018)079 So indeed, e1 is not in quantum cohomology. We know that classical observables should be lifted from cohomology in pairs. Therefore, the combination we got on the right, r1 = (k +1)φk∂−−ψ−−1 2ψ−∂−−φk, should be some classical cohomology class which disappears together with e1. And indeed, it is in the classical cohomology, as it is easy to check. Before, we found classical cohomology classes which had a chance of being superconformal primaries, and this r1 was not among them, which suggests that it should be a descendant. Another computation shows that it is indeed a descendant. The lowest component of J is: j = J | = k + 1 2(k + 2)ψ−ψ−− i k + 2φ∂−−φ, (5.38) (5.38) it is a U(1) current in the N = 2 super-Virasoro. A computation shows that: j−1(φkψ−) =: jφkψ−:= i k + 2  (k + 1)φk∂−−ψ−−ψ−∂−−φk + [Q+, . . . ]. (5.39) (5.39) So this new operator, r1 = (k + 1)φk∂−−ψ−−1 2ψ−∂−−φk, is actually a superconformal descendant of φkψ−. One can ask a similar question: what is this φkψ−? Clearly, it is in the classical cohomology. But in fact, φkψ−= 1 k+1[Q−, φk+1], and recall that we have a relation φk+1 = 0 in the classical cohomology. Therefore φkψ−also vanishes in the classical cohomology. So we have discovered the following: classically, we have cohomology classes e1 and r1, but quantum-mechanically, we have [Q+, e1] = r1. And this r1 is a superconformal descendant of φkψ−, which is actually zero in the classical cohomology. So this new operator, r1 = (k + 1)φk∂−−ψ−−1 2ψ−∂−−φk, is actually a superconformal descendant of φkψ−. One can ask a similar question: what is this φkψ−? Clearly, it is in the classical cohomology. But in fact, φkψ−= 1 k+1[Q−, φk+1], and recall that we have a relation φk+1 = 0 in the classical cohomology. Therefore φkψ−also vanishes in the classical cohomology. So we have discovered the following: classically, we have cohomology classes e1 and r1, but quantum-mechanically, we have [Q+, e1] = r1. And this r1 is a superconformal descendant of φkψ−, which is actually zero in the classical cohomology. 5.2.1 The Ak+1 series Notice that for s ≥k: d1 h Φs−k(∂−−Φ)n+1D−Φ i = iEs,n, (5.29) (5.29) JHEP03(2018)079 so Es,n is exact for s ≥k. On the other hand, for s < k, Es,n is obviously not exact, because, as we can see from the equation (5.13), the image of d1 always contains the field Φ at least k + 1 times, while Es,n contains it s + 1 times. So we conclude that Es,n for s < k indeed represents a non-trivial classical cohomology class. Classical observables Es,n satisfy the following multiplication rule: Es,nEt,m = −iEs+t+1,n+m+1. (5.30) (5.30) They can be combined with the observables Φs, for which we have: ΦsEt,n = Es+t,n. (5.31) (5.31) We see that Φ and Es,n generate a closed sector in the classical cohomology. As we will find soon, these are not all classical cohomology classes, there exist more. But all observables that have a chance of being superconformal primaries in the quantum cohomology are within this sector. The stress-energy supercurrent J that we identified before is of course among these observables: 1 J = 1 k + 2E0,0. (5.32) (5.32) In particular: J Es,n = − i k + 2Es+1,n+1. (5.33) (5.33) This equation implies that the only observables which have a chance of being superconfor- mal primaries at the quantum level are Es,0 and E0,n. But because of: This equation implies that the only observables which have a chance of being superconfor- mal primaries at the quantum level are Es,0 and E0,n. But because of: Es,0 = (k + 2)J Φs, (5.34) (5.34) the former are simply descendants of Φs. So we only have E0,n left. Can E0,n represent cohomology classes in quantum theory? It turns out that only for n = 0. The reason is that for n > 0, the infinite piece that one has to subtract in order to define the composite operator E0,n is not Q+-closed. Consider the simplest operator E0,1. We call its lowest component e1: e1 = (k + 1)∂−−φψ−ψ−−iφ(∂−−φ)2. (5.35) (5.35) – 35 – – 35 – This is a composite operator whose precise definition requires subtraction of singularities: e1(x) = lim ϵ→0  (k + 1)∂−−φ(x)ψ−(x)ψ−(x −ϵ) −i(∂−−φ(x))2φ(x −ϵ) −2ki ϵ−−∂−−φ(x)  . (5.36)  (5.36) We see that the piece that we subtract is not Q+-closed, which already suggests that e1(x) is probably not in the cohomology. 5.2.1 The Ak+1 series This might look confusing — how is it possible that a superconformal descendant of zero is not zero? The resolution of this apparent paradox is that, actually, super-Virasoro algebra does not act in the classical cohomology. It only acts in the quantum cohomology by the OPE with the stress-energy supercurrent J , while there is no notion of OPE in the classical cohomology. Therefore, there is no contradiction between the facts that φkψ− vanishes in the classical cohomology, while its superconformal descendant r1 does not vanish classically. The fact that latter is a descendant of the former is borrowed from the chiral algebra in the quantum cohomology. And in the quantum cohomology, because of this relation, both of them indeed have to vanish. This is quite satisfactory, because it also explains why r1 should be lifted from the classical cohomology — because it vanishes in quantum chiral algebra! In fact, by taking all possible superconformal descendants of the relation φk = 0, we will get a lot of (probably, infinitely many) operators which vanish in the quantum – 36 – cohomology but represent non-vanishing classical cohomology classes. They all should be lifted from the cohomology through the mechanism which we have just described. Also, it is not hard to convince oneself that not only E0,1, but all operators E0,n, n > 0 get lifted from the cohomology at quantum level for the same reasons. Clearly, there is some interesting (or at least non-trivial) mathematical structure in how classical cohomology classes get paired and lifted from the cohomology. It is quite possible that our observables Es,n and superconformal descendants of φk+1 are not the only classical cohomology classes involved in this. However, we are not going to study this question here. We are only interested in the quantum cohomology here, so the conclusion we need now is that the only primary operators in the cohomology are 1, Φ, Φ2, . . . , Φk. They, together with the stress-energy supercurrent J , generate the full chiral algebra in the Q+-cohomology. One can find that: JHEP03(2018)079 J (x1, θ1)Φs(x2, θ2) ∼− 2θ− 12θ − 12 (r12)2 hs + iθ− 12 r12 D−+ 2θ− 12θ − 12 r12 ∂−−+ i r12 qs ! Φs, (5.40) (5.40) where hs = qs 2 = s 2(k+2). We see that dimensions and charges match exactly our expecta- tions for the Ak+1 minimal model. 5.2.2 D and E series of minimal models However, we have an extra primary operator of dimension 1: which are just the relations of the chiral ring, so we get the familiar result (explained in section 2.3) that operators from the chiral ring of the N = (2, 2) theory are primaries of the chiral algebra. However, we have an extra primary operator of dimension 1: JHEP03(2018)079 P = JV −JU, (5.46) (5.46) which is not part of the chiral ring. The existence of this extra primary current in the cohomology was already noticed in [34], where the author also conjectured that every D2n+2 model has, in addition to the generators of the chiral ring, a single dimension-n primary in the cohomology. which is not part of the chiral ring. The existence of this extra primary current in the cohomology was already noticed in [34], where the author also conjectured that every D2n+2 model has, in addition to the generators of the chiral ring, a single dimension-n primary in the cohomology. We are not going to study n > 1 cases here. The only thing we want to mention is that the spectral sequence approach we used for the Ak+1 models can be clearly generalized to the D2n+2 case. For n > 1, the operator D+ will split as a sum of three terms: D+ = d0 + d1 + d2, (5.47) (5.47) where d0 corresponds to the zero superpotential, d1 takes into account the effect of XY 2 term in the superpotential, and d2 encodes the effect of X2n+1 interaction. It should be possible, though more technical than in the Ak+1 case, to compute the cohomology using this splitting and check the conjecture made in [34]. where d0 corresponds to the zero superpotential, d1 takes into account the effect of XY 2 term in the superpotential, and d2 encodes the effect of X2n+1 interaction. It should be possible, though more technical than in the Ak+1 case, to compute the cohomology using this splitting and check the conjecture made in [34]. Finally, a small remark about the E series. The models E6 and E8 correspond to superpotentials X3 + Y 4 and X3 + Y 5. Therefore, their chiral algebras are immediately identified as those of A2 ⊗A3 and A2 ⊗A4 respectively. Therefore, they will also contain extra primary operators, in addition to the chiral ring elements. 5.2.2 D and E series of minimal models We will not go into much details about the chiral algebras of D and E series of minimal models. Instead we will just look at some of their features, leaving a more detailed study for the future. The LG models which are expected to flow to D2n+2 minimal models in the IR are described by the superpotential: W = XY 2 + X2n+1 2n + 1. (5.41) (5.41) Consider the n = 1 theory. It has W = XY 2 + X3 3 . If we make a change of variables V = X + Y √ 2 , U = X −Y √ 2 , (5.42) (5.42) We will get an LG model with W = √ 2 3 V 3 + √ 2 3 U 3. This is just a pair of non-interacting A2 models. Thus the theory in the IR is expected to be just A2 ⊗A2, with the chiral algebra being a tensor product as well. Recall from the previous subsection that, for the W ∝V 3, the chiral algebra has only two primaries: the identity 1 and V , and there is also a stress-energy supercurrent JV . Similarly for the second one: we have 1 and U as primaries, and we have JU. By taking the tensor product of these two, we can identify primaries in the chiral algebra of A2 ⊗A2 as: 1, V, U, V U, JV −JU. (5.43) (5.43) – 37 – Going back to X amd Y , the first four are simply: Going back to X amd Y , the first four are simply: 1, X, Y, X2 −Y 2. (5.44) (5.44) Moreover, since in the cohomology V 2 = U 2 = 0, these X and Y satisfy relations in the chiral algebra: Moreover, since in the cohomology V 2 = U 2 = 0, these X and Y satisfy relations in the chiral algebra: X2 + Y 2 = 0, XY = 0, (5.45) XY = 0, (5.45) (5.45) which are just the relations of the chiral ring, so we get the familiar result (explained in section 2.3) that operators from the chiral ring of the N = (2, 2) theory are primaries of the chiral algebra. 6 Discussions and further directions We have only scratched the surface of the subject, demonstrating some general properties of chiral algebras of N = (0, 2) theories and giving several simple examples. The most general property was the RG invariance of the answer, which makes chiral algebras interesting objects to study in the context of dualities. One obvious extension of this work would be to get a better description of chiral algebras of N = (2, 2) LG models with quasi-homogeneous (or even general) polynomial superpotentials. Our treatment allowed us to find answers in some cases, but it would be much nicer to have a more general result, which would associate chiral algebra to any polynomial superpotential. It would also be useful to find some classes of N = (0, 2) models in which the chiral algebra could be described completely. JHEP03(2018)079 But the most interesting and immediate extension is, of course, the application of chiral algebras to gauge theories. If the LG model has some flavor symmetry, one can gauge it by coupling to gauge multiplets. One can argue that perturbatively, the way this gauging is implemented in the chiral algebra is as follows. If G is the gauge group, one should first take the G-invariant subalgebra of the ungauged chiral algebra, then tensor multiply it by the “small” bc-system of dimension (1, 0) (where “small” means that zero mode of c is excluded from the algebra). The ungauged chiral algebra has a current in it which corresponds to the flavor symmetry we want to gauge. Using this current and the bc-ghosts, one can construct a BRST operator. The condition of its nilpotency is precisely the condition that there is no gauge anomaly, i.e., that the symmetry we want to gauge really can be gauged. Then we have to compute the cohomology of this BRST operator. The answer is the gauged chiral algebra. (This procedure is identical to the gauging of chiral algebras of 4D N = 2 theories from [35].) This procedure seems to hold in perturbation theory. One way to argue it is by writing equations of motion of the gauge theory and, similar to what we did in this paper, computing the cohomology of D+ using perturbation theory (or spectral sequence) in gauge coupling. This approach is somewhat ugly, but it allows to argue that the answer is as we claimed above. 5.2.2 D and E series of minimal models The E7 model has: W = X3 + XY 3, (5.48) (5.48) therefore it has to be studied separately. In this case again we will have: D+ = d0 + d1 + d2, (5.49) (5.49) where d0 is a D+ operator in the theory of two free chiral superfields without any super- potential, d1 takes into account the X3 term and d2 takes care of XY 3. It is clear that at the second step of the spectral sequence computation, when we consider the cohomology of d1, we will essentially get the cohomology of the A2 model multiplied by the free theory described by the chiral superfield Y . Computing the cohomology of d2 at the next step then becomes much simpler, since we already know the answer for A2. – 38 – 6 Discussions and further directions Another, more conceptual proof would be to define the gauge theory using the BRST formalism and the holomorphic gauge v++ = 0. This would give the action: S = S0 + {QB, Ψ} = S0 + Z d2x lAvA ++ + Z d2x bAD++cA, (6.1) (6.1) where lA is the auxiliary field implementing gauge vA ++ = 0, and we added Faddeev-Popov ghosts. One can extend supersymmetry to act trivially on ghosts. Then the supercharge Q+ and the BRST charge QB anticommute: {Q+, QB} = 0, and we really have two commuting complexes. The theory is defined as the cohomology of QB, and within that cohomology we want to find the chiral algebra in the cohomology of Q+. Since the complexes commute, we could first find the cohomology of Q+, and then compute the cohomology of QB. It is quite nice to discover that the gauging procedure we explained above arises in this way. However, some technical details still have to be clarified. A question of utmost importance is to understand how the gauging procedure should be modified to account for non-perturbative effects, such as instantons. – 39 – Another extension, which is also important for gauge theories, is to study models without R-symmetry. We can easily find gauge theories with anomalous R-symmetry. In case they are constructed by gauging some LG models that have (right-handed) R- symmetry, it becomes natural to ask what happens to their chiral algebra during gauging that manifests the breaking of R-symmetry. Acknowledgments The author would like to thank E. Witten for helpful discussions, as well as B. Le Floch, I. Melnikov and E. Sharpe for useful comments on the draft. JHEP03(2018)079 Open Access. 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English
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Feasibility and acceptability of artemisinin-based combination therapy for the home management of malaria in four African sites
Malaria journal
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Received: 22 October 2007 Accepted: 8 January 2008 Received: 22 October 2007 Accepted: 8 January 2008 Published: 8 January 2008 Malaria Journal 2008, 7:6 doi:10.1186/1475-2875-7-6 Malaria Journal 2008, 7:6 doi:10.1186/1475-2875-7-6 This article is available from: http://www.malariajournal.com/content/7/1/6 © 2008 Ajayi et al; licensee BioMed Central Ltd. j y 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 Malaria Journal Open Access Research Open Acc Research Feasibility and acceptability of artemisinin-based combination therapy for the home management of malaria in four African sites Ikeoluwapo O Ajayi*1, Edmund N Browne2, Bertha Garshong3, Fred Bateganya4, Bidemi Yusuf1, Peter Agyei-Baffour2, Leticia Doamekpor3, Andrew Balyeku4, Kaendi Munguti5, Simon Cousens6 and Franco Pagnoni7 p Research Feasibility and acceptability of artemisinin-based combination therapy for the home management of malaria in four African sites Ikeoluwapo O Ajayi*1, Edmund N Browne2, Bertha Garshong3, Fred Bateganya4, Bidemi Yusuf1, Peter Agyei-Baffour2, Leticia Doamekpor3, Andrew Balyeku4, Kaendi Munguti5, Simon Cousens6 and Franco Pagnoni7 Address: 1Malaria Research Laboratories, Institute of Medical Research and Training, College of Medicine, University of Ibadan, Nigeria, 2Department of Community Health, School of Medical Sciences, KNUST, Kumasi, Ghana, 3Health Research Unit, Ghana Health Service, Accra, Ghana, 4Department of Sociology, Makerere University, Kampala, Uganda, 5Institute for Development Studies, College of Humanities and Social Studies, University of Nairobi, Nairobi, Kenya, 6Infectious Diseases Epidemiology Unit, London School of Hygiene and Tropical Medicine, London, UK and 7Implementation Research & Methods Unit, UNICEF/UNDP/World Bank/WHO Special Programme for Research and Training in Tropical Diseases (TDR), Geneva, Switzerland Email: Ikeoluwapo O Ajayi* - ikeajayi2003@yahoo.com; Edmund N Browne - enlbrowne@yahoo.com; Bertha Garshong - Bertha.Garshong@hru-ghs.org; Fred Bateganya - fredb@ss.mak.ac.ug; Bidemi Yusuf - bidemi_yusuf@yahoo.com; Pete Email: Ikeoluwapo O Ajayi* - ikeajayi2003@yahoo.com; Edmund N Browne - enlbrowne@yahoo.com; Bertha Garshong - Bertha.Garshong@hru-ghs.org; Fred Bateganya - fredb@ss.mak.ac.ug; Bidemi Yusuf - bidemi_yusuf@yahoo.com; Peter Agyei- Baffour - agyeibaffour@yahoo.co.uk; Leticia Doamekpor - leticia.Doamekpor@hru-ghs.org; Andrew Balyeku - abalyeku@gmail.com; Kaendi Munguti - kaendim@iconnect.co.ke; Simon Cousens - Simon.Cousens@lshtm.ac.uk; Franco Pagnoni - pagnonif@who.int * Corresponding author * Corresponding author Received: 22 October 2007 Accepted: 8 January 2008 Published: 8 January 2008 Malaria Journal 2008, 7:6 doi:10.1186/1475-2875-7-6 Abstract Background: The Home Management of Malaria (HMM) strategy was developed using chloroquine, a now obsolete drug, which has been replaced by artemisinin-based combination therapy (ACT) in health facility settings. Incorporation of ACT in HMM would greatly expand access to effective antimalarial therapy by the populations living in underserved areas in malaria endemic countries. The feasibility and acceptability of incorporating ACT in HMM needs to be evaluated. Methods: A multi-country study was performed in four district-size sites in Ghana (two sites), Nigeria and Uganda, with populations ranging between 38,000 and 60,000. Community medicine distributors (CMDs) were trained in each village to dispense pre-packaged ACT to febrile children aged 6–59 months, after exclusion of danger signs. A community mobilization campaign accompanied the programme. Artesunate-amodiaquine (AA) was used in Ghana and artemether-lumefantrine (AL) in Nigeria and Uganda. Harmonized qualitative and quantitative data collection methods were used to evaluate CMD performance, caregiver adherence and treatment coverage of febrile children with ACTs obtained from CMDs. Results: Some 20,000 fever episodes in young children were treated with ACT by CMDs across the four study sites. Cross- sectional surveys identified 2,190 children with fever in the two preceding weeks, of whom 1,289 (59%) were reported to have received ACT from a CMD. Coverage varied from 52% in Nigeria to 75% in Ho District, Ghana. Coverage rates did not appear to vary greatly with the age of the child or with the educational level of the caregiver. A very high proportion of children were reported to have received the first dose on the day of onset or the next day in all four sites (range 86–97%, average 90%). The proportion of children correctly treated in terms of dose and duration was also high (range 74–97%, average 85%). Overall, the proportion of febrile children who received prompt treatment and the correct dose for the assigned duration of treatment ranged from 71% to 87% (average 77%). Almost all caregivers perceived ACT to be effective, and no severe adverse events were reported. Conclusion: ACTs can be successfully integrated into the HMM strategy. Page 1 of 9 (page number not for citation purposes) http://www.malariajournal.com/content/7/1/6 Malaria Journal 2008, 7:6 Study design Th d The study was conducted in three phases. The first phase involved advocacy, community mobilization, selection of CMDs and establishment of key baseline indicators. Infor- mation, Education and Communication (IEC) materials were developed and research staff recruited and trained. The intervention phase involved the training of CMDs and distribution of ACT. A year after commencement of ACT distribution, a household survey, interviewing eligi- ble caregivers about child fevers in the past two weeks, was performed to evaluate the intervention. So far, 18 African countries have adopted HMM as part of their malaria control programme [4]. Due to unacceptable levels of resistance to chloroquine by Plasmodium falci- parum, 44 countries in Africa have adopted artemisinin- based combination therapy (ACT) as the first line treat- ment for uncomplicated malaria [5]. However, in most cases, the deployment of ACT is currently limited to health facilities, with large scale implementation of HMM using ACT delayed by concerns about the use of ACT at the community level [6-8]. Background and in Ho district, an urban and a rural site were studied. In Nigeria, two districts Badeku and Ojoku/Ajia in Ona- ara local government were studied. The sites in Ejisu-Jua- ben, Ghana; Ona-ara Local Government, Nigeria; and Buguri and Iganga districts, Uganda were all rural areas. Ghana and Nigeria are located in West Africa, while Uganda is in East Africa. Table 1 describes the location and population characteristics of the different study sites, details of the community-based medicine distributors (CMDs) deployed, their training and supervision proc- esses, and type, dosing schedule and cost to the end user of the antimalarial drug used. g Home Management of Malaria (HMM) for children with uncomplicated malaria in high transmission areas in Africa is an integral part of malaria case management within the overall Roll Back Malaria (RBM) strategy [1]. If the Abuja target of 60% of uncomplicated malaria epi- sodes receiving effective treatment within 24 hours [2] and the Millennium Development Targets 5, 8 and 17 (reducing childhood mortality, halting the increase in malaria incidence and providing access to affordable essential drugs in developing countries) [3] are to be met, there is an urgent need to increase access to effective malaria treatment at the community level, especially in underserved rural areas. Data collection methods A pilot study carried out in Ghana in 2004 [9] provided preliminary evidence for the feasibility and acceptability of the use of artemether-lumefantrine (AL) in the HMM context. However, this study was essentially a qualitative assessment of community acceptability. To provide addi- tional evidence to support the use of ACT at the commu- nity level, the UNICEF-UNDP-WORLD BANK-WHO Special Programme for Research and Training in Tropical Diseases (TDR) funded four studies in sites in Ghana, Nigeria and Uganda, representing different health system and epidemiological settings. This paper reports on the feasibility, acceptability and utilization of ACT provided at the community level. Qualitative and quantitative data collection methods were used. Data collection tools for all the sites were harmo- nized prior to the intervention with some country specific modifications. The tools included the survey question- naire and focus group discussion (FGD) and key inform- ant interview (KII) guides, which were pre-tested and translated into the local languages. The format of CMDs' registers was also harmonized. The household survey focused on the health-seeking behaviour of caregivers of children with fever in the preceding two weeks, with an emphasis on timeliness of treatment, compliance with the prescribed treatment course, and perceptions of effective- ness and drug related adverse events. The FGD and KII guides focused on health-seeking behaviours, beliefs about the aetiology of malaria, treatment practices and community perceptions of the intervention. Sample size A major concern with using ACT at the community level is the potential for poor adherence to the treatment sched- ule by both caregivers and community medicine distribu- tors (CMDs). This could facilitate the development of parasite resistance to these expensive but currently highly efficacious drugs. Other concerns relate to acceptability by the community, the incidence of adverse events, cost and ability to provide adequate storage conditions to ensure drug stability in the community. It was decided that to yield useful results in terms of feasi- bility, the study should cover populations with a mini- mum population of around 40,000 – the size of a typical district. The sample size for the household survey was cal- culated using country-specific fever prevalence rates to provide a precision of ± 5% for the estimates of coverage of ACT treatment through CMDs, assuming a design effect of two. The minimum number of households to be inter- viewed ranged from 700 to 768. Study sites and population This multi-country study was performed in Ghana (two sites), Nigeria and Uganda all in sub-Saharan Africa. In Ghana two districts, Ejisu-Juaben and Ho were studied Page 2 of 9 (page number not for citation purposes) http://www.malariajournal.com/content/7/1/6 Malaria Journal 2008, 7:6 Table 1: Description of study sites and intervention implementation in four sites evaluating home management of malaria with artemisinin combination therapy Ejisu – Juaben District, Ghana Ho District, Ghana Badeku and Ojoku/Ajia Districts, Nigeria Bugiri and Iganga Districts, Uganda Population characteristics 35 rural communities Population c. 38,000 Perennial malaria transmission 49 urban & rural communities Population c. 42,000 Perennial malaria transmission 40 rural communities Population 43,000 Perennial, hyperendemic malaria transmission 56 rural communities Population c. 60,000 Perennial, hyperendemic transmission Community medicine distributors 54 (51 male) trusted members of community (farmers, teachers, drug sellers), chosen by the community 76 (48 male) trusted members of community (farmers, teachers, drug sellers), chosen by the community 60 (4 male) including drug sellers, health workers and mothers chosen by the communities 118 (62 male) pre-existing cadre of CMDs (chosen by the community) 5 days of training 2 days of training 2 days of training 2 days of training Provided with bicycles, boots & $3.50 monthly Provided with T-shirts, watches, raincoats, torches & US$8 quarterly Received commission of 20–30 US cents per pack distributed. Provided with T- shirts, transport reimbursement, Festivity gift, and certificates. Transport refund of USD 1.16 per meeting. T-shirts, baseball caps, certificates Bimonthly supervision by research staff; Monthly supervision by community Health Officers Monthly supervision by health staff and quarterly meetings Monthly supervision by research staff Monthly supervision by health staff CMDs were not obliged to follow up caregivers. However, some of them followed up and some gave drugs under direct observation. No active follow-up of caregivers by CMDs. Program ran within existing home management system. Study sites and population CMDs actively followed up treated children to establish treatment outcome Drugs and dosing schedule Artesunate+Amodiaquine (ASAQ) once daily for 3 days 2 types of blister pack, for children < 1 year and children 1 year and above Artemether-lumefantrine (Coartem®) twice daily for 3 days 2 types of blister pack, for children < 3 years and children 3 years and above 10 US cents for <1 year 20 US cents for 1 year and above 20 US cents for < 3 years 30 US cents for 3 years and above Charged for first 6 months only, thereafter free Free Table 1: Description of study sites and intervention implementation in four sites evaluating home manag artemisinin combination therapy Bimonthly supervision by research staff; Monthly supervision by community Health Officers Monthly supervision by health staff and quarterly meetings CMDs were not obliged to follow up caregivers. However, some of them followed up and some gave drugs under direct observation. No active follow-up of caregivers by CMDs. Program ran within existing home management system. CMDs actively followed up treated children to establish treatment outcome Artemether-lumefantrine (Coartem®) twice daily for 3 days 2 types of blister pack, for children < 3 years and children 3 years and above 20 US cents for < 3 years 30 US cents for 3 years and above Charged for first 6 months only, thereafter free Free Artesunate+Amodiaquine (ASAQ) once daily for 3 days 2 types of blister pack, for children < 1 year and children 1 year and above 10 US cents for <1 year 20 US cents for 1 year and above Community medicine distributors participation. Quarterly monetary allowances varied between USD 4.5 (Uganda), USD 5 (Ejisu-Juaben) and USD 8 (Ho). In Nigeria, the CMDs received a commission of between USD 0.20 and USD 0.30 for each pack of AL dispensed in addition to periodic gifts and reimburse- ment of transportation fares. Community medicine distributors were trusted members of the community who were chosen by the community from a range of backgrounds, including farmers, chemi- cal/medicine sellers, teachers, traders, community health workers, artisans, 'mother trainers', and opinion leaders (Table 1). In Ghana and Nigeria nurses and community health officers at first-level formal health facilities were counted as CMDs. 'Mother trainers' were lay mothers who were selected from within the communities and trained to distribute drugs. Drugs and dosing schedule Ethics Ethical approval for conduct of this study was obtained from the WHO Ethics Review Committee and at national level from the appropriate Ethical Review Boards. Informed consent was obtained from community heads, household heads and the caregivers who participated in the study. Supervision Monthly supervision was undertaken in all sites, by health staff or by the research team (Table 1). In each site, super- vision included checking drug stocks, their storage condi- tions, and the CMDs' registers. The research team provided fuel and travel allowances to supervisors. In Nigeria, unscheduled inspections were periodically undertaken. Utilization of CMDs Based on the records maintained by the CMDs, some 20,000 fever episodes in young children were treated with ACTs by CMDs across the four study sites (Table 2). The total number of episodes treated varied considerably between sites as did the average number of episodes treated per CMD (from 17 per CMD in Nigeria to 93 in Uganda). The higher number of episodes treated in Uganda may be explained by the fact that home-based management of fevers has been implemented there since 2001. A higher attrition rate of CMDs in Nigeria com- pared to other study sites may also have contributed to the lower figures recorded there. Slightly more girls (4,928) than boys (4,595) were treated across the three sites in which this information was recorded. In this study, the accurate population denominators were not available. However, taking into account the width of the age groups, utilization appeared to decline somewhat with age (from 3,571 children in the 6-month age band from 6–11 months to 6,627 children in the two year age band from 36–59 months). Utilization was generally prompt with at least half of all children presenting on the day of onset or Drugs used in the study Two different ACTs were used in accordance with the national drug policies of the participating countries; AL was used in Uganda and Nigeria, and artesunate-amodi- aquine (AS/AQ) was used in Ghana. Selection criteria for CMDs included being a permanent resident (at least one year), trusted and respected by the community, able to keep simple records, and a willing- ness to serve. Uganda had a functioning pre-existing cadre of CMDs who were co-opted into the study. The number of CMDs per community depended on the community's population size. An average of two CMDs per community was used across the four sites (one CMD per 600 popula- tion). For the AS/AQ combination, blister packs containing co- packed artesunate and amodiaquine tablets of 50 and 153 mgs respectively were used. For children aged 12 to 59 months the recommended dose of AS/AQ was one tablet of each drug once per day. However, for children of six to 11 months of age, tablets had to be broken in half and repackaged to comply with the recommended AS/AQ dose in that age group. Although CMDs were not paid a salary, various motiva- tion mechanisms were used. Some of these included rain coats, bicycles, boots, watches, T-shirts and certificates of In Nigeria and Uganda, a fixed combination of 20 mg of artemether and 120 mg of lumefantrine (AL) in two types of blister pack, one for children below three years of age Page 3 of 9 (page number not for citation purposes) http://www.malariajournal.com/content/7/1/6 http://www.malariajournal.com/content/7/1/6 Malaria Journal 2008, 7:6 notes, the responses to questions asked to explore issues of interest were grouped together, coded, categorized and analysed according to emerging themes. The findings were interpreted and reported in the form of narratives. and another for children three years and above, were used. The recommended dose for children aged six to 35 months was one tablet twice daily for three days, and for children aged 36 to 59 months two tablets twice daily for three days. Caregivers were advised to administer the drug after meals, preferably fatty food. Drug supply I ll h i In all the sites, drugs were provided through existing pub- lic health structures, at health district or sub-district level. In general, the distribution points for drugs were the local health facilities. In Uganda, CMDs replenished their stock at monthly meetings at health facility level. In Nigeria and Ghana, CMDs replenished their stocks from the health facilities as the need arose. In some instances drugs were delivered by the research team or by health staff during supervisory visits. A young woman in Nigeria said: within 24 hours of onset in all four sites. In the three sites which recorded promptness as "same day" or "next day" 73–90% of children presented on the day of onset or the next day. 'CMDs are always available. It is good we have two in our community, when one is out the village the other one attends to caregivers'. Data analysis EPI info version 6.02, SPSS version 11.0 and STATA ver- sion 9.2 were used by the four sites to enter and analyse data. A set of indicators and quantitative analyses to be performed were agreed in discussion with research staff from each site. Each research team then undertook the analysis of their own data using standard statistical meth- ods. The FGD and KII information was transcribed and content analysis performed. From the transcripts and field Page 4 of 9 (page number not for citation purposes) Table 2: Utilization of CMDs for treatment of children with fever in 4 sites (source: CMD registers) Ejisu – Juaben District, Ghana Ho District, Ghana Badeku and Ojoku/Ajia Districts, Nigeria Bugiri and Iganga Districts, Uganda Totals Number of CMDs 54 76 60 118 308 Total number febrile episodes in children less than 5 treated with ACTs by CMDs 4522 3958 1044 11039 20563 Number (%) treated by age 6–11 months 789 (17%) 474 (12%) 99 (9%) 2209 (20%) 3571 (17%) 12–23 months 1531 (34%) 1157 (29%) 186 (18%) 2571 (23%) 5445 (26%) 24–35 months 1082 (24%) 1054 (27%) 236 (23%) 2548 (23%) 4920 (24%) 36–59 months 1120 (25%) 1273 (32%) 523 (50%) 3711 (34%) 6627 (32%) Number (%) treated by sex Female 2458 (54%) 1977 (50%) 493 (47%) NA 4928 (52%) Male 2064 (46%) 1980 (50%) 551 (53%) NA 4595 (48%) Number (%) treated by promptness of utilization Same day 2636 (58%) 2125 (54%) 479 (49%) 5445 (49%) within 24 hours 14124 (69%) Next day 1886 (42%) 1313 (33%) 240 (24%) Later than next day 0 (0%) 520 (13%) 263 (27%) 5594 (51%) 6377 (31%) Table 2: Utilization of CMDs for treatment of children with fever in 4 sites (source: CMD registers) http://www.malariajournal.com/content/7/1/6 Malaria Journal 2008, 7:6 http://www.malariajournal.com/content/7/1/6 Caregiver adherence From the survey data, a very high proportion of children were reported to have been treated promptly in all four sites (Table 5). The proportion of febrile children who received prompt treatment and received the correct dose for the assigned duration of treatment ranged from 71% in Uganda to 87% in Ghana (P < 0.001; Table 5). Adher- ence by caregivers was linked to better treatment outcome. A woman participating in a FGD in Ejisu-Juaben district said: Treatment coverage of febrile episodes by CMDs In two of the four sites there was some turnover of CMDs but the attrition rate in these sites was relatively low. The need for incentives for CMDs, however, was often men- tioned as an issue. An opinion leader in Ho, Ghana, said: Data from the community-based cross-sectional surveys were used to estimate the proportion of febrile children receiving ACT from CMDs. A total of 2,190 children with fever in the two weeks preceding the survey were identi- fied of whom 1,289 (59%) were reported to have received ACT from a CMD (Table 3). Coverage varied from 52% in Nigeria to 75% in Ho District, Ghana (P < 0.001). Cover- age rates did not appear to vary greatly with age, except perhaps in Ejisu-Juaben District, Ghana, where coverage appeared lower in the six to 11 months age group than in older age groups (Table 3). Overall, across the four sites, treatment coverage did not appear to vary strongly with the educational level of the caregiver. There is this saying that "the one who feeds a child will surely put her hands in her mouth"; our volunteers should be appreciated so that they can enjoy the work they are doing. For this, the community and I will think about it and compensate them by giving some allowance because some- times they have to go and call them from wherever they are. CMD performance Based on the information recorded by the CMDs in their registers, a very high proportion of children received the correct dose of ACTs (97% or greater in all sites; Table 4). From the survey data, CMDs were reported to have explained the dosing schedule on a very high proportion of occasions (>90% in all sites), but performed less well with respect to explaining danger signs or possible adverse events (Table 4). This was particularly pronounced in Ejisu-Juaben District in Ghana. Availability of CMDs was reported to be good, with 85% or more of caretakers reporting that they found the CMD at the first time of vis- iting. "When I went in for the drug he gave me instructions on how to give the drug to the child. I was told to give it to him continuously for three days – two tablets (white & yellow) each day. I did that and afterwards the child was well again. Happiness was then restored in my home". CMD performance Page 5 of 9 (page number not for citation purposes) Table 3: Treatment coverage of febrile children aged 6–59 months with ACTs obtained from CMDs (source: household survey) Ejisu – Juaben District, Ghana Ho District, Ghana Badeku and Ojoku/Ajia Districts, Nigeria Bugiri and Iganga Districts, Uganda Totals Total number of febrile children identified 428 124 551 1087 2190 Number (%) treated with ACTs from a CMD 289 (68%) 93 (75%) 288 (52%) 619 (57%) 1289 (59%) 95% CI 61–74% 63–87% 44–61% 51–63% Number treated (% coverage) by age in months 6–11 20 (45%) 13 (76%) 17 (53%) 16 (52%) 66(53%) 12–23 89 (74%) 21 (81%) 49 (49%) 179 (56%) 338 (60%) 24–35 64 (73%) 25 (76%) 69 (52%) 142 (56%) 300 (59%) 36–59 116 (66%) 28 (72%) 153 (54%) 282 (58%) 579 (59%) Number treated (% coverage) by sex Female 137 (65%) 46 (72%) 154 (54%) NA 337 (60%) Male 152 (70%) 47 (78%) 134 (50%) NA 333 (61%) Number treated (%) coverage by educational level of caregiver None 71 (75%) 20 (77%) 80 (47%) 192 (54%) 363 (56%) Primary 74 (68%) 28 (80%) 155 (56%) 315 (56%) 572 (58%) Secondary 144 (64%) 45 (76%) 53 (52%) 112 (65%) 354 (63%) Number treated (% coverage) by marital status of caregiver Married 219 (67%) 87 (75%) 256 (54%) 571 (56%) 914 (47%) Not married 70 (69%) 6 (75%) 30 (41%) 48 (63%) 127 (49%) e children aged 6–59 months with ACTs obtained from CMDs (source: household survey) Table 3: Treatment coverage of febrile children aged 6–59 months with ACTs obtained from CMDs (sou ment coverage of febrile children aged 6–59 months with ACTs obtained from CMDs (source: household sur http://www.malariajournal.com/content/7/1/6 Malaria Journal 2008, 7:6 Table 4: Measures of CMD performance in delivering ACTs in 4 sites (source: CMD registers + household survey) Ejisu – Juaben District, Ghana Ho District, Ghana Badeku and Ojoku/Ajia Districts, Nigeria Bugiri and Iganga Districts, Uganda Totals Correctness of prescription (from CMD register) Number (%) of all children correctly dosed 4473 (99%) 3900 (98%) 1019 (98%) 10,671 (97%) 20063 (98%) Number (%) of young children receiving an over dose 30 (0.7%) 15 (3.2%) 11 (2%) 27 (0.4%) 83 (0.7%) Number (%) of older children receiving an under dose 8 (0.2%) 43 (1.2%) 12 (3%) 54 (1.5%) 117 (1%) Number (%) of occasions on which CMD explained (from survey): Treatment schedule 281 (97%) 93 (100%) 264 (92%) 569 (92%) 1207 (94%) Danger signs 53 (18%) 83 (89%) 240 (83%) 462 (75%) 838 (65%) Possible adverse events 44 (15%) 86 (93%) 241 (84%) N/A 371 (55%) Availability of CMDs (from survey) Number (%) of mothers who did not find the CMD at the first attempt 36 (12%) 5 (5%) 36 (12.5%) 86 (14%) 163 (12.5%) Storage of ACTs (from supervision records) Number (%) of supervisory visits at which drugs were stored appropriately 523 (99%) 720 (100%) 960 (100%) 420 (100%) 2623 (99.8%) Attrition of CMDs Number (%) of CMDs withdrawing from role 0 (0%) 3 (4%) 6 (10%) 0 (0%) 9 (3%) Table 4: Measures of CMD performance in delivering ACTs in 4 sites (source: CMD registers + household survey) mance in delivering ACTs in 4 sites (source: CMD registers + household survey) A local council chairperson of Bwalula village in Uganda explained: child's condition becomes worse. If you are lucky to get to the hospital early, after treatment you walk the same dis- tance back or referred to the big hospital. The unlucky ones either die before they get to the clinic or die at the clinic; but ever since the introduction of this program, we have seen a change. For a year now, since the introduction of the new drug, no child has died and we have not sent any of them to the hospital". child's condition becomes worse. If you are lucky to get to the hospital early, after treatment you walk the same dis- tance back or referred to the big hospital. The unlucky ones either die before they get to the clinic or die at the clinic; but ever since the introduction of this program, we have seen a change. For a year now, since the introduction of the new drug, no child has died and we have not sent any of them to the hospital". From 1–2 weeks during Homapak, it now takes about three months before my child gets sick. It is better than CQ when you complete the dose. "As for the drug, it works like magic!" "As for the drug, it works like magic!" Safety and perceived effectiveness of ACTs The proportion of children for whom an adverse event was reported to the CMD was low (circa 1%) in both sites in which this information was available (Table 6). At the survey the proportion of children reported to have experi- enced an adverse event following treatment with ACT was somewhat higher (4–8%) but none of these adverse events were reported to be serious. In all sites almost all caretakers perceived ACT to be effective. In Ho, Ghana, a community member argued that: Discussion There are two major findings from this study. First, it showed, at a larger scale than previously [9], that making ACT available at the community level through trained CMDs results in a high degree of adherence by sensitized caregivers. Second, that community-based strategy can deliver high coverage of febrile episodes in children with prompt and adequate treatment. Importantly, these find- "The change is that, previously, when children fall sick, we take some time to look for money and walk all the distance to go to the hospital. Sometimes before we get there the Table 5: Adherence of caregivers to treatment schedule Ejisu – Juaben District, Ghana Ho District, Ghana Badeku and Ojoku/Ajia Districts, Nigeria Bugiri and Iganga Districts, Uganda Totals Number of episodes treated with ACTs from a CMD 289 93 288 619 1289 Number (%) of children correctly treated (dose and duration) 281 (97%) 69 (74%) 256 (89%) 490 (79%) 1096 (85%) Number (%) of children treated promptly (receiving first dose on the same or next day) 259 (90%) 89 (96%) 278 (97%) 531 (86%) 1157 (90%) Number (%) of children treated promptly AND correctly 252 (87%) 69 (74%) 231 (80%) 438 (71%) 990 (77%) Table 5: Adherence of caregivers to treatment schedule http://www.malariajournal.com/content/7/1/6 Malaria Journal 2008, 7:6 Table 6: Safety and perceived effectiveness of treatment of ACTs obtained from CMDs Ejisu – Juaben District, Ghana Ho District, Ghana Badeku and Ojoku/Ajia Districts, Nigeria Bugiri and Iganga Districts, Uganda Totals CMD registers Number of episodes treated with ACTs from a CMD (CMD registers) 4522 3958 1044 11039 20563 Number (%) of children reporting an adverse event to a CMD 63 (1%) 10 (<1%) NA NA Number (%) children reported recovered NA NA NA NA Survey data Number of episodes treated with ACTs from a CMD (surveys) 289 93 288 619 1289 Number (%) of children reporting an adverse event at survey 24 (8%) 7 (8%) 10 (4%) 34 (6%) 75 (6%) Number (%) children reported recovered NA NA 284 (99%) NA 284 (99%) Number of caregivers (%) who perceived treatment to be effective 286 (99%) 93 (100%) 280 (97%) NA 659 (98%) Table 6: Safety and perceived effectiveness of treatment of ACTs obtained from CMDs ings are consistent in four different sites in sub-Saharan Africa, both in West and East Africa. These studies were designed for the CMDs to provide treatment on the basis of a clinical diagnosis. Discussion The possibil- ity of moving away from a symptom based treatment to more complex therapeutic protocols based on parasito- logical confirmation is currently being investigated. To bring parasitological diagnosis to the community level will require reliable diagnostic tools, and evidence of fea- sibility and acceptability by the community. There was high utilization of CMDs. In an estimated pop- ulation of children aged 6 to 59 months across the sites of 27,450, the CMDs treated 20,563 episodes of fever during the year of implementation (0.75 episodes of fever per child on average)). This contrasts with the figure of 0.12–0.34 episodes per year reported from African health facilities in available literature [10]. Furthermore, the majority of caregivers reported that CMDs were available at the first time of asking, and very little attrition of CMDs was reported. This demonstrates the potential of CMDs to increase access to care in underserved rural areas in Africa, where government health facilities are often under-uti- lized [11,12]. While every effort was made to ensure the implementation of the study as close as possible to real life conditions, its short duration and the research setting are likely to have contributed to the low attrition rate of CMDs observed in this study. Indeed, the need to provide CMDs with incentives was repeatedly mentioned in inter- views and FGDs, and financial motivation is known to be a key factor determining long term performance of CMDs [13]. It is clear that this need must be addressed to ensure the long-term sustainability of CMDs performance. Adherence to the full treatment course has been high- lighted as a matter of concern [14-16]. AL has to be taken twice a day for three days (one or two tablets depending on the age group), AS/AQ once daily for three days but two different tablets (or half tablets) each time. Both of these regimens are different from those for older antima- larials like chloroquine or sulphadoxine-pyrimethamine (SP). Nonetheless, reported adherence by caregivers to the correct treatment schedule in terms of dose and duration was high for both treatment regimes in all sites, ranging from 79% in Uganda to 97% in Ejisu, Ghana (85% on average across the sites). These findings are reassuring with regard to the potential for development of resistance, as exposure to sub-therapeutic drug levels is known to be a major factor i Page 7 of 9 (page number not for citation purposes) n the selection of resistant parasites [17]. Concerns have been raised by several authors about the potential misuse of ACT when deployed beyond the health system [6,7], which could lead to wastage of resources and the development of resistance to ACT. How- ever, our findings indicate that there was appropriate pre- scription in terms of dose by CMDs and correct use by caregivers. In all study sites, 97% or more of the CMDs prescribed the correct dose of ACTs, and almost all of them explained the treatment schedule to the caregiver. Drug storage conditions were satisfactory in all sites. Furthermore, the majority of caregivers obtaining ACT from a CMD reported that they treated their children promptly (ranging from 86% to 97%, 90% on average across the sites, Table 5). Data from CMDs' registers (Table 2) support this finding, albeit with slightly lower proportions of children recorded to be treated on the day of onset or the next day in three sites. In Uganda, CMDs reported treating only half of the episodes within 24 hours, a proportion which is still substantially higher than previously reported by Nsungwa-Sabiiti [16]. The design Page 7 of 9 (page number not for citation purposes) Page 7 of 9 (page number not for citation purposes) http://www.malariajournal.com/content/7/1/6 http://www.malariajournal.com/content/7/1/6 http://www.malariajournal.com/content/7/1/6 Malaria Journal 2008, 7:6 of this study does not provide robust data on pharma- covigilance. However, both CMDs and caregivers report a very low incidence of adverse drug effects, none of which were reported to be serious. While further studies are necessary to develop mecha- nisms for ensuring long-term performance of CMDs and to evaluate the effectiveness and safety of ACTs used within the context of HMM programmes, the findings of this study provide evidence to support scaling-up imple- mentation of HMM with ACTs. Access to prompt and effective treatment is a cornerstone of the current malaria control strategy [18]. Several strate- gies have been developed to address the issue of access to antimalarials in Africa, including home management of malaria [1], improving the services of private medicine sellers [19], a set of integrated interventions including social marketing approaches [20] and improving the qual- ity of health service performance [21]. n the selection of resistant parasites [17]. Our study provides evidence that once the option of treating children with effective antimalarials close to home is made available to caregivers, a majority of them (ranging from 52% in Nigeria to 75% in Ho, Ghana) make use of it, in most cases in an appropriate and timely manner. This option should be regarded as a complementary to, rather than competing with, health facility delivery of care. As high- lighted by Unger et al [21], interventions aiming at improving use rates of general health services have also a high potential of increasing malaria cure rates. Competing interests The author(s) declare that they have no competing inter- ests. Authors' contributions All the authors except SC conceived the study; IA, ENB, BG and FB were principal investigators for their respective country's study site and together with FP, LD, BY and AB participated in the research designed and supervised data collection from the field. BY, PAB and SC performed most of the quantitative data analysis while KM analysed the qualitative data. FP, the WHO/TDR HMM research pro- gram manager monitored the four sites. All authors con- tributed to the draft. All authors read and approved the final manuscript. Acknowledgements Overall effectiveness of drugs distributed at the commu- nity level is known to be influenced by several factors [16,22,23], not all of them addressed in this study. How- ever, the high level of correct prescription by CMDs, the promptness of treatment seeking and adherence to the treatment schedule by caregivers, and the coverage (59% on average across the sites) achieved in this study show that some key factors determining the effectiveness of antimalarial treatment can be successfully addressed. Thus, this approach has great potential to contribute to reaching the Abuja target that "at least 60% of those suffer- ing from malaria have prompt access to, and are able to cor- rectly use, affordable and appropriate treatment within 24 hours of the onset of symptoms" [2]. The authors acknowledge the immense role of the community members, opinion leaders, CMDs, the health workers, research assistants, field super- visors and workers in Ejisu – Juaben and Ho district, Ghana, Ona-ara Local Government, Nigeria and Bugiri and Iganga Districts, Uganda. We also acknowledge the valuable contribution of Dr. Josephine Namboze (WHO/AFRO) and Wilson Were (WHO/GMP) who facilitated the proto- col development workshops. This investigation received technical and financial support from the UNICEF/UNDP/World Bank/WHO Special Pro- gramme for Research and Training in Tropical Diseases. References 1. World Health Organization: The Roll Back Malaria Strategy for Improving Access to Treatment through Home Manage- ment of malaria. WHO/HTM/MAL/2005.1101 . 2. World Health Organization: The African Summit on Roll Back Malaria, Abuja, Nigeria. Geneva. WHO/CDS/RBM/2000.17 . 25 April 2000 Previous studies have shown that use of the correct dose of chloroquine in uncomplicated malaria correlates with improvements in clinical condition [24], and that HMM with chloroquine has been effective in reducing both mor- tality[25] and severe malaria morbidity [26]. Studies in Uganda and Tanzania [27,28] have recently shown that the high parasitological cure rate of AL is not affected by unsupervised administration under routine clinical prac- tice. A sub-study evaluating parasitological efficacy of the ACTs used in this study is being undertaken and will be reported on separately. 3. Millennium goals: [http://www.un.org/millenniumgoals/_links.html]. accessed 20 August 2007. g 4. World Health Organization: WHO Global Malaria Programme, personal communication 2007. 4. World Health Organization: WHO Global Malaria Programme, personal communication 2007. 5. World Health Organization: WHO Global Malaria Programme, personal communication 2007. 5. World Health Organization: WHO Global Malaria Programme, personal communication 2007. 6. Charlwood D: The paradox of home management of malaria with artemisinin combinations. Trends Parasitol 2004, 20:405-406. 7. D'Alessandro U, Talisuna A, Boelaert M: Should artemisinin- based combination treatment be used in the home-based management of malaria? Trop Med Int Health 2005, 10:1-2. g p 8. Pagnoni F, Kengeya-Kayondo J, Ridley R: Artemisinin-based com- bination treatment in home-based management of malaria. Trop Med Int Health 2005, 10:621-22. p 9. Chinbuah MA, Gyapong JO, Pagnoni F, Wellington EK, Gyapong M: Feasibility and Acceptability of the use of artemether-lume- fantrine in the Home-management of uncomplicated malaria in children 6–59 months old in Ghana. Trop Med Int Health 2006, 11:1003-1016. This study shows that ACT use can be successfully inte- grated in the HMM strategy. 10. 10. Levy-Bruhl D, Soucat A, Osseni R, Ndiaye JM, Dieng B, De Bethune X, Diallo AT, Conde M, Cisse M, Moussa y, Drame K, Knippenberg R: 0. Levy-Bruhl D, Soucat A, Osseni R, Ndiaye JM, Dieng B, De Bethune X, Diallo AT, Conde M, Cisse M, Moussa y, Drame K, Knippenberg R: 10. References Unger J-P, d'Alessandro U, De Paepe P, Green A: Can malaria be controlled where basic health services are not used? Trop Med Int Health 2006, 11:314-322. 22. Krause G, Sauerborn R: Comprehensive community effective- ness of health care. A study of malaria treatment in children and adults in rural Burkina Faso. Ann Trop Paed 2000, 20(4):273-282. ( ) 23. Mueller O, Razum O, Traore C, Kouyate B: Community effective- ness of chloroquine and traditional remedies in the treat- ment of young children with falciparum malaria in rural Burkina Faso. Malar J 2004, 3:36. doi:10.1186/1475-2875-3-36 J 24. Okonkwo PO, Akpala CO, Okafor HU, Mbah AU, Nwaiwu O: Com- pliance to correct dose of chloroquine in uncomplicated malaria correlates with improvement in the condition of rural Nigerian children. Trans R Soc Trop Med Hyg 2001, 95:320-324. 25. Kidane G, Morrow RH: Teaching mothers to provide home management of malaria in Tigray, Ethopia: a randomized trial. Lancet 2000, 356:550-55. 26. Sirima SB, Konaté A, Tiono AB, Convelbo , Cousens S, Pagnoni F: Early treatment of childhood fevers with pre-packed antima- larial drugs in the home reduces severe malaria morbidity in Burkina Faso. Trop Med Int Health 2003, 8:133-139. Publish with BioMed Central and every scientist can read your work free of charge "BioMed Central will be the most significant development for disseminating the results of biomedical research in our lifetime." Sir Paul Nurse, Cancer Research UK Your research papers will be: available free of charge to the entire biomedical community peer reviewed and published immediately upon acceptance cited in PubMed and archived on PubMed Central yours — you keep the copyright Submit your manuscript here: http://www.biomedcentral.com/info/publishing_adv.asp BioMedcentral Page 9 of 9 (page number not for citation purposes) Publish with BioMed Central and every scientist can read your work free of charge "BioMed Central will be the most significant development for disseminating the results of biomedical research in our lifetime." Sir Paul Nurse, Cancer Research UK Your research papers will be: available free of charge to the entire biomedical community peer reviewed and published immediately upon acceptance cited in PubMed and archived on PubMed Central yours — you keep the copyright Submit your manuscript here: http://www.biomedcentral.com/info/publishing_adv.asp BioMedcentral Publish with BioMed Central and every scientist can read your work free of charge 27. References Levy-Bruhl D, Soucat A, Osseni R, Ndiaye JM, Dieng B, De Bethune X, Diallo AT, Conde M, Cisse M, Moussa y, Drame K, Knippenberg R: Page 8 of 9 (page number not for citation purposes) Page 8 of 9 (page number not for citation purposes) (page number not for citation purposes) Malaria Journal 2008, 7:6 http://www.malariajournal.com/content/7/1/6 http://www.malariajournal.com/content/7/1/6 The Bamako Initiative in Benin and Guinea: improving the effectiveness of primary health care. Int J Health Plann Manag 1997:S49-S79. 11. Mutabingwa TK: Artemisinin-based combination therapies (ACTs): Best hope for malaria treatment but inaccessible to the needy! Acta Trop 2005, 95:305-315. y p 12. Breman JG, Alilio RS, Mills A: Conquering the Intolerable Bur- den of Malaria: What's new, what's needed: a summary. Am J Trop Med Hyg 2004:1-15. 13. Batega DW, Greer GJ, Plowman BA: Home-based management of fever strategy in Uganda: Assessment of implementation and operation at district and community levels. In Ministry of Health Uganda, the World Health Organization, and BASICS II for the United States Agency for International Development Arlington, Va; 2004. g y p g 14. McCombie SC: Self-treatment for malaria: the evidence and methodological issues. Health Policy Plan 2002, 17:333-344. g y 15. Yeung S, White NJ: How do patients use antimalarial drugs? A review of the evidence. Trop Med Int Health 2005, 10:121-138. 16 N S bii i J T G P i G O l Ok J P S 16. Nsungwa-Sabiiti J, Tomson G, Pariyo G, Ogwal-Okeng J, Peterson S: Community effectiveness of malaria treatment in Uganda – a long way to Abuja targets. Ann Trop Paed 2005, 25:91-100. g y j g p 17. White NJ: Antimalarial drug resistance. J Clin Invest 2004, 113:1084-1092. 18. World Health Organization/UNICEF Africa malaria report 2003 WHO, Geneva 2003 [http://mosquito.who.int/amd2003/ amr2003/pdf/amr2003.pdf]. 19. Abuya TO, Mutemi W, Karisa B, Ochola SA, Fegan G, Marsh V: Use of over-the-counter malaria medicines in children and adults in three districts in Kenya: implications for private medicine retailer interventions. Malar J 2007, 6:57. doi:10.1186/1475-2875- 6-57 20. Hetzel MW, Iteba N, Makemba A, Mshana C, Lengeler C, Obrist B, Schulze A, Nathan R, Dillip A, Alba S, Mayumana I, Khatib RA, Njau JD, Mshinda H: Understanding and improving access to prompt and effective malaria treatment and care in rural Tanzania: the ACCESS Programme. Malar J 2007, 6:83. doi:10.1186/1475-2875-6-83 21. References Piola P, Fogg C, Bajunirwe F, Biraro S, Grandesso F, Ruzagira E, Babi- gumira J, Kigozi I, Kiguli J, Kyomuhendo J, Ferradini L, Taylor W, Checchi F, Guthmann JP: Supervised versus unsupervised intake of six-dose artemether-lumefantrine for treatment of acute, uncomplicated Plasmodium falciparum malaria in Mbarara, Uganda: a randomised trial. Lancet 2005, 365:1467-1473. g 28. Mutabingwa TK, Anthony D, Heller A, Hallett , Ahmed J, Drakeley C, Greenwood BM, Whitty CJM: Amodiaquine alone, amodiaquine_sulphadoxinepyrimethamine, amodiaquine artesunate, and artemether-lumefantrine for outpatient treatment of malaria in Tanzanian children: a four-arm ran- domised effectiveness trial. Lancet 2005, 365:1474-1480.
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Malaria policies versus practices, a reality check from Kinshasa, the capital of the Democratic Republic of Congo
BMC public health
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* Correspondence: mavoko@yahoo.com 1Département de Médecine Tropicale, Université de Kinshasa, B.P. 747, Kin XI, Kinshasa, République Démocratique du Congo 2International Health Unit, Department of Epidemiology, University of Antwerp, Campus Drie Eiken, Universiteitsplein 1, 2610 Kinshasa, République Démocratique du Congo Full list of author information is available at the end of the article RESEARCH ARTICLE Open Access Malaria policies versus practices, a reality check from Kinshasa, the capital of the Democratic Republic of Congo Hypolite Muhindo Mavoko1,2*, Gillon Ilombe1, Raquel Inocêncio da Luz2, Albert Kutekemeni3, Jean-Pierre Van geertruyden2 and Pascal Lutumba1 Muhindo Mavoko et al. BMC Public Health (2015) 15:352 DOI 10.1186/s12889-015-1670-0 Study setting and design y This was an observational study embedded in a two- stage cluster randomized survey conducted in Kinshasa, the capital city of the Democratic Republic of Congo (DRC). This city is divided into 35 health zones among them 12 were randomly selected: Bandalungwa, Binza ozone, Bumbu, Kimbanseke, Kingabwa, Kokolo, Limete, Makala, Maluku 1, Masina 1, Police and Selembao. In each health zone, one public or private HC was ran- domly selected among those reporting to the National Health Information System. The former study consisted of evaluating the accuracy of Optimal-IT®, as well as Paracheck-Pf® malaria rapid diagnostic tests (RDTs) [5]. RDTs were performed and microscopy slides prepared at recruitment. HC laboratory technicians had to read the slides and were blinded to the RDTs results. The same slides were read by expert microscopists for quality control (QC) and they were blinded to previous results. The QC results constituted the gold standard to assess the accuracy of RDTs and microscopy at PHC level. The sample size of 624 was calculated for the main study based on the accuracy of Optimal-IT® RDT and the same number was involved in these supplementary analyses. In Sub Saharan Africa, which holds the gross of the malaria burden, the proportion of people treated for malaria with a confirmed diagnosis is low compared to other regions of the world [4]. In addition, the accuracy of malaria diagnosis at primary health care has been re- ported to be poor in many areas [5-8]. This may explain the fact that health practitioners tend to prescribe antimalarial drugs regardless of laboratory results. Fur- thermore, patients can purchase antimalarial drugs dir- ectly on the informal market without any medical prescription [9,10]. In 2010, only a few months after its publication, the Congolese National Malaria Control Program (NMCP) adopted the second edition of the WHO malaria treatment guidelines [2]. The major novelty was the recommendation to give antimalarial treatment upon parasitological evidence of malaria infection even in children under the age of five years. The purpose was to improve targeting of treatment and better quality of care, in particular in an environment of in- creased malaria control and or declining malaria en- demicity [2]. Indeed non malaria cases would be identified and may benefit from timely treatment thereby reducing morbidity and mortality. Abstract BMC Public Health (2015) 15:352 Page 2 of 7 Methods Approximately half of the global population is exposed to malaria [1]. Early parasitological diagnosis and effica- cious treatment in all age groups are recommended by the World Health Organization (WHO) as key strategies to control malaria [2]. However, next to policies, avail- ability of good quality drugs and parasite based diagnos- tic is essential [3]. Other important factors are access to health care services as well as the health seeking behav- iour of the population. Policies are principally based on high quality research evidence, but implementation re- mains challenging on various levels because of some field realities. Abstract Background: Artemisinin-based combination therapy (ACT) following a confirmed parasitological diagnosis is recommended by the World Health Organization (WHO) and the Congolese National Malaria Control Program (NMCP). However, commitment and competence of all stakeholders (patients, medical professionals, governments and funders) is required to achieve effective case management and secure the “useful therapeutic life” of the recommended drugs. The health seeking behaviour of patients and health care professionals’ practices for malaria management were assessed. Methods: This was an observational study embedded in a two-stage cluster randomized survey conducted in one health centre (HC) in each of the 12 selected health zones in Kinshasa city. All patients with clinical malaria diagnosis were eligible. Their health seeking behaviour was recorded on a specific questionnaire, as well as the health care practitioners’ practices. The last were not aware that their practices would be assessed. Results: Six hundred and twenty four patients were assessed, of whom 136 (21.8%) were under five years. Three hundred and thirty five (55%) had taken medication prior to the current consultation (self -medication with any product or visiting another HC) of whom 47(14%) took an antimalarial drug, and 56 (9%) were treated presumptively. Among those, 53.6% received monotherapy either with quinine, artesunate, phytomedicines, sulfadoxine-pyrimethamine or amodiaquine. On the other side, when clinicians were informed about laboratory results, monotherapy was prescribed in 39.9% of the confirmed malaria cases. Only 285 patients (45.7%) were managed in line with WHO and NMCP guidelines, of whom 120 (19.2%) were prescribed an ACT after positive blood smear and 165 (26.4%) received no antimalarial after a negative result. Conclusion: This study shows the discrepancy between malaria policies and the reality on the field in Kinshasa, regarding patients’ health seeking behaviour and health professionals’ practices. Consequently, the poor compliance to the policies may contribute to the genesis and spread of antimalarial drug resistance and also have a negative impact on the burden of the disease. Keywords: Malaria, Policies, Practices, Health seeking behaviour, Phytomedicines, Democratic Republic of Congo © 2015 Muhindo Mavoko et al.; licensee BioMed Central. This is an Open Access article distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/by/4.0), which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly credited. The Creative Commons Public Domain Dedication waiver (http://creativecommons.org/publicdomain/zero/1.0/) applies to the data made available in this article, unless otherwise stated. Muhindo Mavoko et al. Study setting and design In DRC, confirmed uncomplicated malaria cases are recommended to be treated either with artesunate+amodiaquine (ASAQ) or artemether+lumefantrine (AL) as the first line, and quinine+antibiotic having antimalarial activity as second line [11]. Data were collected from May to June 2011. All the patients attending the health facility and clinically diag- nosed with malaria and to whom a blood smear (BS) was requested, were eligible. Clinical diagnosis was established by clinicians based on unspecific known mal- aria symptoms like fever, history of fever, headache, chills, weakness, muscle and joint pain and anorexia. Data was collected in 2 phases. First, a questionnaire was filled during an interview. Socio-demographic data were recorded and the patient/guardians’ health seeking behaviour was assessed i.e. current illness, action taken and health seeking practices prior to the current visit. In a second phase, the medications (antimalarial and others) prescribed by the clinicians before and after the BS results as well as the laboratory results were also re- corded. Clinicians were not informed on the data collec- tion to avoid observer’s bias. Antimalarial drugs taken before seeking care and/or prescribed at the PHC were identified using the list of antimalarial drugs available on the market in Kinshasa (e.g. Artemether+lumefantrine: Coartem®, Coartesiane®, Lonart®, Luther®, etc.). Improving malaria case management requires the involvement of all stakeholders and detection of the operational bottle necks. Inspired by the Piot Model [12], the health seeking behaviour of the patients and practices of the health care providers were assessed going from the first presumed malaria symptoms until the delivery of care in formal health centres (HC). The objective was to assess health seeking be- haviour of patients and practices related to the man- agement of uncomplicated malaria by the health professional at primary health care level (PHC) com- pared to the policies. Laboratory examination y The collected BSs were stained with 10% Giemsa for 10 minutes. Thin BSs were fixed with methanol prior to the staining. Microscopy reading was first performed by the HC laboratory technicians and result was given to clinicians, who were supposed to manage the cases according to parasitological diagnosis and National Page 3 of 7 Muhindo Mavoko et al. BMC Public Health (2015) 15:352 age groups (p < 0.001) (Table 1). At PHC, 361 patients (57.9%) tested positive for malaria, whereas only 123 (19.7%) were confirmed by the experts’ reading. guidelines and policies [11]. Slides were subsequently transported to the parasitology unit, Kinshasa University for QC. Prescribing practice ( The study was approved by the Committee for Medical Ethics of the Antwerp University Hospital, Belgium (approval reference: 14/36/236) and the Ethical Committee of the School of Public Health, Kinshasa University, DRC (approval reference: ESP/CE/082/10). Before inclusion, written informed consent was obtained from all patients or their legal guardians if minors. Prescribing practice Only 285 cases (45.7%) were managed in line with WHO guidelines as ACTs were prescribed to 120 positive cases and nothing to 165 negative cases (Figure 1). ACTs consisted of ASAQ (100 cases, 30.0%), AL (63 cases, 19.0%) and artesunate+sulfadoxine-pyrimeth- amine (AS+SP) (7 cases, 2.1%). Monotherapy consisted of quinine (88 cases, 26.7%), artesunate (31 cases, 9.4%), phy- tomedicines (24 cases, 7.3%), sulfadoxine-pyrimethamine (16 cases, 4.8%) and amodiaquine (1 case, 0.3%). Data analysis g Among 373 patients who reported history of fever, 24 (6.4%) attended the HC the same day when fever started, 55 (14.8%) the following day, 154 (41.3%) after 2–3 days, 95 (25.5%) after 4–7 days, whereas 45 (10.1%) waited for more than a week. Lowest proportion of malaria was confirmed (both at PHC and by experts) among those who came more than a week after the fever onset (Table 2). At the same time, malaria was less confirmed in the group that attended the HC directly (Table 2). Be- fore the current consultation, 50 patients (8%) had taken traditional treatment, 276 (44.2%) resorted to self- treatment with modern drugs, 16 (2.8%) visited another HC previously. Two hundred and eighty two (45.2%) did not undertake any action prior to the current visit (Figure 1). Before attending the HC, 100 patients (16%) had taken an antimalarial drug, 288 (46.2%) took an- other type of drug (antibiotics, antipyretic, etc.), and 235 (37.8%) took no drugs. Patients who had taken no anti- malarial drugs more likely attended the HC during the first 3 days of fever onset (p = 0.02). Data were double-entered and validated in Epi info ver- sion 3.5.1 software and analysed using Stata version 11 (Stata Corp, Lakeway, College Station, Texas, USA). The primary outcome was the proportion of patients to whom a recommended antimalarial drug was prescribed after a positive test and those for whom no prescription was made after a negative test. Secondary outcomes in- cluded description of patients’ health seeking behaviour (time for consultation regarding the illness episode on- set, medication taken before consultation) and malaria testing outcome compared to patients’ health seeking behaviour and clinicians’ practices. Descriptive statistics were used to get frequencies and percentages. Cross tab- ulations were performed to check relationship between variables of interest using the Chi-square test of Pearson. Predictors of malaria confirmation were assessed by a logistic regression, referring to experts’ microscopy read- ing result. The cluster effect was checked and did not influence the results. *out of 623; **out of 487. Baseline characteristics The lack of compliance with NMCP guidelines has been reported elsewhere in sub-Saharan Africa [15-20]. Malaria control programs need to elaborate an efficient strategy for the follow up of adherence to guidelines by practitioners. In addition, a dialogue between policy makers, researchers and program managers is needed to discuss and address the gaps in the implementation of malaria management policies [21]. The overtreatment reported in this survey could be the result of unsatisfactory diagnostic accuracy at PHC [5,22]. This may explain why clinicians seldom relied on the BS results. Another explanation could be related to the mistrust of policies evidence, as reported in other studies [23,24]. In Tanzania, Reyburn et al. [25] found that almost half of patients with a negative malaria test were treated with an antimalarial drug. The responsibil- ity of the overtreatment is to be shared with the patients who can influence health practitioners’ practice [26,27]. In Ghana, participants to a focus group discussion claimed that a test should result in a diagnosis, even fol- lowing a negative result [28]. In this aspect, clinicians may choose to prescribe antimalarial drugs, mostly when laboratory diagnoses for other infections are not available. most prescribed antimalarials in these cases. Clinicians changed the prescription for 7 of them in light of the BS results from SP to ACT. Prior treatment before attending the HC did not influence prescription be- havior (p = 0.09). Clinicians tended to prescribe more antimalarial drugs to children younger than five, even when laboratory re- sults were negative. Clear guidelines for management of non-malaria febrile illnesses as well as appropriate tests and drugs are urgently needed [29]. By the time this survey was conducted, ASAQ and AL were the recom- mended first line ACTs. It was noticed that clinicians tended to prescribe more ASAQ when BS result was positive. When prescription was done before BS or in spite of negative BS result, either Quinine or AL was preferred. Assuming that they were not aware about the introduction of AL in the national guidelines some months prior to this assessment, the hypothesis is that they had preference for non-recommended antimalarial drugs in absence of malaria confirmation. For 330 patients (53.0%), antimalarial prescription was done based on BS results. In that case, ASAQ was most frequently prescribed (30.3%), followed by quinine monotherapy (26.7%) and AL (19.1%). Baseline characteristics Fifty six patients (9.0%) received an antimalarial pre- scription while BS results were still unknown and this was observed more frequently in children under five years, but the difference was not significant (p = 0.54). Quinine (34.0%), AL (34.0%) and SP (10.7%) were the A minimum of 50 patients were enrolled from each HC, with a total of 624. Of those, 136 were under five years (21.8%). In general, history of fever was the most fre- quent symptom (59.8%), and it was significantly more reported in children under five years compared to other Table 1 Baseline characteristics of 624 patients in primary health care setting, Kinshasa, DRC, 2011 Variable All age group <5 years ≥5 year p (N = 624) (N = 136) (N = 488) n (%) n (%) n (%) Female (n, %) 362 (58.0) 62 (45.6) 300 (61.5) 0.001 History of fever 373 (59.8) 123 (90.4) 250 (51.2) <0.001 Ownership mosquito net 333 (53.4) 76 (55.8) 257 (52.7) 0.51 Slept under mosquito net last night 224 (35.9) 56 (41.1) 168 (34.4) 0.15 Malaria self-treatment prior current consultation using any antimalarial drug 100 (16.0)* 18 (13.2) 82 (16.8)** 0.31 * t f 623 ** t f 487 ics of 624 patients in primary health care setting, Kinshasa, DRC, 2011 Page 4 of 7 Muhindo Mavoko et al. BMC Public Health (2015) 15:352 Table 2 Diagnosis outcome compared to the patients’ health seeking practice in primary health care setting, Kinshasa, DRC, 2011 Patients’ practice Malaria cases confirmed at PHC Malaria cases confirmed by experts n (%) n (%) Days from fever onset to the current consultation (n/ N) 0 (24/ 373) 17 (70.8) 7 (29.2) 1 (55/ 373) 30 (54.6) 15 (27.3) 2 to 3 (154/ 373) 102 (66.2) 50 (32.5) 4 to 7 (95/ 373) 61 (64.2) 21 (22.1) >7 (45/ 373) 26 (57.8) 9 (20.0) Care seeking bahaviour Self-treatment with traditional products (50/ 624) 33 (66.0) 12 (24.0) Self-treatment with modern products (276/ 624) 166 (60.1) 72 (26.1) Visited another HC (16/ 624) 9 (56.3) 2 (12.5) No action (282/ 624) 153 (54.3) 37 (13.1) Table 2 Diagnosis outcome compared to the patients’ health seeking practice in primary health care setting, Kinshasa, DRC, 2011 drugs and the patient/ care giver’s compliance to the treat- ment [13,14]. Nevertheless, this study did not explore those aspects. Baseline characteristics Monotherapy was prescribed to 39.8% of patients who tested positive for malaria at PHC. Malaria was more likely confirmed by experts’ reading when the patients had reported history of fever (p < 0.001), undertook no treatment before attending the HC (p < 0.001) and lacked mosquitonet (p = 0.05). In the multivariate analysis, history of fever, treatment seeking behaviour and not sleeping under mosquitonet were significantly associated with malaria confirmation (Table 3). Combination treatment using ACTs is the key strategy to reduce the impact of failing monotherapies [30,31]. Unfortunately and against the national guidelines, this survey highlighted a worrying proportion of pre- scribing monotherapy, including artemisinin mono- therapy (AMT), a practice that could increase treatment failures and contribute to the spread of resistance. Despite the WHO’s call to stop AMT production and marketing to refrain the development of drug resistance [31], the field reality is still different. However, a trend towards their disappearance from the market in Kinshasa is notably ob- served. In 2009 they constituted 10% and 41% of antimal- arial drugs stocked in public/ Not-for-Profit and private *Malaria confirmed by experts. Acknowledgement We thank the study patients and the staff of the involved health centres. We are also grateful to Vito Baraka for proofreading of the manuscript. This study was funded by VLIR-UOS through project number ZRDC2008MP053, which aimed to create a clinical research unit and the capacity building at the Tropical Medicine Department, Kinshasa University. Apparently, none of the clinicians involved was aware, 6 months after the NMCP guidelines update, that quin- ine should not be used as a monotherapy. This survey did not focus on the knowledge nor the background of practitioners and further research will be needed to as- sess explanatory causes and identify the bottlenecks where training or adapted measurements can be taken. The lack of this information is a limitation of the study, but these findings may be reproducible in other settings. References Directives nationales de traitement du paludisme en République Démocratique du Congo. Kinshasa: Ministère de la Santé; 2010. 12. Mumba M, Visschedijk J, van Cleeff M, Hausman B. A Piot model to analyse case management in malaria control programmes. Trop Med Int Health. 2003;8(6):544–51. 12. Mumba M, Visschedijk J, van Cleeff M, Hausman B. A Piot model to analyse case management in malaria control programmes. Trop Med Int Health. 2003;8(6):544–51. Authors’ contributions HMM, PL and JPV designed the study protocol. HMM and GI supervised the activities on the field. HMM performed the statistical analysis. RIL, HMM and JPV drafted the manuscript. HMM, JPV, PL, GI, RIL and AK reviewed the paper and all authors approved the final version. Competing interests Competing interests The authors declare that they have no competing interests. It is worth mentioning the ongoing initiative in the coun- try supported by the Global Fund that aims to supply RDTs as well as recommended medications. The support is pres- ently available in 60% of the health zones in DRC. At the time this survey was conducted, two of the twelve HC in- volved were supplied, although on an irregular basis. References 1. WHO. World Malaria Report 2011. Geneva: World Health Organization; 2011. 2. WHO. Guidelines for the treatment of malaria. Geneva: World Health Organization; 2010. p g ; 2. WHO. Guidelines for the treatment of malaria. Geneva: World Health Organization; 2010. 3. Bate R, Coticelli P, Tren R, Attaran A. Antimalarial drug quality in the most severely malarious parts of Africa – a six country study. PLoS One. 2008;3(5):2132. 3. Bate R, Coticelli P, Tren R, Attaran A. Antimalarial drug quality in the most severely malarious parts of Africa – a six country study. PLoS One. 2008;3(5):2132. 4. WHO. World Malaria Report 2008. Geneva: World Health Organization; 2008. 5. Muhindo MH, Ilombe G, Meya R, Mitashi PM, Kutekemeni A, Gasigwa D, et al. Accuracy of malaria rapid diagnosis test Optimal-IT® in Kinshasa, the Democratic Republic of Congo. Malar J. 2012;11:224. 6. Mukadi P, Gillet P, Lukuka A, Atua B, Kahodi S, Lokombe J, et al. External quality assessment of malaria microscopy in the Democratic Republic of the Congo. Malar J. 2011;10:308. g 7. McKenzie FE, Sirichaisinthop J, Miller RS, Gasser RA, Wongsrichanalai C. Dependence of malaria detection and species diagnosis by microscopy on parasite density. Am J Trop Med Hyg. 2003;69:372–6. 8. Zurovac D, Midia B, Ochola SA, English M, Snow RW. Microscopy and outpatient malaria case management among older children and adults in Kenya. Trop Med Int Health. 2006;11:432–40. 9. Luxemburger C, Nosten F, Kyle DE, Kiricharoen L, Chongsuphajaisiddhi T, White NJ. Clinical features cannot predict a diagnosis of malaria or differentiate the infecting species in children living in an area of low transmission. Trans R Soc Trop Med Hyg. 1998;92:45–9. Approximately a third (35.9%) of those owning a mos- quitonet slept under it the night before consultation. The protective impact on sleeping under mosquitonet was shown by the multivariate analysis. Hence the cam- pains are highly needed to sensitize the mass on the benefits of using the mosquitonet. 10. Mbonye AK, Bygbjerg IC, Magnussen P. Prevention and treatment practices and implications for Malaria control in Mukono District Uganda. J Biosoc Sci. 2008;40:283–96. 10. Mbonye AK, Bygbjerg IC, Magnussen P. Prevention and treatment practices and implications for Malaria control in Mukono District Uganda. J Biosoc Sci. 2008;40:283–96. 11. Programme National de Lutte contre le Paludisme. Directives nationales de traitement du paludisme en République Démocratique du Congo. Kinshasa: Ministère de la Santé; 2010. 11. Programme National de Lutte contre le Paludisme. Author details 1 1Département de Médecine Tropicale, Université de Kinshasa, B.P. 747, Kin XI, Kinshasa, République Démocratique du Congo. 2International Health Unit, Department of Epidemiology, University of Antwerp, Campus Drie Eiken, Universiteitsplein 1, 2610 Kinshasa, République Démocratique du Congo. 3Programme National de Lutte contre le Paludisme, Ministère de la Santé Publique, Kinshasa, République Démocratique du Congo. Almost half of the patients (44.2%) undertook a treat- ment prior to the current consultation and they tended to be more infected with malaria. This means that either the medication taken was not effective or the treatment course was not appropriate. Most of them used antimal- arial, antibiotics and antipyretics. Such inappropriate use of antibiotics and antimalarials is dangerous as it may contribute to the emergence and spread of drug resistant bacteria and Plasmodium [29,33]. Self-treatment has been reported to be common, mainly in rural areas of Africa [9]. People should be encouraged to address to the health facilities as soon as the clinical episode starts, for proper diagnosis and treatment. In the urban settings like Kinshasa, geographical access is not the main obs- tacle but affordability of the health services can be the main limiting factor. This could explain why few people with history of fever attended the HC the same day. Ac- cess to health may be increased by increasing the health insurance coverage either through the Government or the private stakeholders. Received: 14 June 2014 Accepted: 20 March 2015 Discussion Results of this survey highlight the alarming situation of malaria management at PHC in the DRC because of a huge discrepancy between policy and field reality. Almost half of the patients (45.7%) were managed in line with NMCP guidelines. However, 7 patients (2.1%) received a prescription for AS+SP which is not offi- cially recommended in DRC but still in line with the WHO recommendations. Besides, a proper prescription is not enough to ensure accurate malaria management. The steps forward are availability of quality-assured antimalarial Page 5 of 7 Muhindo Mavoko et al. BMC Public Health (2015) 15:352 Figure 1 Flow chart of the patients’ and clinicians’ practices in primary health care setting, Kinshasa, DRC, 2011. igure 1 Flow chart of the patients’ and clinicians’ practices in primary health care setting, Kinshasa, DRC, 2011. confirmed by experts’ reading based on practices of patients and medicals in primary health care a, DRC, 2011 Table 3 Malaria confirmed by experts’ reading based on practices of patients and medicals in primary health care setting, Kinshasa, DRC, 2011 Variable (n) Blood smear positive (%)* OR (95% CI) p Adj OR* (95% CI) p Age group <5 years (136) 33 (24.3) 1.4 (0.9-2.3) 0.13 - NS ≥5 years (488) 90 (18.4) 1 History of fever Yes (373) 102 (27.4) 4.1 (2.5-7.1) <0.001 3.6 (2.1-6.1) <0.001 No (251) 21 (8.4) 1 1 Antimalarial treatment prior current consultation Yes (100) 17 (17) 1 - No (523) 106 (20) 0.8 (0.4-1.4) 0.45 NS Treatment seeking practice Yes (342) 86 (25.2) 2.2 (1.4-3.5) <0.001 1.6 (1.0-2.5) 0.04 No (282) 37 (13.1) 1 Mosquitonet ownership Yes (333) 56 (16.8) 1 0.05 - NS No (291) 67 (23.0) 1.4 (1.0-2.4) Slept under mosquito net last night Yes (224) 35 (15.6) 1 0.40 1 0.02 No (109) 21 (19.3) 1.3 (0.7-2.4) 1.7 (1.0-2.6) Malaria treatment prescribed before blood smear result Yes (56) 16 (28.6) 0.6 (0.3-1.2) 0.08 - NS No (568) 107 (18.9) 1 *Malaria confirmed by experts. Muhindo Mavoko et al. BMC Public Health (2015) 15:352 Page 6 of 7 Page 6 of 7 sector outlets respectively [13]. Four years later their avail- ability decreased below 1% in both sectors [32]. Competing interests Conclusion Data of this study indicate that neither the health seek- ing behaviour of the patients nor the treatment practices of the health care providers were appropriate. This may have a negative impact on the burden of the disease and contribute to the genesis and spread of drug resistance. However, national malaria related health policies are in line with WHO recommendations and regularly up- dated. Health education, including health promotion and training on fever management is urgently needed. 13. O’Connell KA, Gatakaa H, Poyer S, Njogu J, Evance I, Munroe E, et al. Got ACTs? Availability, price, market share and provider knowledge of anti-malarial medicines in public and private sector outlets in six malaria-endemic countries. Malar J. 2011;10:326. 13. O’Connell KA, Gatakaa H, Poyer S, Njogu J, Evance I, Munroe E, et al. Got ACTs? Availability, price, market share and provider knowledge of anti-malarial medicines in public and private sector outlets in six malaria-endemic countries. Malar J. 2011;10:326. 14. Banek K, Lalani M, Staedke SG, Chandramohan D. Adherence to artemisinin- based combination therapy for the treatment of malaria: a systematic review of the evidence. Malar J. 2014;13:7. 14. Banek K, Lalani M, Staedke SG, Chandramohan D. Adherence to artemisinin- based combination therapy for the treatment of malaria: a systematic review of the evidence. Malar J. 2014;13:7. 15. Sayang C, Gausseres M, Vernazza-Licht N, Malvy D, Bley D, Millet P. Treatment of malaria from monotherapy to artemisinin-based combination therapy by health professionals in rural health facilities in southern Cameroon. Malar J. 2009;8:174. 15. Sayang C, Gausseres M, Vernazza-Licht N, Malvy D, Bley D, Millet P. Treatment of malaria from monotherapy to artemisinin-based combination therapy by health professionals in rural health facilities in southern Cameroon. Malar J. 2009;8:174. Page 7 of 7 Page 7 of 7 Muhindo Mavoko et al. BMC Public Health (2015) 15:352 16. Krause G, Sauerborn R. Comprehensive community effectiveness of health care. A study of malaria treatment in children and adults in rural Burkina Faso. Ann Trop Paediatr. 2000;20:273–82. 17. Rowe AK, Ponce de León GF, Mihigo J, Santelli AC, Miller NP, Van-Dúnem P. Quality of malaria case management at outpatient health facilities in Angola. Malar J. 2009;8:275. 18. Manirakiza A, Njuimo SP, Le Faou A, Malvy D, Millet P. Availability of antimalarial drugs and evaluation of the attitude and practices for the treatment of uncomplicated malaria in Bangui, Central African Republic. J Trop Med. 2010;2010:510834. MOH. Conclusion Health Sector Strategic Plan III. Kampala, Uganda: Ministry of Health; 2 20. Ahmed ME, Yousif MA. Impact of the national protocol for malaria treatment on prescribing patterns in Gezira state, Sudan. East Mediterr Health J. 2004;10:566–72. 21. Mbonye AK, Magnussen P. Translating health research evidence into policy and practice in Uganda. Malar J. 2013;12:274. 22. Mbonye MK, Burnett SM, Colebunders R, Naikoba S, Van geertruyden JP, Weaver MR, et al. Disease diagnosis in primary care in Uganda. BMC Fam Pract. 2014;15(1):165. 23. Kramera AR, Dickinson KL, Anderson RM, Fowler VG, Miranda ML, Mutero CM, et al. Using decision analysis to improve malaria control policy making. Health Policy. 2009;92:133–40. y 24. Amin AA, Zurovac D, Kangwana BB, Greenfield J, Otieno DN, Akhwale WS, et al. The challenges of changing national malaria drug policy to artemisinin-based combinations in Kenya. Malar J. 2007;6:72. 25. Reyburn H, Mbakilwa H, Mwangi R, Mwerinde O, Olomi R, Drakeley C, et al. Rapid diagnostic tests compared with malaria microscopy for guiding outpatient treatment of febrile illness in Tanzania: randomised trial. BMJ. 2007;334(7590):403. 26. Greer AL, Goodwin JS, Freeman JL, Wu ZH. Bringing the patient back in. Int J Technol Assess Health Care. 2002;18:747–61. 27. Howteerakul N, Higginbotham N, Freeman S, Dibley MJ. ORS is never enough: physician rationales for altering standard treatment guidelines when managing childhood diarrhoea in Thailand. Soc Sci Med. 2003;57:1031–44. 28. Ansah EK, Reynolds J, Akanpigbiam S, Whitty CJM, Chandler CIR. “Even if the test result is negative, they should be able to tell us what is wrong with us”: a qualitative study of patient expectations of rapid diagnostic tests for malaria. Malar J. 2013;12:258. 29. WHO. Informal consultation on fever management in peripheral health care settings: a global review of evidence and practice. Geneva: World Health Organization; 2013. 29. WHO. Informal consultation on fever management in peripheral health care settings: a global review of evidence and practice. Geneva: World Health Organization; 2013. g 30. White NJ. Malaria – time to act. N Eng J Med. 2006;55:1956–7. 30. White NJ. Malaria – time to act. N Eng J Med. 2006;55:1956–7. 31. WHO. Guidelines for the Treatment of Malaria. Geneva: World Health Organization; 2006. 31. WHO. Guidelines for the Treatment of Malaria. Geneva: World Health Organization; 2006. 32. ACTWatch Evidence for Malaria Medicines Policy. Résultats de l’enquête ACTWatch en République Démocratique du Congo, 2013. http://www. actwatch.info/sites/default/files/content/publications/attachments/ACTwatch %20DRC%20brief%2015Jan15%20.pdf. Accessed 12 Mar 2015. 32. Conclusion ACTWatch Evidence for Malaria Medicines Policy. Résultats de l’enquête ACTWatch en République Démocratique du Congo, 2013. http://www. actwatch.info/sites/default/files/content/publications/attachments/ACTwatch %20DRC%20brief%2015Jan15%20.pdf. Accessed 12 Mar 2015. 33. White NJ. Delaying antimalarial drug resistance with combination chemotherapy. Parassitologia. 1999;41:301–8. 33. White NJ. Delaying antimalarial drug resistance with combination chemotherapy. Parassitologia. 1999;41:301–8. Submit your next manuscript to BioMed Central and take full advantage of: • Convenient online submission • Thorough peer review • No space constraints or color figure charges • Immediate publication on acceptance • Inclusion in PubMed, CAS, Scopus and Google Scholar • Research which is freely available for redistribution Submit your manuscript at www.biomedcentral.com/submit Submit your next manuscript to BioMed Central and take full advantage of: • Convenient online submission • Thorough peer review • No space constraints or color figure charges • Immediate publication on acceptance • Inclusion in PubMed, CAS, Scopus and Google Scholar • Research which is freely available for redistribution Submit your manuscript at www.biomedcentral.com/submit Submit your next manuscript to BioMed Central and take full advantage of: • Convenient online submission • Thorough peer review • No space constraints or color figure charges • Immediate publication on acceptance • Inclusion in PubMed, CAS, Scopus and Google Scholar • Research which is freely available for redistribution Submit your manuscript at www.biomedcentral.com/submit Submit your next manuscript to BioMed Central and take full advantage of: Submit your next manuscript to BioMed Central and take full advantage of: • Convenient online submission
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Key role for lipids in cognitive symptoms of schizophrenia
Translational psychiatry
2,020
cc-by
9,766
2020, Article / Letter to editor (Translational Psychiatry, 10, 1, (2020), pp. 1-12, article 399) Doi link to publisher: https://doi.org/10.1038/s41398-020-01084-x Version of the following full text: Publisher’s version Downloaded from: https://hdl.handle.net/2066/227649 Download date: 2024-10-24 Version of the following full text: Publisher’s version Downloaded from: https://hdl.handle.net/2066/227649 Download date: 2024-10-24 © The Author(s) 2020 OpenAccessThisarticleislicensedunderaCreativeCommonsAttribution4.0InternationalLicense,whichpermitsuse,sharing,adaptation,distributionandreproduction in any medium or format, as long as you give appropriate credit to the original author(s) and the source, provide a link to the Creative Commons license, and indicate if changes were made. The images or other third party material in this article are included in the article’s Creative Commons license, unless indicated otherwise in a credit line to the material. If material is not included in the article’s Creative Commons license and your intended use is not permitted by statutory regulation or exceeds the permitted use, you will need to obtain permission directly from the copyright holder. To view a copy of this license, visit http://creativecommons.org/licenses/by/4.0/. Abstract Schizophrenia (SZ) is a psychiatric disorder with a convoluted etiology that includes cognitive symptoms, which arise from among others a dysfunctional dorsolateral prefrontal cortex (dlPFC). In our search for the molecular underpinnings of the cognitive deficits in SZ, we here performed RNA sequencing of gray matter from the dlPFC of SZ patients and controls. We found that the differentially expressed RNAs were enriched for mRNAs involved in the Liver X Receptor/Retinoid X Receptor (LXR/RXR) lipid metabolism pathway. Components of the LXR/RXR pathway were upregulated in gray matter but not in white matter of SZ dlPFC. Intriguingly, an analysis for shared genetic etiology, using two SZ genome-wide association studies (GWASs) and GWAS data for 514 metabolites, revealed genetic overlap between SZ and acylcarnitines, VLDL lipids, and fatty acid metabolites, which are all linked to the LXR/RXR signaling pathway. Furthermore, analysis of structural T1-weighted magnetic resonance imaging in combination with cognitive behavioral data showed that the lipid content of dlPFC gray matter is lower in SZ patients than in controls and correlates with a tendency towards reduced accuracy in the dlPFC-dependent task-switching test. We conclude that aberrations in LXR/RXR-regulated lipid metabolism lead to a decreased lipid content in SZ dlPFC that correlates with reduced cognitive performance. A R T I C L E O p e n A c c e s s Key role for lipids in cognitive symptoms of schizophrenia Dorien A. Maas 1,2,3, Marijn B. Martens4, Nikos Priovoulos5, Wieteke A. Zuure1, Judith R. Homberg3, Brahim Nait-Oumesmar 2 and Gerard J. M. Martens1,4 Correspondence: Dorien A. Maas (d.a.maas@outlook.com) 1Faculty of Science, Centre for Neuroscience, Department of Molecular Animal Physiology, Donders Institute for Brain, Cognition and Behavior, Radboud University Nijmegen, Geert Grooteplein Zuid 26-28, 6525 GA Nijmegen, The Netherlands 2Sorbonne Université, Paris Brain Institute – ICM, Inserm U1127, CNRS UMR 7225, Hôpital Pitié-Salpêtrière, Paris, France Full list of author information is available at the end of the article Note: To cite this publication please use the final published version (if applicable). Maas et al. Translational Psychiatry (2020) 10:399 https://doi.org/10.1038/s41398-020-01084-x Translational Psychiatry Introduction negative and cognitive symptoms of SZ8. Cognitive symp- toms and related changes in the prefrontal cortex (PFC) of SZ patients are already present before disease onset9 and contribute negatively to functional outcome10–13. Cognitive deficits are found in individuals at high risk to develop SZ14 and family members of SZ patients15, albeit to a lower degree. The various subregions of the PFC are involved in deficits in specific cognitive domains16. For example, although ventro-lateral PFC functioning remains largely unaffected, impaired dorsolateral (dl)PFC-dependent processes are thought to underlie a range of cognitive deficits in SZ17–19. In addition, dlPFC activation during the performance of cognitive tasks is decreased in SZ patients18,20,21. Schizophrenia (SZ) is a psychiatric disorder with a con- voluted etiology and a lifetime prevalence of 0.84%. It is thought that an interplay between genetic, epigenetic, and environmental risk factors is involved in SZ etiology1. Symptoms of SZ include positive, negative, and cognitive symptoms2. The positive symptoms comprise delusions and hallucinations3, the negative symptoms are a loss of typical affective functions2, and the most prominent cog- nitive symptoms of SZ are deficits in attention4 and executive functioning5–7. There are currently no effective pharmacological treatment strategies that target the Correspondence: Dorien A. Maas (d.a.maas@outlook.com) 1Faculty of Science, Centre for Neuroscience, Department of Molecular Animal Physiology, Donders Institute for Brain, Cognition and Behavior, Radboud University Nijmegen, Geert Grooteplein Zuid 26-28, 6525 GA Nijmegen, The Netherlands Transcriptomic studies on the PFC of SZ patients have increased our understanding of the molecular mechan- isms contributing to the PFC-dependent cognitive impairment in SZ. The majority of transcriptomic studies performed on SZ dlPFC (RNA sequencing22–29 or © The Author(s) 2020 © The Author(s) 2020 OpenAccessThisarticleislicensedunderaCreativeCommonsAttribution4.0InternationalLicense,whichpermitsuse,sharing,adaptation,distributionandreproduction in any medium or format, as long as you give appropriate credit to the original author(s) and the source, provide a link to the Creative Commons license, and indicate if changes were made. The images or other third party material in this article are included in the article’s Creative Commons license, unless indicated otherwise in a credit line to the material. If material is not included in the article’s Creative Commons license and your intended use is not permitted by statutory regulation or exceeds the permitted use, you will need to obtain permission directly from the copyright holder. To view a copy of this license, visit http://creativecommons.org/licenses/by/4.0/. Maas et al. Translational Psychiatry (2020) 10:399 Page 2 of 12 microarray30–32 analyses) have been conducted on a mix of gray and white matter. However, gray and white matter display discrete gene expression patterns33, and therefore investigating the transcriptome of a gray and white-matter mix does not allow the detection of gene expression dif- ferences that arise from and are specific to either gray or white matter. One transcriptomic study has been per- formed on SZ PFC gray matter, but did not specify the PFC subregion that was used34. Yet, spatial differences in gene expression patterns exist throughout the cortex35 and PFC subregions have distinct contributions to the cognitive deficits in SZ16. Only two transcriptomic studies published to date have analyzed solely the gray matter of the SZ dlPFC subregion, with one study reporting dif- ferences in the axon guidance pathway36 and the other analyzing the expression of only the delta 4-desaturase, sphingolipid 2 (DEGS2) gene37. mapped reads. Gene expression levels (fragments per kilobase of transcript per million mapped reads) were calculated using RSEM and NOIseq algorithms were then used to determine genes differentially expressed in SZ patients and controls. Significantly differentially expressed genes (probability > 0.8) were used for analysis with the Ingenuity Pathway Analysis software package (Qiagen). RNA sequencing and RNA-sequencing data analysis were performed by researchers that were blinded for disease state. RNA-sequencing data are freely available through https://doi.org/10.6084/m9.figshare.12640460.v1 Samples and RNA sequencing Human post-mortem dlPFC brain tissue from four chronic SZ patients and four control individuals was obtained from the Dutch Brain Bank (Amsterdam, The Netherlands). Sample size was based on tissue availability. Sections of 300 µm were obtained in a cryostat (Leica) at −15 °C and two to three punches were collected from different places in the gray matter and in the white matter using a 2.00 mm punch needle (Harris). Punches were frozen on dry ice and stored at −80 °C until RNA isolation using RNeasy lipid tissue mini kit (74804 Qiagen). Isolated RNA was sent for quality control, RNA sequencing, and bioinformatics data analysis to BGI Genomics. Agilent 2100 Bio Analyzer was used to determine RNA quality and RNA integrity numbers of all RNA samples were 6.7 or higher. RNA sequencing was performed using BGISEQ-500 platform generating 6.71 Gb bases per sample. Using hierarchical indexing for spliced alignment of transcripts or HISAT, clean reads were mapped to the reference genome UCSC HG38 with an average of 92.06% Quantitative real-time PCR For quantitative real-time PCR (qPCR) analysis, per sample 350 μg RNA was treated with DNase I (Fermentas) and cDNA was synthesized using the Revert Aid H-minus first-strand cDNA synthesis kit (Thermo Scientific). cDNA was subsequently diluted 1 : 20 in MilliQ H2O and stored at −20 °C until qPCR analysis. qPCR samples contained 2.0 μL diluted cDNA, 0.8 μL 5 μM forward primer, 0.8 μL 5 μM reverse primer, 5 μL SybrGreen mix (Roche), and 1.8 μL MilliQ H2O. qPCR was performed with a Rotor Gene 6000 Series (Corbett Life Sciences) using a three-step paradigm with a fixed gain of 8. Fifty cycling steps of 95, 60, and 72 °C were applied and fluorescence was acquired after each cycling step. Primers were designed with NCBI Primer-Blast and synthesized by Sigma (for primer pair sequences, see Supplementary Table S1). Melting temperature was used to check whe- ther a single PCR product was generated and the take off and amplification values of the housekeeping genes (Ppia and Gapdh) were used to determine the normalization factor with GeNorm38 after which normalized mRNA expression levels were calculated. qPCR data were ana- lyzed using Levene’s test for equality of variances and two- tailed independent samples T-tests in SPSS Statistics 21. Individual data points and means were plotted using Graphpad Prism 4. Researchers were blinded for disease state during qPCR analysis. In the current study, we sequenced the transcriptome of the gray matter of dlPFC in SZ and controls. As we found that the differentially expressed genes were enriched in Liver X Receptor/Retinoid X Receptor (LXR/RXR)- mediated lipid metabolism genes, we next investigated whether SZ has a genetic link with lipid metabolism. We indeed identified shared genetic etiology between SZ and among other acylcarnitines, very-low-density lipoprotein (VLDL) lipids, and fatty acid metabolites. Finally, exploratory analyses of structural magnetic resonance imaging (MRI) data were in accordance with a lower lipid content of the dlPFC gray matter in SZ patients as com- pared to controls and correlated with reduced cognitive performance. Thus, distortions in lipid homeostasis play a key role in the cognitive symptoms of SZ. Materials and methods Samples and RNA sequencing Samples and RNA sequencing Shared genetic etiology Using PRSice, single-nucleotide polymorphisms (SNPs) were clumped to remove linkage disequilibrium (LD) with an LD threshold of 0.1, a distance threshold of 250 kb, and the 1000 Genomes Project data as genotype reference47. A range of SNP significance thresholds was used (pT < 0.01, 0.05, 0.1, 0.2, 0.3, 0.4, and 0.5) to calculate shared genetic etiology and the p-values obtained using these thresholds were corrected with Bonferroni multiple comparisons correction for the number of metabolites tested. dlPFC was extracted from the atlas and projected to each individual scan. The dlPFC regions of interest were cor- rected at the individual level with a gray-matter mask made with FSL-FAst and the output was visually verified. The average MP-RAGE signal in the dlPFC gray matter of SZ patients and controls was examined. Two linear models were fitted including the average left and right gray-matter dlPFC MP-RAGE signal as the dependent variable and age, sex and group as the independent vari- ables. A retrospective motion-estimate (Average Edge Strength) was also calculated with the homonymous Matlab toolbox51 and entered as an independent variable. The analyses were repeated for data acquired at both 3T scanners (Trio, Siemens Healthineers). We then utilized a linear model to analyze the correlation between dlPFC gray-matter MP-RAGE signal and accuracy in the task- switching test in SZ patients accounting for age and motion. For details on the task-switching test, see ref. 48. Individual data points and means were plotted using Graphpad Prism 4. Researchers were blinded for disease state during data analysis. RNA sequencing reveals LXR/RXR activation as the top- enriched canonical pathway in gray matter of SZ dlPFC RNA sequencing reveals LXR/RXR activation as the top- enriched canonical pathway in gray matter of SZ dlPFC The Consortium for Neuropsychiatric Phenomics made available an MRI dataset including 125 healthy individuals (median age = 28 years old, 53% female) and 50 indivi- duals (median age = 37.5 years old, 76% female) diag- nosed with SZ or schizoaffective disorder. This dataset includes a T1-weighted magnetization prepared–rapid gradient echo (MP-RAGE) sequence (repetition time = 1.9 s, echo time = 2.26 ms, field-of-view = 250 mm, matrix = 256 × 256, slice thickness = 1 mm, 176 slices), as well as cognitive behavioral data from the task-switching test. For details on the dataset, see ref. 48. For all MRI analyses, open source code was used. The MP-RAGEs were corrected for B0/B1 inhomogeneities using the N4 algorithm. A study-specific template of the MP-RAGE scans was created in the common space between the scans with an iterative diffeomorphic warp estimate using the ANTS package49. The template was diffeomorphically registered to the MarsAtlas50. A segmentation of the enriched canonical pathway in gray matter of SZ dlPFC RNA sequencing was performed on gray matter from dlPFC of four SZ patients and four controls (see Supple- mentary Table S2 for subject and tissue characteristics). Gene expression density was similar for all samples (Supplementary Fig. S1a) and differential expression analysis showed 132 significantly upregulated genes and 5 significantly down-regulated genes in SZ dlPFC gray matter (Supplementary Fig. S1b). Ingenuity pathway analysis of the significantly differentially expressed genes revealed that “LXR/RXR activation” was the most sig- nificantly enriched canonical pathway in the dlPFC of SZ patients (p = 3.89E-07 in Benjamini–Hochberg corrected T-test; see Table 1 for the top five canonical pathways with statistical values and molecules involved); the other canonical pathways were at least 30 times less enriched. The LXR/RXR pathway regulates cholesterol homeostasis in the brain. The increased abundance of transcripts that Table 1 Ingenuity pathway analysis of genes differentially expressed in SZ vs. control dlPFC gray matter. Canonical pathway p-value (Benjamini–Hochberg corrected) Genes LXR/RXR activation 3.89E −07 AGT, APOC2, C4A/C4B, IL1RL1, S100A8, SERPINA1, TNFRSF11B Complement system 1.15E −05 C1QA, C1QB, C1QC, C4A/C4B Antigen presentation pathway 1.43E −05 HLA-DMA, HLA-DQB1, HLA-DRB3, HLA-DRB5 PD1-PD-L cancer immunotherapy pathway 5.45E −05 HLA-DMA, HLA-DQB1, HLA-DRB3, HLA-DRB5, TNFRSF11B T-helper cell differentiation 1.71E −04 HLA-DMA, HLA-DQB1, HLA-DRB5, TNFRSF11B Table 1 Ingenuity pathway analysis of genes differentially expressed in SZ vs. control dlPFC gray matter. Shared genetic etiology Two SZ genome-wide association studies (GWASs) and four metabolite GWAS datasets were used to calculate shared genetic etiology between SZ and metabolite levels. We first calculated shared genetic etiology between 561 metabolites and SZ using previously published SZ GWAS data that was obtained from 33,426 SZ patients from European ancestry39. We then replicated the calculation using a second SZ GWAS dataset, namely the GWAS data from 36,989 SZ patients as provided by the Psychiatric Genomics Consortium40, which includes the same patients from European descent, but also includes indi- viduals with East-Asian ancestry. The metabolite GWAS data were obtained from Rhee et al.41 including 268 Maas et al. Translational Psychiatry (2020) 10:399 Page 3 of 12 Page 3 of 12 metabolite GWASs, Draisma et al.42 including 129 metabolite GWASs, Kettunen et al.43 including 123 metabolite GWASs, and Ahola-Olli et al.44 including 41 cytokine GWASs, and included 2076, 7478, and 24,925 participants of European decent, and 2019 Finnish parti- cipants, respectively. Shared genetic etiology was calcu- lated using the freely available program PRSice version 1.2345 with PLINK version 1.9 and based on the method of Johnson et al.46. Metabolite GWAS data were taken as base samples and SZ GWAS data as the target sample, and correlation results were weighted by the SZ group size. Using PRSice, single-nucleotide polymorphisms (SNPs) were clumped to remove linkage disequilibrium (LD) with an LD threshold of 0.1, a distance threshold of 250 kb, and the 1000 Genomes Project data as genotype reference47. A range of SNP significance thresholds was used (pT < 0.01, 0.05, 0.1, 0.2, 0.3, 0.4, and 0.5) to calculate shared genetic etiology and the p-values obtained using these thresholds were corrected with Bonferroni multiple comparisons correction for the number of metabolites tested. metabolite GWASs, Draisma et al.42 including 129 metabolite GWASs, Kettunen et al.43 including 123 metabolite GWASs, and Ahola-Olli et al.44 including 41 cytokine GWASs, and included 2076, 7478, and 24,925 participants of European decent, and 2019 Finnish parti- cipants, respectively. Shared genetic etiology was calcu- lated using the freely available program PRSice version 1.2345 with PLINK version 1.9 and based on the method of Johnson et al.46. Metabolite GWAS data were taken as base samples and SZ GWAS data as the target sample, and correlation results were weighted by the SZ group size. RNA sequencing reveals LXR/RXR activation as the top- enriched canonical pathway in gray matter of SZ dlPFC control dlPFC gray and white matter. a Normalized mRNA expression of angiotensinogen (Agt), apolipoprotein C2 (Apoc2), and complement 4b (C4b) in SZ vs. control dlPFC gray matter. These mRNAs are components of the canonical pathway “LXR/RXR activation” in the Ingenuity pathway analysis. b Normalized mRNA expression of the LXR/RXR signaling cascade components Liver X receptor β (Lxrβ), retinoid X receptor β (Rxrβ), ATP-binding cassette transporter A1 (Abca1), peripheral myelin protein 22 (Pmp22), apolipoprotein E (ApoE), and sterol regulatory element-binding protein 1 (SREBP1) in SZ vs. control dlPFC gray matter. c, d Normalized mRNA expression of the same genes as in a and b in the white matter of dlPFC. n = 4 samples per group, #p < 0.1, *p < 0.05 in independent samples T-test. are associated with activation of the LXR/RXR pathway indicates a change in cholesterol metabolism in SZ dlPFC gray matter. p = 0.885, df = 3.378). The LXRβ/RXRβ pathway acti- vates the transcription factor sterol regulatory element- binding proteins (e.g., SREBP1) and as such stimulates cholesterol and oxysterol efflux from the cell via ATP- binding cassette transporter A1 (Abca1), which is regulated by peripheral myelin protein 22 (Pmp22)52–54. Upon efflux from the cell, cholesterol is packed in the brain in high-density lipoprotein (HDL)-like particles containing apolipoproteins, predominantly apolipopro- tein E (ApoE)52. Abca1 and Pmp22, but not Srebp1 and Apoe, mRNA expression were upregulated in SZ vs. control dlPFC gray matter (Fig. 1b; independent samples T-test Srebp1 t = 1.047, p = 0.335, df = 6, Apoe t = 1.606, p = 0.206, df = 3.032, Abca1 t = 3.836, p = 0.023, df = 3.538, Pmp22 t = 2.219, p = 0.068, df = 6), indicat- ing increased cholesterol efflux in SZ dlPFC gray matter. Notably, in the dlPFC white matter, no changes in LXR/ RXR-related mRNA expression were found (Fig. 1c, d), highlighting the importance of studying mRNA g y Upregulation of the “LXR/RXR activation” canonical pathway components angiotensinogen (Agt), apolipopro- tein C2 (Apoc2), and complement 4b (C4b) in SZ vs. control dlPFC gray matter was confirmed by qPCR (Fig. 1a; independent samples T-test t = 2.407, p = 0.053, df = 6, t = 2.673, p = 0.056, df=3.986, t = 2.155, p = 0.083, df=3.059, respectively; see Supplementary Table S1 for primer sequences). We next investigated whether other mRNAs related to LXR/RXR activation were also differentially expressed in SZ dlPFC gray matter. RNA sequencing reveals LXR/RXR activation as the top- enriched canonical pathway in gray matter of SZ dlPFC Canonical pathway p-value (Benjamini–Hochberg corrected) Genes LXR/RXR activation 3.89E −07 AGT, APOC2, C4A/C4B, IL1RL1, S100A8, SERPINA1, TNFRSF11B Complement system 1.15E −05 C1QA, C1QB, C1QC, C4A/C4B Antigen presentation pathway 1.43E −05 HLA-DMA, HLA-DQB1, HLA-DRB3, HLA-DRB5 PD1-PD-L cancer immunotherapy pathway 5.45E −05 HLA-DMA, HLA-DQB1, HLA-DRB3, HLA-DRB5, TNFRSF11B T-helper cell differentiation 1.71E −04 HLA-DMA, HLA-DQB1, HLA-DRB5, TNFRSF11B Table 1 Ingenuity pathway analysis of genes differentially expressed in SZ vs. control dlPFC gra Maas et al. Translational Psychiatry (2020) 10:399 Page 4 of 12 Fig. 1 Expression of LXR/RXR-related mRNAs in SZ vs. control dlPFC gray and white matter. a Normalized mRNA expression of angiotensinogen (Agt), apolipoprotein C2 (Apoc2), and complement 4b (C4b) in SZ vs. control dlPFC gray matter. These mRNAs are components of the canonical pathway “LXR/RXR activation” in the Ingenuity pathway analysis. b Normalized mRNA expression of the LXR/RXR signaling cascade components Liver X receptor β (Lxrβ), retinoid X receptor β (Rxrβ), ATP-binding cassette transporter A1 (Abca1), peripheral myelin protein 22 (Pmp22), apolipoprotein E (ApoE), and sterol regulatory element-binding protein 1 (SREBP1) in SZ vs. control dlPFC gray matter. c, d Normalized mRNA expression of the same genes as in a and b in the white matter of dlPFC. n = 4 samples per group, #p < 0.1, *p < 0.05 in independent samples T-test. Fig. 1 Expression of LXR/RXR-related mRNAs in SZ vs. control dlPFC gray and white matter. a Normalized m gray and white matter. a Normalized mRNA expression of Fig. 1 Expression of LXR/RXR-related mRNAs in SZ vs. control dlPFC gray and white matter. a Normalized mRNA expression of angiotensinogen (Agt), apolipoprotein C2 (Apoc2), and complement 4b (C4b) in SZ vs. control dlPFC gray matter. These mRNAs are components of the canonical pathway “LXR/RXR activation” in the Ingenuity pathway analysis. b Normalized mRNA expression of the LXR/RXR signaling cascade components Liver X receptor β (Lxrβ), retinoid X receptor β (Rxrβ), ATP-binding cassette transporter A1 (Abca1), peripheral myelin protein 22 (Pmp22), apolipoprotein E (ApoE), and sterol regulatory element-binding protein 1 (SREBP1) in SZ vs. control dlPFC gray matter. c, d Normalized mRNA expression of the same genes as in a and b in the white matter of dlPFC. n = 4 samples per group, #p < 0.1, *p < 0.05 in independent samples T-test. /RXR-related mRNAs in SZ vs. control dlPFC gray and white matter. a Normalized mRNA expression of Fig. 1 Expression of LXR/RXR-related mRNAs in SZ vs. RNA sequencing reveals LXR/RXR activation as the top- enriched canonical pathway in gray matter of SZ dlPFC LXRβ is the isoform of LXR that is expressed most abundantly in the brain and forms heterodimers with RXRβ52. We found an upregulation of Rxrβ, but no changes in the mRNA expression of Lxrβ in the SZ dlPFC gray matter as com- pared to controls (Fig. 1b; independent samples T-test Rxrβ t = 2.202, p = 0.070, df = 6, Lxrβ t = 0.156, Maas et al. Translational Psychiatry (2020) 10:399 Page 5 of 12 Page 5 of 12 free-water pool and the macromolecule pool. Following the saturation, the macromolecular pool quickly relaxes and subsequently accelerates the relaxation of the free- water pool in a process termed magnetization transfer. We hypothesized that a difference in lipid content and thus macromolecular pool would contribute to a change in magnetization transfer. We tested this by comparing the dlPFC gray-matter MP-RAGE signal between SZ patients and controls. We found that the MP-RAGE sig- nal was significantly decreased in the dlPFC gray matter of SZ patients as compared to controls, both in the left and right hemispheres, and accounting for age, sex, motion, and scanning site (Fig. 2a and Supplementary Table S6; linear model left dlPFC estimate = −26.025, t = −4.433, p < 0.001, right dlPFC estimate = −25.249, t = −4.319, p < 0.001; Supplementary Fig. S2). These results are in accordance with a lower macromolecular content and thus a lower lipid content of the SZ dlPFC gray matter. Notably, we found a correlation between the accuracy on the dlPFC-dependent task-switching test and the MP- RAGE signal in both the left and right dlPFC accounting for age and motion (Fig. 2b; linear model left dlPFC estimate = 4.286, t = 1.946, p = 0.0579, right dlPFC esti- mate = 4.330, t = 1.969, p = 0.0551). These data are con- sistent with a lower lipid content of the SZ dlPFC gray matter that correlates with a reduced accuracy in the dlPFC-dependent task-switching test and as such is in line with an important role for a distorted lipid metabolism in the cognitive deficits of SZ. expression patterns in the gray and white matter separately. In addition to the LXR/RXR activation pathway, the canonical pathway analysis revealed significant enrich- ment of four immune-related pathways in SZ dlPFC gray matter (Table 1), in line with the dysregulation of mRNA expression of genes related to inflammation and the immune system in SZ PFC25,38. Discussion SZ is a psychiatric disorder with an unknown etiology and its cognitive deficits are associated with the dlPFC. Here we performed RNA sequencing of post-mortem dlPFC gray matter of SZ patients and controls to gain insight into the molecular mechanisms contributing to the cognitive dysfunction in SZ. We found an enrichment of differentially expressed genes in the LXR/RXR activa- tion pathway and validated upregulation of components of the LXR/RXR lipid metabolism pathway in SZ dlPFC gray, but not white, matter. We further revealed shared genetic etiology between SZ and a number of lipid-related metabolites, confirming a genetic link between SZ and lipid metabolism. Finally, the results obtained with the MP-RAGE signals from structural MRI data are in accordance with a decreased lipid content in the dlPFC gray matter of SZ patients and correlated with reduced performance in the task-switching cognitive test. Shared genetic etiology between SZ and lipid metabolism Shared genetic etiology between SZ and lipid metabolism S a ed ge et c et o ogy bet ee S a d p d etabo s To further investigate the role of lipid metabolism in SZ, we analyzed the shared genetic etiology between SZ and 514 circulating metabolites, including amino acids, nutrients, organic compounds, cytokines, growth factors, and lipids. Following Bonferroni correction (Table 2 and Supplementary Table S4), we found significant overlap between genetic risk for SZ and 35 metabolites (Table 2 and Supplementary Table S4; p < 0.05) using the results of the 2018 SZ GWAS study published by the Bipolar and Schizophrenia working group of the Psychiatric Genomics Consortium39. Using a second 2014 SZ GWAS dataset provided by the Psychiatric Genomics Consortium40, the genetic association between SZ risk and 25 of the 35 metabolites was replicated, and 21 additional metabolites that share genetic etiology with SZ were identified (Table 2 and Supplementary Table S5). Strikingly, the 56 metabolites that share significant genetic etiology with SZ are all related to lipids (except for IP10 and IL16) and fall within three themes: acyl- carnitines, VLDL lipids, and fatty acid metabolites. We conclude that disruptions in lipid homeostasis are genetically associated with SZ. The finding that two immune-related metabolites IP10 and IL16 (Table 2) share genetic etiology with SZ is in line with the invol- vement of the immune system in the disorder. RNA sequencing reveals LXR/RXR activation as the top- enriched canonical pathway in gray matter of SZ dlPFC Furthermore, the top upstream regulator in the Ingenuity pathway analysis was interferon-γ (Supplementary Table S3; p = 2.22E −16) and this and other proinflammatory cytokines are asso- ciated with SZ55–57. Lipid content of dlPFC gray matter is lower in SZ than in controls and correlates with reduced accuracy in the task- switching test We further investigated the effect of the disrupted lipid homeostasis in SZ dlPFC gray matter using a publicly available dataset from the Consortium for Neu- ropsychiatric Phenomics. This dataset contains amongst others structural MRI scans and performance in the task- switching cognitive test of 50 SZ patients and 125 control individuals48. From this dataset, we analyzed the T1- weighted MP-RAGE signal. The macromolecular pool in the brain consists mainly of lipids, as illustrated by the typical gray–white matter contrast obtained in T1- weighted MRI scans. The T1 inversion pulse saturates the Gray and white matter have a different cellular com- position and function, and distinct transcriptomes33. Gray matter of the cortex consists mainly of neurons and glial cells, while white matter consists primarily of myelinated axons. Previous RNA-sequencing studies on mixes of gray and white matter from the dlPFC of SZ patients and Maas et al. Translational Psychiatry (2020) 10:399 Page 6 of 12 Table 2 Metabolites that share significant genetic etiology with SZ. Metabolite Lowest significant p-value treshold Bonferroni- corrected p-value Lowest significant p-value treshold Bonferroni- corrected p-value SZ GWAS 201840 SZ GWAS 201448 C5.1.DC1 0.1 0.000246 0.05 0.015176 IP105 0.3 0.000997 0.05 0.048767 CH2.DB.ratio2 0.1 0.00153 0.1 0.014121 LPE16_0_LIPID4 0.001 0.001988 0.001 0.000768 XS.VLDL.TG3 0.2 0.002406 0.3 0.039835 C14.1.OH1 0.05 0.002534 0.05 0.008502 DB.in.FA2 0.1 0.003962 0.1 0.010729 XS.VLDL.P3 0.3 0.004685 0.2 0.008044 IDL.C3 0.05 0.006454 NA NA PC38_2_LIPID4 0.3 0.007062 0.2 0.000573 CH2.in.FA2 0.2 0.008944 0.1 0.006678 Bis.DB.ratio2 0.2 0.009453 0.05 0.018628 DHA2 0.4 0.010114 0.05 0.00935 SM.C26.04 0.1 0.010245 0.05 0.002634 XS.VLDL.L3 0.05 0.010881 0.2 7.49E-05 TAG54_6_LIPID4 0.001 0.016009 0.001 0.009793 SM.OH.C24.14 0.1 0.017865 0.05 0.006078 FAw34 0.05 0.01807 0.05 0.002175 fumarate_maleate_valerat_CMH 0.05 0.019523 0.05 0.001032 Ratio_PC3806_LPC2206_LIPID4 0.05 0.023256 NA NA IDL.FC3 0.4 0.024663 NA NA LPC20_3_LIPID4 0.3 0.026304 NA NA XS.VLDL.PL3 0.05 0.027834 0.2 0.00178 XL.VLDL.TG3 0.3 0.032077 0.3 0.001178 PC32_0_LIPID4 0.1 0.032928 0.1 0.000627 PC.ae.C44.34 0.1 0.034516 NA NA IDL.L3 0.1 0.034726 NA NA S.VLDL.C3 0.4 0.035649 0.4 0.047852 IDL.P3 0.1 0.036891 NA NA lysoPC.a.C20.44 0.3 0.039526 0.2 0.019734 Bis.FA.ratio2 0.1 0.040777 0.05 0.005426 S.VLDL.L3 0.5 0.0408 NA NA GROa 0.001 0.042828 NA NA MCP1 0.001 0.045366 0.001 0.001432 LPC22_6_LIPID4 0.1 0.048797 NA NA Cit NA NA 0 05 0 00031 Table 2 Metabolites that share significant genetic etiology with SZ. Page 7 of 12 Maas et al. Lipid content of dlPFC gray matter is lower in SZ than in controls and correlates with reduced accuracy in the task- switching test Translational Psychiatry (2020) 10:399 Table 2 continued Metabolite Lowest significant p-value treshold Bonferroni- corrected p-value Lowest significant p-value treshold Bonferroni- corrected p-value SZ GWAS 201840 SZ GWAS 201448 PC38_6_LIPID4 NA NA 0.1 0.002651 CE20_5_LIPID3 NA NA 0.2 0.002948 TAG56_6_LIPID4 NA NA 0.2 0.005974 PC40_6_LIPID4 NA NA 0.3 0.008719 PC.aa.C24.04 NA NA 0.1 0.010428 TAG56_8_LIPID4 NA NA 0.2 0.012203 IL165 NA NA 0.05 0.01252 TAG58_10_LIPID4 NA NA 0.3 0.014247 TAG56_6_LIPID4 NA NA 0.3 0.020381 XXL.VLDL.PL3 NA NA 0.3 0.027232 L.VLDL.P3 NA NA 0.05 0.030895 XL.HDL.L3 NA NA 0.5 0.032042 aconitate_CMH NA NA 0.1 0.032102 XL.VLDL.L3 NA NA 0.4 0.03703 TAG58_11_LIPID4 NA NA 0.3 0.037887 PC38_4_LIPID4 NA NA 0.4 0.038142 LDL.D3 NA NA 0.001 0.042824 FAw64 NA NA 0.05 0.044601 ADP_CMH NA NA 0.4 0.049512 1A l i i hallucinations, but not between controls and SZ patients without auditory hallucinations36. This highlights that mRNA expression in dlPFC gray matter might differ among subgroups of SZ patients. As in the present study we did not compare subgroups of SZ patients, we may have missed more subtle mRNA expression differences between patients and controls. In addition, for unknown reasons we did not find the previously reported decreased mRNA expression of the SZ-associated DEGS2 gene in SZ dlPFC gray matter37 nor the decreased expression of sodium channel subunit SCN2A, the latter probably due to the fact that we studied a different PFC subregion34. Thus, future transcriptomic studies should include SZ patient subgroups and various PFC subregions. controls have shown among others altered abundance of transcripts involved in glucocorticoid signaling29, pre- synaptic function32, inflammation25, nuclear receptor signaling23, synaptic vesicle recycling, transmitter release, and cytoskeletal dynamics31. Our RNA-sequencing study on only the dlPFC gray matter confirms the dysregulation of inflammation-related genes in SZ. More importantly, among the differentially expressed dlPFC gray-matter genes the most enriched pathway involved LXR/RXR- mediated cholesterol lipid homeostasis. LXR/RXR-related genes were upregulated in the dlPFC gray matter of SZ patients, but were unaltered in the dlPFC white matter. The previous transcriptomic studies on a mix of SZ dlPFC gray and white matter have likely missed the enrichment of this pathway because of the relatively high contribution of the lipid content of the white matter. The LXR/RXR pathway is activated by binding of oxy- sterols to LXR. Oxysterols are metabolites produced during the breakdown of cholesterol and able to cross the blood brain barrier. Lipid content of dlPFC gray matter is lower in SZ than in controls and correlates with reduced accuracy in the task- switching test In the brain, LXRβ forms hetero- dimers with RXRβ and their activation leads to increased Interestingly, dlPFC gray-matter mRNA expression differences in the axon guidance pathway are known to exist between controls and SZ patients with auditory Maas et al. Translational Psychiatry (2020) 10:399 Page 8 of 12 Fig. 2 MP-RAGE signal in SZ and control dlPFC gray matter and correlation with task-switching accuracy. a Left: schematic representation of the dlPFC gray matter in the left and right brain hemispheres. Right: average MP-RAGE signal from the left and right dlPFC gray matter in SZ versus control (CON). **p < 0.001 in a linear model corrected for sex, age, motion, and scanning site. b Scatter plot of the accuracy in the task-switching test and the MP-RAGE signal from the dlPFC gray matter in the left and right hemisphere of SZ patients. Fig. 2 MP-RAGE signal in SZ and control dlPFC gray matter and correlation with task-switching accuracy. a Left: schematic representation of the dlPFC gray matter in the left and right brain hemispheres. Right: average MP-RAGE signal from the left and right dlPFC gray matter in SZ versus control (CON). **p < 0.001 in a linear model corrected for sex, age, motion, and scanning site. b Scatter plot of the accuracy in the task-switching test and the MP-RAGE signal from the dlPFC gray matter in the left and right hemisphere of SZ patients. HDL, and apolipoprotein levels64,65, and lower short- chain acylcarnitine levels in the blood66. Moreover, in SZ patients using antipsychotic medication the occurrence of metabolic syndrome is increased and cholesterol levels are correlated with cognitive impairment63,67, implicating a role for peripheral lipid metabolism in brain functioning and cognitive deficits in SZ. In the present study, we find that SZ shares genetic etiology with a number of meta- bolites, most of which were replicated using a second SZ GWAS study. Among the metabolites that share genetic etiology with SZ, we found an enrichment of acylcarni- tines, VLDL lipids and fatty acid metabolites. A previous polygenic risk score analysis has revealed that the severity of cognitive deficits is linked to genetic variations in genes involved in retinoid signaling68, a pathway that, similar to the LXR/RXR pathway, is linked to lipid metabolism. Lipid content of dlPFC gray matter is lower in SZ than in controls and correlates with reduced accuracy in the task- switching test Therefore, our results together with this earlier finding highlight a genetic contribution to the observed altera- tions in lipid homeostasis in SZ that are thus likely not solely caused by antipsychotic treatment. efflux of cholesterol via ABCA1- and PMP22-regulated mechanisms into HDL-like particles containing apolipo- protein, inhibition of cholesterol uptake by the cell and stimulation of fatty acid synthesis52–54. We indeed found moderate upregulation of Rxrβ, Apoc2, Abca1, and Pmp22 in SZ dlPFC gray matter as compared to controls. Inter- estingly, LXR signaling is involved in the development of ventral midbrain dopaminergic neurons52,58 and there is a genetic association between PMP22 and SZ59. In vitro studies have shown contradictory effects of antipsychotics on LXR signaling in that one study has reported an increased mRNA expression of Abca1 and Apoe60, whereas a second study has shown that antipsychotics reduce cholesterol synthesis and export from the endo- plasmic reticulum, and do not induce LXR activation61. Nevertheless, a disturbance of LXR-mediated cholesterol homeostasis appears to play a role in SZ etiology, but further studies are necessary. A number of links exist between a distorted lipid homeostasis and SZ. For example, a meta-analysis has revealed that metabolic syndrome in SZ patients, a con- dition in which cholesterol and triglyceride levels are abnormal, is associated with a high degree of cognitive impairment62. Metabolic syndrome also impairs cognition in otherwise healthy individuals63. Blood triglyceride levels are correlated with positive symptom severity and blood HDL levels with global functioning of SZ patients64. Unmedicated SZ patients have lower total cholesterol, Notably, acylcarnitines, fatty acid production, choles- terol efflux into HDL-like particles, and LXR/RXR acti- vation share a common molecular pathway (Fig. 3). During fatty acid oxidation, unsaturated fatty acids esterify with acyl-CoA to form acylcarnitine that is sub- sequently transported into the mitochondrial inner membrane. Once inside the inner mitochondrial mem- brane, acylcarnitines are subjected to β-oxidation, which Page 9 of 12 Maas et al. Translational Psychiatry (2020) 10:399 Fig. 3 Molecular pathways involving LXR/RXR signaling, acylcarnitines, VLDL lipids, and fatty acid metabolites. Dark green rectangles represent genes upregulated in SZ dlPFC. Light green ovals represent lipid-related metabolites that share significant genetic etiology with SZ. Arrows indicate the nature of molecular interactions (see legend in the figure for details). References54,68,84–94 have been used to construct the molecular pathways. Fig. 3 Molecular pathways involving LXR/RXR signaling, acylcarnitines, VLDL lipids, and fatty acid metabolites. Lipid content of dlPFC gray matter is lower in SZ than in controls and correlates with reduced accuracy in the task- switching test Dark green rectangles represent genes upregulated in SZ dlPFC. Light green ovals represent lipid-related metabolites that share significant genetic etiology with SZ. Arrows indicate the nature of molecular interactions (see legend in the figure for details). References54,68,84–94 have been used to construct the molecular pathways. involving fatty acid oxidation and cholesterol efflux, pro- duction, and transport may well play a role in SZ. involving fatty acid oxidation and cholesterol efflux, pro- duction, and transport may well play a role in SZ. Using a publicly available dataset48, we show that the T1-weighted MP-RAGE signal is significantly decreased in SZ dlPFC gray matter. The T1-weighted MP-RAGE signal creates contrast between gray and white matter, which is thought to be due to magnetization transfer effects, where produces acetyl-CoA that can either enter the citric acid cycle, or is transported to the cytosol where it participates in lipid biosynthesis (fatty acid and cholesterol synthesis). Cholesterol can be transported out of the cell via HDL- like particles. Based on our transcriptomic study and the shared genetic etiology between SZ and several lipid- related metabolites, we conclude that lipid homeostasis Using a publicly available dataset48, we show that the T1-weighted MP-RAGE signal is significantly decreased in SZ dlPFC gray matter. The T1-weighted MP-RAGE signal creates contrast between gray and white matter, which is thought to be due to magnetization transfer effects, where Using a publicly available dataset48, we show that the T1-weighted MP-RAGE signal is significantly decreased in SZ dlPFC gray matter. The T1-weighted MP-RAGE signal creates contrast between gray and white matter, which is thought to be due to magnetization transfer effects, where Maas et al. Translational Psychiatry (2020) 10:399 Page 10 of 12 Page 10 of 12 increased lipid content results in increased signal. Our results are therefore consistent with a decreased lipid content in the gray matter of the dlPFC of SZ patients. This conclusion does not take into account the added effects from differences in spin density or in the relaxation rate of the free-water pool itself to the MP-RAGE signal. However, the relaxation rate of the free-water pool itself is largely homogeneous across the brain69,70. Our finding of a decreased lipid content in SZ dlPFC gray matter thus warrants future validation with quantitative magnetiza- tion transfer methods. Notably, the decreased MP-RAGE signal in SZ dlPFC gray matter correlates with decreased accuracy in the task-switching test in SZ patients. Publisher’s note Springer Nature remains neutral with regard to jurisdictional claims in published maps and institutional affiliations. Springer Nature remains neutral with regard to jurisdictional claims in published maps and institutional affiliations. References 1. Modai, S. & Shomron, N. Molecular risk factors for schizophrenia. Trends Mol. Med. 22, 242–253 (2016). 1. Modai, S. & Shomron, N. Molecular risk factors for schizophrenia. Trends Mol. Med. 22, 242–253 (2016). 2. Tandon, R., Nasrallah, H. A. & Keshavan, M. S. Schizophrenia, “just the facts” 4. Clinical features and conceptualization. Schizophrenia Res. 110, 1–23 (2009). 2. Tandon, R., Nasrallah, H. A. & Keshavan, M. S. Schizophrenia, “just the facts” 4. Clinical features and conceptualization. Schizophrenia Res. 110, 1–23 (2009). 3. Arndt, S., Andreasen, N. C., Flaum, M., Miller, D. & Nopoulos, P. A longitudinal study of symptom dimensions in schizophrenia. Prediction and patterns of change. Arch. Gen. 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Lipid content of dlPFC gray matter is lower in SZ than in controls and correlates with reduced accuracy in the task- switching test The task-switching test examines executive functioning and relies on the dlPFC71–73. In SZ patients, reduced accu- racy74 and reaction time differences75 in this test have been reported. We find that in SZ altered performance in the task-switching test might arise from a decreased lipid content in the dlPFC. Kapittelweg 29, 6525 EN Nijmegen, The Netherlands. 4NeuroDrug Research Ltd, Toernooiveld 1, 6525 ED Nijmegen, The Netherlands. 5Spinoza Centre for Neuroimaging, Meibergdreef 75, Amsterdam-Zuidoost, 1105 BK Amsterdam, The Netherlands Kapittelweg 29, 6525 EN Nijmegen, The Netherlands. 4NeuroDrug Research Ltd, Toernooiveld 1, 6525 ED Nijmegen, The Netherlands. 5Spinoza Centre for Neuroimaging, Meibergdreef 75, Amsterdam-Zuidoost, 1105 BK Amsterdam, The Netherlands Author contributions D.A.M. and G.J.M.M. designed the project and wrote the manuscript. D.A.M. performed data acquisition and analysis, and drafted the manuscript. W.A.Z. performed data acquisition of RNA sequencing and qPCR experiments. M.B.M. performed data analysis of shared genetic etiology, N.P. performed data analysis of structural MRI data. J.R.H. and B.N.O. contributed to supervision of the project. G.J.M.M. supervised the project. Supplementary Information accompanies this paper at (https://doi.org/ 10.1038/s41398-020-01084-x). Supplementary Information accompanies this paper at (https://doi.org/ 10.1038/s41398-020-01084-x). Received: 17 February 2020 Revised: 2 October 2020 Accepted: 26 October 2020 Received: 17 February 2020 Revised: 2 October 2020 Accepted: 26 October 2020 About half of the dry weight of the brain is attributable to lipids and about 80% of brain lipids are part of myelin sheaths. In SZ PFC, abnormalities in myelination are evident and decreased PFC myelin content contributes to disease symptomatology76,77. 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Associations between cognitive function, schizo- phrenic symptoms, and functional outcome in early-onset schizophrenia with and without a familial burden of psychosis. Isr. J. Psychiatry Relat. Sci. 52, 6–12 (2015). Acknowledgements g This study was supported by grants from the program “Top talent” Donders Centre for Medical Neuroscience (to D.A.M.) and a Van Gogh travel grant (to D. A.M., B.N.O., and G.J.M.M.). 12. Bhagyavathi, H. D. et al. Cascading and combined effects of cognitive deficits and residual symptoms on functional outcome in schizophrenia - a path- analytical approach. Psychiatry Res. 229, 264–271 (2015). Conflict of interest The authors declare that they have no conflict of interest. 1Faculty of Science, Centre for Neuroscience, Department of Molecular Animal Physiology, Donders Institute for Brain, Cognition and Behavior, Radboud University Nijmegen, Geert Grooteplein Zuid 26-28, 6525 GA Nijmegen, The Netherlands. 2Sorbonne Université, Paris Brain Institute – ICM, Inserm U1127, CNRS UMR 7225, Hôpital Pitié-Salpêtrière, Paris, France. 3Department of Cognitive Neuroscience, Donders Institute for Brain, Cognition and Behavior, Donders Centre for Medical Neuroscience, Radboud University Medical Center, Author details 1 l f 1Faculty of Science, Centre for Neuroscience, Department of Molecular Animal Physiology, Donders Institute for Brain, Cognition and Behavior, Radboud University Nijmegen, Geert Grooteplein Zuid 26-28, 6525 GA Nijmegen, The Netherlands. 2Sorbonne Université, Paris Brain Institute – ICM, Inserm U1127, CNRS UMR 7225, Hôpital Pitié-Salpêtrière, Paris, France. 3Department of Cognitive Neuroscience, Donders Institute for Brain, Cognition and Behavior, Donders Centre for Medical Neuroscience, Radboud University Medical Center, 1Faculty of Science, Centre for Neuroscience, Department of Molecular Animal Physiology, Donders Institute for Brain, Cognition and Behavior, Radboud University Nijmegen, Geert Grooteplein Zuid 26-28, 6525 GA Nijmegen, The Netherlands. 2Sorbonne Université, Paris Brain Institute – ICM, Inserm U1127, CNRS UMR 7225, Hôpital Pitié-Salpêtrière, Paris, France. 3Department of Cognitive Neuroscience, Donders Institute for Brain, Cognition and Behavior, Donders Centre for Medical Neuroscience, Radboud University Medical Center, 13. 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Susceptibilidad de afección por enjambres de medusas (Pelagia noctiluca) en las playas del litoral occidental de la provincia de Málaga
Investigaciones geográficas/Investigaciones geográficas
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Investigaciones Geográficas Instituto Interuniversitario de Geografía Universidad de Alicante Nº 77, enero-junio de 2022, pp. 239-258. ISSN: 0213 - 4691. eISSN: 1989 - 9890. DOI: 10.14198/INGEO.18723 Cita bibliográfica: de la Fuente Roselló, A., Sortino Barrionuevo, J.F., Reyes Corredera, S. J., & Perles Roselló, M.J. (2022). Susceptibilidad de afección por enjambres de medusas (Pelagia noctiluca) en las playas del litoral occidental de la provincia de Málaga. Investigaciones Geográficas, (77), 239-258. https://doi.org/10.14198/INGEO.18723 Susceptibilidad de afección por enjambres de medusas (Pelagia noctiluca) en las playas del litoral occidental de la provincia de Málaga Susceptibility of affection by jellyfish swarms (Pelagia noctiluca) on the beaches of the western coast of the province of Malaga Ana de la Fuente Roselló1* Juan Francisco Sortino Barrionuevo2 Sergio Jesús Reyes Corredera3 María Jesús Perles Roselló4 Resumen En el artículo se analiza y cartografía la susceptibilidad de afección por enjambres de medusas (Pelagia noctiluca) en las playas del litoral occidental de la provincia de Málaga. Los indicadores de peligrosidad de aparición de enjambres se elaboran a partir de un inventario de datos empíricos de avistamientos de medusas, registrados a través de la aplicación InfoMedusa (datos diarios para los meses estivales del periodo 2015-2020). Se proponen distintos indicadores de peligrosidad (frecuencia y severidad), y se calculan parámetros específicos que corrijan el problema del número desigual de observaciones en las playas. Los resultados cartográficos muestran que el patrón de distribución espacial no es aleatorio. Se observa una mayor frecuencia de episodios en las playas más occidentales del tramo estudiado, y el análisis preliminar de los factores condicionantes en cada playa permiten deducir una asociación de las playas con más propensión a recibir enjambres con la amplitud del ángulo de apertura de la misma, así como con la orientación al suroeste del tramo de costa en el que se sitúan las playas. Estas condiciones facilitan la entrada de las corrientes de levante procedentes del mar de Alborán, origen predominante de los enjambres. Palabras clave: Costa del Sol; Infomedusa; cartografía de susceptibilidad; playas. Abstract The article analyses and maps the hazard of jellyfish swarms (Pelagia Noctiluca) on the western coast of Malaga province. Hazard indicators are based on empirical data from jellyfish sightings recorded with ‘InfoMedusa’ (daily observations for the summer months of the 2015-2020 period). Various hazard indicators are proposed (frequency and severity) and specific parameters are calculated to correct the problem of unequal numbers of beach observations and its repercussions on the representativeness of the results. The cartographic results show that the spatial distribution pattern is not random. A higher frequency of episodes is observed on the westernmost beaches of the area under study and a preliminary analysis of the conditioning factors in each beach allows us to associate the beaches with the greatest propensity to receive swarms with the width of the opening angle of these beaches – as well as the southwest orientation of the stretch of coast where the beaches are located. These conditions facilitate the entry of eastern currents from the Alboran sea (the main origin of the swarms). Keywords: Costa del Sol; Infomedusa; susceptibility mapping; beaches. 1 Departamento de Geografía, Universidad de Málaga, España. anadelafuente@uma.es.* Autora para correspondencia 2 Departamento de Geografía, Universidad de Málaga, España. francis.sortino@uma.es 3 Departamento de Geografía, Universidad de Málaga, España. sergioreyes@uma.es 4 Departamento de Geografía, Universidad de Málaga, España. mjperles@uma.es Recepción: 14/01/2021. Aceptación: 29/07/2021. Publicación: 26/01/2022 (en prensa: 25/11/2021). Este trabajo se publica bajo una licencia de Creative Commons Reconocimiento 4.0 Internacional. © Ana de la Fuente Roselló, Juan Francisco Sortino Barrionuevo, Sergio Jesús Reyes Corredera, María Jesús Perles Roselló de la Fuente Roselló, A., Sortino Barrionuevo, J.F., Reyes Corredera, S. J. y Perles Roselló, M.J. 1. Introducción Las costas y playas son elementos de indiscutible importancia para el desarrollo económico del territorio en el que se localizan, constituyendo un pilar básico en actividades como el turismo de sol y playa, cuyo atractivo resulta muy sensible a la degradación de estos espacios y a cualquier cambio que genere incertidumbre en su uso (Enríquez y Bujosa, 2020). En las últimas décadas, probablemente motivado por la influencia del ser humano en el funcionamiento del clima y sus consecuencias sobre de los mares y océanos, se han incrementado de una forma alarmante la presencia de floraciones de medusas que acaban por llegar hasta la costa (Dong, 2019; Mills, 2001; Purcell, 2011). En el entorno mediterráneo, estos problemas están especialmente motivados por la medusa clavel (Pelagia noctiluca), que supone la especie potencialmente más dañina de esta área (Bellido el al., 2020). Se trata de una de las especies de medusas más importantes en el Mediterráneo debido a su amplia distribución, abundancia, función ecológica y también especialmente motivado por su interacción negativa con la población (Canepa, Fuentes, Sabatés, Piraino y Boero, 2014), siendo además una de las especies más usuales en los enjambres de medusas en la provincia de Málaga. El incremento de la frecuencia de aparición de floraciones es analizado por distintos autores. Brotz, Cheung, Keisner, Pakhomov y Pauly (2012) constatan cambios en las poblaciones de medusas y analizan la tendencia al aumento de la presencia de éstas en las costas a partir del año 1950. Asimismo, Purcell, Uye y Lo (2007) pronostica un progresivo incremento de poblaciones de medusas en áreas costeras asociado a la presión antropogénica sobre el mar. Kogovsek, Bogunovic y Malej (2010) analizan las ocurrencias históricas en el mar Adriático en los últimos 200 años combinadas con variables ambientales (temperatura, salinidad, pH, clorofila, peso seco de zooplancton y descargas principales en los ríos), concluyendo que la periodicidad de aparición en forma de plaga se ha acortado en las últimas décadas, aumentando así la recurrencia de las floraciones de esta especie. Los archivos de la Station Zoologique, en el Observatorio Oceanográfico Villefranche-sur-Mer, contienen registros de años con presencia de Pelagia noctiluca y años sin visitas de ésta, indicando que en el periodo de 200 años que va desde 1785 a 1985, se produjeron apariciones masivas de esta especie, con una periodicidad aproximada de doce años. En el caso concreto de la Costa del Sol, el Aula del Mar observa un incremento de frecuencia y magnitud de apariciones de medusas en el litoral malagueño, que parece indicar un cambio de tendencia. En la última década, ha registrado proliferaciones en periodos cíclicos de 2-4 años, cuando con anterioridad los ciclos esperados se estimaban en 10-20 años (Aula del Mar, 2021). La llegada de enjambres de medusas a las playas de un determinado litoral en cantidad y frecuencia extraordinaria constituye una anomalía con consecuencias negativas sobre la sociedad, que permite clasificarlo, según la Oficina de las Naciones Unidas para la Reducción del Riesgo de Desastres (UNISDR), como un riesgo ambiental de tipo biológico. Estos enjambres acarrean una gran problemática para las costas que lo sufren puesto que, aparte de la pérdida de biodiversidad que conllevan, traen consigo importantes efectos negativos en sectores como la pesca (Sabatés et al., 2010) pero sobre todo en el sector turístico (Ghermandi, Galil, Gowdy y Nunes, 2015; Nunes et al., 2015; Rubio y Gutiérrez, 2020). El enfoque territorial de análisis del riesgo (Cutter, 1994; Calvo, 2001; Olcina, 2008; Perles y Mérida, 2010; Veyret, Beucher y Bonnard, 2005) destaca la importancia de la posición de los elementos causantes y receptores del daño a la hora de evaluar el riesgo. Perles y Cantarero (2010), de forma más concreta, insisten en la importancia de conocer el patrón espacial con el que se comporta el evento peligroso. Proponen articular el patrón espacial de peligrosidad en áreas causantes de la peligrosidad, líneas o superficies transmisoras de los flujos, y áreas receptoras de los impactos del proceso. Para que un peligro incida sobre un área receptora tiene que existir algún vector de transferencia de materia y energía que los una, y además, el área causante ha de estar situada en una posición topológica que permita esta transferencia. Este enfoque es idóneo para analizar la peligrosidad de llegada de los enjambres de medusas a los distintos puntos del litoral. Existen investigaciones que se centran en el análisis y predicción del comportamiento espacial y/o temporal de los enjambres, y en la búsqueda de factores condicionantes del proceso. En este sentido, Bellido et al. (2020) indagan en la relación entre los índices atmosféricos y la incidencia de enjambres costeros y encuentran una conexión entre años de incidencia alta de proliferación de enjambres de medusas y lluvias copiosas durante invierno y primavera. (Bellido et al., 2020; Gutiérrez-Estrada et al., 2021). Kogovsek et al. (2010) analizan las ocurrencias históricas en el mar Adriático en los últimos 200 años, combinadas con variables ambientales (temperatura, salinidad, pH, clorofila, peso seco de zooplancton y descargas principales en los ríos), concluyendo que la periodicidad de aparición en forma de plaga se ha acortado en las últimas décadas, aumentando así la recurrencia de las floraciones de esta especie. Asimismo, Goy, Morand y Etienne (1988) manifiestan que, usando un modelo de pronóstico (variables climáticas, temperatura, lluvia y presión atmosférica), se pueden predecir periodos con Pelagia noctiluca. 240 Investigaciones Geográficas, nº 77, pp. 239-258. Susceptibilidad de afección por enjambres de medusas (Pelagia noctiluca) en las playas del litoral occidental de la provincia de Málaga Los estudios orientados a conocer y cartografiar el comportamiento espacial de la especie con un sentido más aplicado, puede citarse el Proyecto Medusa, que se crea en 2007, por la Agencia Catalana del Agua en colaboración con el Instituto de Ciencias del Mar de Barcelona (ICM-CSIC), con el objeto de controlar la presencia de medusas y entender su funcionamiento en el espacio y en el tiempo a lo largo de la costa catalana. Este proyecto ha realizado sondeos diarios en época estival en diferentes playas, donde se mapean las mayores concentraciones de medusas varadas. Asimismo, se incorporan en las conclusiones de su estudio la importancia de factores como la producción de medusas en alta mar, así como las estructuras artificiales que propician las condiciones idóneas para su crecimiento y reproducción. También se incluyen los factores que propician su debilitamiento, como son los vientos del sureste (Canepa et al., 2014). Con respecto a los antecedentes más próximos al área de estudio, cabe señalar el análisis realizado en este ámbito (Rubio y Gutiérrez, 2020) que, para analizar la distribución del fenómeno, utiliza como fuente la recurrencia de búsquedas de información sobre medusas en internet, la publicación de noticias relacionadas de los principales periódicos de Andalucía y encuestas a usuarios de playas en la Costa del Sol. Con este material concluye un patrón de distribución en el que destacan los avistamientos estivales, espacialmente concentrados entre la Costa del Sol Oriental (Málaga) y la Costa Tropical (Granada). La escala de trabajo del citado informe, a diferencia del desarrollado en la presente investigación, no se acerca de forma expresa al litoral, ni desciende al nivel de detalle de la playa, sino se desarrolla a escala regional. Gutiérrez-Estrada et al. (2021) enfocan su estudio al desarrollo de un sistema predictivo de la llegada de medusas a tierra en la Costa del Sol. A una escala más detallada, el trabajo de Olmedo (2019) constituye un referente del comportamiento y peligrosidad de los enjambres de medusas en las playas del sector oriental de la provincia de Málaga. A nivel de la Costa del Sol (Málaga), un antecedente directo de este trabajo es el informe presentado por el Aula del Mar basado en la aplicación móvil InfoMedusa (https://infomedusa.es). Esta aplicación fue creada por la Diputación Provincial en colaboración con el Aula del Mar para el seguimiento de la aparición masiva de Pelagia noctiluca en las distintas playas que componen este litoral. Suministra datos informativos sobre el comportamiento, evolución, trayectoria e intensidad de la plaga, suponiendo por ello una importante base para la elaboración de modelos predictivos (Bellido, Souviron, Báez, Ferri-Yañez y Salas, 2018). En el ámbito español existen otras plataformas de recopilación de datos tales como MedusApp, Grumering o iMedJelly, con diferentes periodos temporales y escalas espaciales en el fenómeno de avistamiento de medusas, aunque ninguna de ellas tan abundante ni extensa en el área de estudio como InfoMedusa. A partir de la problemática causada por la llegada de enjambres a la costa, en la realización de esta investigación, se ha considerado de interés analizar cuáles son los patrones espaciales de comportamiento de las medusas a nivel litoral. Se pretende identificar las playas más proclives a recibir medusas como paso previo para la identificación de factores relacionados con esta distribución, y poder proponer, en última instancia, medidas de mitigación idóneas para estas zonas. De forma específica, se abordan los siguientes objetivos: 1) Explotar la base de datos proporcionada por el programa InfoMedusa, para generar un inventario básico de avistamientos de medusas en las playas del litoral occidental de la provincia de Málaga, para el periodo 2015-2020. 2) Analizar la frecuencia de aparición de medusas en cada playa de la zona de estudio (recurrencia diaria en periodo estival durante seis años), así como su severidad. Ajustar los indicadores de peligrosidad a las particularidades y sesgos de la fuente de datos y del inventario. 3) Elaborar una cartografía de peligrosidad por llegada de enjambres en las playas de la Costa del Sol occidental. 4) Comprobar que la distribución espacial de la peligrosidad de afección muestra un patrón aleatorio o, por el contrario, permite identificar tendencias estructurales en la mayor susceptibilidad de algunas playas. 5) Proponer hipótesis preliminares sobre los factores que pueden estar condicionando la mayor predisposición a recibir medusas manifestada en algunas playas. 2. Metodología Para la elaboración de la cartografía de peligrosidad se han desarrollado las siguientes fases de trabajo: 1) Delimitación espacial y cartografía de las playas del litoral occidental de la provincia de Málaga. Identificación de sus principales características fisiográficas y otros factores que puedan relacionarse como posibles factores condicionantes de la mayor o menor presencia de medusas. 2) Volcado y tratamiento inicial de depuración de la base de datos proporcionada por el programa InfoMedusa. Tránsito de la información cualitativa (comentarios) hacia cuantitativa (dato numérico) y asignación del número de registros a cada playa. Investigaciones Geográficas, nº 77, pp. 239-258. 241 de la Fuente Roselló, A., Sortino Barrionuevo, J.F., Reyes Corredera, S. J. y Perles Roselló, M.J. 3) S elección para el estudio de las playas con un número de registros válidos que garanticen la fiabilidad de los datos. 4) Análisis de la frecuencia y severidad de aparición de medusas en cada playa de la zona de estudio (recurrencia diaria durante los meses estivales durante un periodo de seis años). 5) Elaboración de la cartografía de susceptibilidad mediante el software de procesamiento geoespacial ArcGis. 6) Análisis de posibles factores explicativos del patrón espacial de peligrosidad observado. 2.1. Selección del área de estudio El análisis de la frecuencia y severidad de la aparición de medusas se ha aplicado al conjunto de playas del litoral occidental malagueño. Tras una primera fase de revisión y depuración de los datos en todas las playas, se han seleccionado, para una segunda fase de estudio, las 12 playas que reúnen una totalidad de registros suficiente como para ser consideradas fiables, además de una casuística variada en lo que se refiere a factores que puedan resultar explicativos de una mayor o menor propensión a recibir enjambres. (playas de Guadalhorce, Guadalmar, Los Álamos, La Carihuela, Las Gaviotas, Los Boliches, Dunas de Artola, El Cable, La Rada, El Cristo, Playa Ancha y Sabinillas, recogidas en la Figura 1). Se han seleccionado playas de extensión diversa, que se distribuyen a lo largo de unos 90 km del litoral (Tabla 1). Se ha procurado que en la muestra exista una casuística variada en función de los criterios recogidos en la guía de playas del Ministerio para la Transición Ecológica y el Reto Demográfico (MITECO). Se ha atendido principalmente a características como el carácter, clasificando las playas en urbana, cuando esta se encuentra integrada en el entramado urbano, semiaislada, si tiene acceso a alguna urbanización de baja densidad edificativa y aislada, cuando no existen urbanizaciones en el frente costero. También se han tenido en cuenta las condiciones de baño (oleaje moderado y aguas tranquilas), el tipo y composición de la arena el cual, aunque muy similar en la totalidad del tramo de costa analizado, puede distinguir entre arena y grava, y si se localizan en zonas protegidas. Además, en la selección, se ha procurado que las playas abarquen la práctica totalidad de los términos municipales que componen el tramo de costa. En la Figura 2 se muestran imágenes en las que se observa las mencionadas características. De forma añadida, la alta dependencia de este sector del territorio respecto a la actividad turística justifica sobradamente el interés del análisis en la zona. Se ha determinado que la unidad de análisis y representación de la peligrosidad en esta investigación sea la playa, frente a otras opciones utilizadas en otras investigaciones similares, como por ejemplo el municipio. La playa es la delimitación fisiográfica mínima en las que las características morfológicas y los procesos funcionales costeros se comportan de forma sistémica, y constituyen una unidad. Por este motivo, la playa constituye la unidad especial idónea a la hora de interpretar la mayor susceptibilidad a recibir medusas de algunos sectores del litoral, y las causas que pueden explicar esta mayor afección. Figura 1. Playas seleccionadas como área de estudio Elaboración propia 242 Investigaciones Geográficas, nº 77, pp. 239-258. Susceptibilidad de afección por enjambres de medusas (Pelagia noctiluca) en las playas del litoral occidental de la provincia de Málaga Tabla 1. Características de las playas objeto de estudio Playa Término municipal Longitud (metros) Anchura (metros) Grado de urbanización Paseo marítimo Condiciones de baño Fachada litoral Tipo de arena Composición Zona protegida Descripción Guadalhorce Málaga 950 60 Aislada No Oleaje moderado Dunas / Humedal Oscura Arena RENPA Paraje natural Playa junto a la desembocadura del río Guadalhorce, con escasa afluencia de público. Guadalmar Málaga 400 30 Semiurbana No Oleaje moderado Urbana / Dunas Oscura Arena No Forma parte de la zona conocida como playa de San Julian, cerca de la desembocadura del río Guadalhorce. Presenta un alto grado de urbanización La Carihuela Torremolinos 2100 30 Urbana Sí Oleaje moderado Urbana Oscura Arena No Playa con un alto nivel de servicios, ambiente familiar y muy frecuentada. Los Álamos Torremolinos 1500 60 Semiurbana No Oleaje moderado Semiurbana Dorada Arena No Playa familiar. La más alejada del centro de Torremolinos. Las Gaviotas Fuengirola 1200 40 Urbana Sí Aguas tranquilas Urbana Oscura Arena No Buen nivel de servicios y alta ocupación. Los Boliches Fuengirola 1100 40 Urbana Sí Aguas tranquilas Urbana Oscura Arena No Playa muy popular, bien equipada, en el centro urbano de Fuengirola. Dunas de Artola Marbella 1200 30 Aislada No Oleaje moderado Semiurbana Dorada Arena RENPA Parque Natural Playa situada en una zona protegida denominada Dunas de Artola. El Cable Marbella 400 40 Semiurbana No Oleaje moderado Dunas / Semiurbana Oscura Arena No Playa formada por una franja de arena estrecha, con poca afluencia de público. El Cristo Estepona 700 25 a 10 Semiurbana No Aguas tranquilas Semiurbana / Humedal Oscura Arena Zona protegida de interés ornitológico Playa del casco urbano, aunque algo retirada del centro, junto al Puerto Deportivo de Estepona. Forma una bonita cala, regenerada recientemente, configurando una zona abrigada y resguardada de los vientos. La Rada Estepona 2300 80 a 50 Urbana Sí Aguas tranquilas Urbana Oscura Arena No Playa situada frente al casco urbano, ha sido recientemente regenerada. Muy extensa y concurrida, cuenta con gran número de servicios. Urbana Oscura Grava Zona protegida de interés ornitológico Aguas tranquilas y gran afluencia de público. Dotada con un buen número de servicios. Urbana Oscura Arena No Se extiende entre el Puerto Deportivo y el río Manilva. El frente litoral está ocupado por el núcleo de San Luis de Sabinillas. Playa Ancha Casares 1300 50 Urbana Sí Aguas tranquilas Sabinillas Manilva 1700 50 Urbana Sí Aguas tranquilas Fuente: MITECO, 2021. Elaboración propia Investigaciones Geográficas, nº 77, pp. 239-258. 243 de la Fuente Roselló, A., Sortino Barrionuevo, J.F., Reyes Corredera, S. J. y Perles Roselló, M.J. Figura 2. Imágenes de playas seleccionadas Guadalhorce Guadalmar Dunas de Artola Fuente: Diario Sur (s.f.) Fuente: Turismo y Planificación Costa del Sol SLU (s.f.) Fuente: Consejería de Agricultura, Ganadería, Pesca y Desarrollo Sostenible (s.f.) Los álamos Las Gaviotas Los Boliches Fuente: Diario Sur (s.f.) Fuente: Diputación de Málaga (s.f.) Fuente: Narrogeographic (16/09/2011) La Carihuela El Cable La Rada Fuente: Aforo Costa del Sol (s.f.) Fuente: Costalsur (s.f.) Fuente: Diario Sur (s.f.) El Cristo Playa Ancha Sabinillas Fuente: Turismo y Planificación Costa del Sol SLU (s.f.) Fuente: Ayuntamiento de Casares (s.f.) Fuente: Inspain (s.f.) 2.2. Características de la base de datos InfoMedusa y tratamiento y depuración de los datos La investigación se fundamenta en los datos ofrecidos por la aplicación informática InfoMedusa (https://infomedusa.es), activada en 2013 por parte de la Diputación Provincial en colaboración con 244 Investigaciones Geográficas, nº 77, pp. 239-258. Susceptibilidad de afección por enjambres de medusas (Pelagia noctiluca) en las playas del litoral occidental de la provincia de Málaga el Aula del Mar. La aplicación tiene como objetivo informar sobre la actividad de las medusas en las playas del litoral malagueño y es realizada gracias a la colaboración ciudadana, que participa mediante la introducción de comentarios acerca de la presencia o no de medusas clasificadas por playas. La base de datos asociada constituye una importante fuente a tratar y explotar, pero requiere una labor previa de transformación de los registros, expresados en forma de comentarios generales sobre el estado de la mar, en datos fehacientes y concretos sobre ausencia o presencia de enjambres. Para la utilización de la base de datos en cuestión, ha sido necesario tomar una serie de decisiones iniciales, y aplicar una serie de procesos de depuración: 1) Interpretación y filtrado de comentarios, con la eliminación de errores y clasificación de los comentarios relevantes o útiles para el estudio. Hay que señalar que los comentarios son expresados de forma libre por los usuarios, por lo que existe un variado patrón de observaciones. 2) Delimitación del periodo de información en el que los datos poseen la suficiente calidad como para poder ser utilizados para un propósito científico, que se concreta en la serie temporal que transcurre desde el año 2015 al 2020 durante los meses de junio a septiembre. 3) Selección de las playas a analizar en el tramo de estudio seleccionado en razón de la totalidad de registros de datos de cada playa. Se han eliminado del análisis las playas del tramo occidental de la Costa del Sol con menos de 500 registros. Este valor de exclusión se ha obtenido tomando como referencia el total de anotaciones del año 2018, el cual supone, con diferencia, el que posee un mayor número total de comentarios. A partir del registro total de datos del año de referencia sobre el total de playas de la costa occidental de Málaga presentes en la aplicación (170), se ha obtenido el valor medio de 576, 4, que se ha redondeado a la baja para facilitar el análisis. Total comentarios año 2018: 98.004. Total playas: 170. Promedio: 576, 49. 4) Diseño de matriz de volcado de datos diarios depurados por playas, que contiene la serie temporal comentada, las playas objeto de análisis y las variables seleccionadas. Para sistematizar los datos obtenidos de los comentarios de los usuarios de la aplicación, la información se ha clasificado en cuatro categorías (Tabla 2). Tabla 2. Clasificación de los comentarios de InfoMedusa Clasificación de medusas Comentario 0 Comentario irrelevante 1 No presencia de medusas 2 Presencia de medusas dispersas 3 Presencia de enjambres Elaboración propia Se han consultado de forma directa y manual un total de 16.726 comentarios para las playas objeto de este estudio. Tras los procesos de filtrado y depuración de la base de datos han resultado un total de 1.885 registros útiles para el estudio. 2.3. Análisis de la frecuencia y severidad de aparición de medusas. Indicadores calculados Para obtener datos diarios de la presencia o ausencia de medusas en cada playa en un formato binario (sí/no), los dos grados de severidad diferenciados en el inventario (Presencia de medusas dispersas/ presencia de enjambres) han sido unificados en uno sólo, ya que ambos indican la presencia de medusas. Se han calculado distintos parámetros indicadores de la frecuencia de medusas en la playa: por una parte, se ha procedido a ponderar el número de registro por la superficie útil de la playa, entendiendo que la superficie de la playa puede resultar un indicador aproximado de la afluencia o potencial de visitas de cada una de ellas. El indicador obtenido se ha denominado Número de días con medusas ponderado por la superficie de la playa. Asimismo, para evitar el problema de la diferente afluencia en función de la localización, se ha aplicado un coeficiente de minoración para las playas alejadas de los circuitos de acceso urbano, multiplicando por 0,5 la superficie de la playa. Paralelamente, para el análisis comparativo de las anotaciones diarias de presencia/ausencia de medusas entre playas, se han calculado datos porcentuales relativos al total de registros en cada playa (número Investigaciones Geográficas, nº 77, pp. 239-258. 245 de la Fuente Roselló, A., Sortino Barrionuevo, J.F., Reyes Corredera, S. J. y Perles Roselló, M.J. de días con y sin medusas para cada playa en relación al total de registros de la misma). Se han obtenido dos indicadores: Porcentaje de días con medusas respecto al total de registros de la playa y Porcentaje de días sin medusas respecto al total de registros de la playa. A continuación, para clasificar en intervalos los distintos indicadores de peligrosidad calculados y asignar los correspondientes grados de gravedad, se han utilizados dos criterios: intervalos de naturaleza estadística (media y desviación estándar) en el caso de las variables Número de días con y sin medusas ponderado por la superficie de la playa, e intervalos regulares para el indicador Porcentaje de días con y sin medusas. En este último caso, se ha optado por estandarizar los intervalos para el conjunto de las playas a partir del valor máximo presente en la muestra, esto es, el porcentaje de días con medusas máximo en las playas del área de estudio, a fin de observar con mayor claridad las diferencias entre playas y afinar la comparación entre ellas. Las Tablas 3, 4 y 5 recogen los intervalos asignados. Tabla 3. Media y desviación estándar de las variables número de días con y sin medusas ponderado por la superficie de la playa Cálculos Número de días con medusas Número de días sin medusas Ʃ superficie de la playa 248,54 366,91 20,71 30,58 σ 24,38 38,47 1/2 σ 12,19 19,24 Elaboración propia En razón de los estadísticos obtenidos, se han definido tres intervalos de gravedad del peligro (Tabla 4). Tabla 4. Días con medusas ponderado por la superficie de la playa. Intervalos de peligrosidad Intervalos en función de la superficie de la playa Días con medusas Días sin medusas 1 <9 <12 2 10-31 12-50 3 >31 >50 Elaboración propia Tabla 5. Porcentaje de días con y sin medusas. Intervalos asignados Intervalos en función de datos relativos Días con medusas Días sin medusas 1 < 38 <52 2 38-47 52-61 3 >47 >61 Elaboración propia La representación gráfica y cartográfica de estos indicadores y sus intervalos se ha realizado siguiendo la semiótica de color del semáforo. La representación cartográfica se ha ejecutado mediante el programa de procesamiento geoespacial ArcGis. 2.4. Asignación de intervalos de peligrosidad a los distintos indicadores Como se ha citado anteriormente, el número de registros (comentarios en InfoMedusa, 2020) no es homogéneo entre las distintas playas. Para discriminar las playas que son más y menos fiables para este estudio en razón del número de anotaciones de usuarios disponible, se han calculado los valores medios de comentarios para el conjunto del área de estudio y su desviación estándar (Tabla 7). A partir de estos datos, se han considerado, por una parte, fiables aquellas playas que tienen un número de registros en 246 Investigaciones Geográficas, nº 77, pp. 239-258. Susceptibilidad de afección por enjambres de medusas (Pelagia noctiluca) en las playas del litoral occidental de la provincia de Málaga torno a la media menos ½ desviación estándar y/o superiores a ella. Por otra parte, se consideran playas poco fiables las que disponen de un número inferior a la media menos ½ desviación estándar (Tabla 6). Tabla 6. Número de registros por playa. Media y desviación estándar Cálculos Registros de datos Ʃ 594,90 49,58 σ 57,37 1/2 σ 28,68 Elaboración propia Tabla 7. Intervalos de fiabilidad de la playa en función del número de registros Intervalos de fiabilidad de las playas Días con medusas Poco fiable < 21 Fiable 21-78 Muy fiable >78 Elaboración propia En una fase posterior, para evaluar la fiabilidad de los datos ofrecidos por la playa, se ha utilizado una estrategia añadida. Se ha procedido a constatar que, si una playa presenta un elevado número de días con medusas, no coincida con la observación contraria, esto es, que tenga a la vez un número elevado de días sin medusas, lógica que invalidaría la consistencia de la afirmación primera. Se ha considerado que, si la playa ofrece un número elevado de días con medusas y a la vez un número elevado de días sin medusas, la contradicción hace perder fiabilidad a ambas observaciones. A partir de este análisis, la fiabilidad de los datos ofrecidos por cada playa se rige por el razonamiento que se muestra en la Tabla 8. Tabla 8. Criterios aplicados para deducir la fiabilidad de los datos en cada playa Playa fiable Se considera que una playa es fiable cuándo tiene un más de 21 de registros (dato ponderado por la superficie de la playa) y una diferencia entre los días con y sin medusas mayor al 60%. Playa fiable proclive a las medusas Playa con un porcentaje de registro de días con medusas mayor al 60%. Playa fiable no proclive a las medusas Playa con un porcentaje de registro de días sin medusas mayor al 60%. Playa no fiable Se considera que una playa no es fiable cuándo tiene un número de registros por debajo de la media y/o una escasa diferencia entre los días con medusas y los días sin medusas (del 50 al 60%) Elaboración propia 3. Resultados Tras el proceso de consulta directa y manual de un total de 16.726 comentarios referentes a las playas seleccionadas, los comentarios han sido depurados y filtrados en razón de su calidad y representatividad estadística, ofreciendo como resultado un total de 1.885 registros útiles para el estudio, datos que centran el análisis en un total de 12 playas del litoral occidental. Como puede observarse en la Figura 3, los resultados muestran que existe una notable disparidad en el número de comentarios útiles (nº de registros) entre las distintas playas, que oscila entre 57 en Guadalhorce y 264 en La Carihuela. Los valores de frecuencia expresados en valores absolutos son más elevados en las playas con más registros, tanto para el indicador de días con medusas como para su contrario, días sin medusas (Figuras 4 y 5). Este hecho resta valor comparativo a los indicadores de peligrosidad expresados en valores absolutos, tales como el Número absoluto de días con medusas representado en la Figura 6, en la que, como puede observarse, las playas Los Álamos, La Carihuela, Los Boliches y La Rada presentan los valores más elevados de frecuencia de afección por medusas. Investigaciones Geográficas, nº 77, pp. 239-258. 247 de la Fuente Roselló, A., Sortino Barrionuevo, J.F., Reyes Corredera, S. J. y Perles Roselló, M.J. Figura 3. Número de días con registros de medusas Fuente: InfoMedusa, 2020. Elaboración propia Figura 4. Días con medusas por playas. Medias del periodo de análisis y datos absolutos Fuente: InfoMedusa, 2020. Elaboración propia Figura 5. Días sin medusas por playas. Medias del periodo de análisis y datos absolutos Fuente: InfoMedusa, 2020. Elaboración propia 248 Investigaciones Geográficas, nº 77, pp. 239-258. Susceptibilidad de afección por enjambres de medusas (Pelagia noctiluca) en las playas del litoral occidental de la provincia de Málaga Figura 6. Días con medusas por playas. Datos absolutos Fuente: InfoMedusa, 2020. Elaboración propia Al objeto de matizar el problema que introduce en la interpretación el hecho de que las playas tengan un número de registro tan dispar, se ha calculado el indicador de frecuencia ponderado por la superficie de la playa. En la Tabla 9 se muestra el resultado de la ponderación de los días con medusas con respecto a la superficie total de la playa, así como los cálculos realizados para la obtención del valor medio y los intervalos de gravedad establecidos con relación al mismo. Los valores superiores a la media aparecen resaltados en la Tabla 9. Tabla 9. Número de días con medusas ponderado por la superficie de la playa Playa Días con medusas Guadalhorce 3,81 Guadalmar 23,49 Los Álamos 7,50 La Carihuela 4,52 Las Gaviotas 9,90 Los Boliches 81,00 Dunas de Artola 42,38 El Cable 9,31 La Rada 7,29 El Cristo 28,79 Playa Ancha 13,05 Sabinillas 9,51 Ʃ 248,54 σ 24,38 1/2 σ 12,19 20,71 Elaboración propia Investigaciones Geográficas, nº 77, pp. 239-258. 249 de la Fuente Roselló, A., Sortino Barrionuevo, J.F., Reyes Corredera, S. J. y Perles Roselló, M.J. Como resultado de la aplicación de la ponderación por superficie se ha obtenido el resultado mostrado en la Figura 7. Figura 7. Número de días con medusas ponderado por la superficie total de la playa Fuente: InfoMedusa, 2020. Elaboración propia Considerando la superficie de cada playa, los datos varían notablemente, destacando sobre todo la playa de Los Boliches y, en menor medida, Dunas de Artola, como las que más porcentaje de medusas han registrado en función de su superficie, siendo éstas las únicas playas que se sitúan en el intervalo 3, por encima de la media considerada. El mapa resultante clasificado en intervalos se muestra en la Figura 8. Figura 8 Intervalos de días con medusas ponderado por la superficie total de la playa Fuente: InfoMedusa, 2020. Elaboración propia Sin un patrón de continuidad, se pueden distinguir tres niveles de presencia de medusas: inferior a la media considerada (playas de Guadalhorce, Los Álamos, La Carihuela y La Rada), playas en la media considerada (Las Gaviotas, El Cable, El Cristo, Playa Ancha y Sabinillas) y playas con valores superiores a la media considerada (Los Boliches y Dunas de Artola). 250 Investigaciones Geográficas, nº 77, pp. 239-258. Susceptibilidad de afección por enjambres de medusas (Pelagia noctiluca) en las playas del litoral occidental de la provincia de Málaga Una vez comprobado que con el cálculo de intervalos regulares no se aprecia de manera conveniente las diferencias entre playas, para una mejor apreciación de las diferencias entre playas y en un intento de homogeneizar los datos de presencia de medusas, se ha optado por la realización de intervalos relativos al máximo y mínimo porcentaje de la muestra. El resultado del análisis previo comparativo entre datos registrados de presencia y ausencia de medusas en base 100 se puede ver en la Figura 9. Figura 9. Porcentaje de presencia/ausencia de medusas por playas. Datos relativos Fuente: InfoMedusa, 2020. Elaboración propia Los resultados de los cálculos relativos de los días con medusas se presentan en la Tabla 10, en la cual se resaltan los valores superiores a la media. Tabla 10. Porcentaje de días con medusas con intervalos relativos a la muestra de la zona de estudio Playa Presencia de medusas Guadalhorce 36,84 Guadalmar 29,37 Los Álamos 34,48 La Carihuela 35,61 Las Gaviotas 39,58 Los Boliches 39,38 Dunas de Artola 43,75 El Cable 54,74 La Rada 50,00 El Cristo 40,74 Playa Ancha 57,03 Sabinillas 53,79 Ʃ 515,32 42,94 σ 8,95 1/2 σ 4,48 Elaboración propia En el análisis del porcentaje de días con medusas con intervalos relativos a la muestra de la zona de estudio se han definido tres intervalos. En la Figura 10 se representan los resultados ordenados de menor a mayor y por tonalidades de color. En este análisis se observan, a grandes rasgos, menos diferencias entre playas que en la ponderación por superficies. De entre los intervalos definidos, como playas con menos presencia de medusas, se encuentran Guadalmar, Los Álamos y La Carihuela. A continuación, en el intervalo intermedio se presentan cinco playas, estas son, Guadalhorce, Los Boliches, Las Gaviotas, Investigaciones Geográficas, nº 77, pp. 239-258. 251 de la Fuente Roselló, A., Sortino Barrionuevo, J.F., Reyes Corredera, S. J. y Perles Roselló, M.J. El Cristo y Dunas de Artola. En el último intervalo, con más días de medusas por playas, están La Rada, Sabinillas, El Cable y Playa Ancha. Figura 10. Porcentaje de días con medusas por playa con intervalos relativos a la muestra de la zona de estudio Fuente: InfoMedusa, 2020. Elaboración propia El análisis con datos porcentuales relativos permite observar, con mayor claridad, un patrón de distribución espacial de la peligrosidad bastante definido, con una disposición este-oeste en la que aumenta progresivamente la presencia de medusas (Figura 11). Figura 11. Intervalos con datos relativos de presencia de días con medusas por playas Fuente: InfoMedusa, 2020. Elaboración propia 4. Discusión La bibliografía sobre los ciclos de aparición de floraciones de medusas en el mediterráneo y su llegada a la costa está poniendo de manifiesto un acortamiento progresivo de los ciclos. La mayoría de los autores, a la hora de buscar causas explicativas al incremento de la frecuencia en zonas costeras, han trabajado 252 Investigaciones Geográficas, nº 77, pp. 239-258. Susceptibilidad de afección por enjambres de medusas (Pelagia noctiluca) en las playas del litoral occidental de la provincia de Málaga a escala regional. Entre las causas aludidas para esta tendencia, se citan la presión antropogénica sobre las zonas litorales y su impacto directo en los mares y océanos (Condon et al., 2013; Duarte et al., 2013; Purcell, 2011). Otros autores apuntan como los efectos del cambio climático inciden directamente en este hecho (Purcell, 2011) con lo que podría entenderse que es un efecto global y común en todo el entorno Mediterráneo. El motivo del presente estudio es observar si existen diferencias apreciables entre áreas, ante un hecho universal, en esta tendencia generalizada al aumento de frecuencia y severidad de las floraciones de medusas, en cuyo caso las razones de la variabilidad entre zonas habría que investigarlas en las características estructurales de la costa. Se considera fundamental entender el comportamiento de estas floraciones de medusas, ya que afectan negativamente a actividades relacionadas con estos espacios litorales como la pesca, la acuicultura o el turismo (Richardson, Bakun, Hays y Gibbons, 2009). Para analizar la incidencia y severidad se ha utilizado una aplicación de registro de medusas, InfoMedusa, abordando el problema mediante la colaboración de la población, la cual aporta, en esta y otras materias, conocimiento con uso científico que puede ser incorporado en las políticas públicas y, en este caso particular, puede ayudar en la implantación de medidas preventivas y/o paliativas del riesgo. Además, la contribución de la ciudadanía, puede ser muy beneficiosa para la comunidad, ya que siente formar parte del proceso (Danielsen, Burgess, Jensen y Pirhofer-Walzl, 2010). Los resultados que aportan este tipo de metodologías se equiparan a otro tipo de técnicas, más tradicionales, en las que intervienen profesionales del tema en cuestión, lo que argumenta a favor de esta técnica en estudios científicos (Cohn, 2008; Irwin, 1995; Theobald et al., 2015). Silvertown (2009) matiza la utilidad de esta técnica de investigación, indicando la necesidad de validar los datos que aporta el público participante y la necesidad de un buen diseño y estandarización para que los resultados no pierdan calidad. En lo que se refiere a la calidad de la fuente utilizada en la investigación en relación a otras fuentes con similar aplicación, un análisis comparado de las prestaciones y limitaciones permite observar ventajas notables de InfoMedusa sobre otras opciones. Sus principales ventajas son la totalidad de observaciones por playa que aporta y la extensión temporal de los registros válidos (periodo 2015 a 2020). Como inconveniente puede mencionarse la detallada labor de interpretación de los datos que requiere, hecho que se relaciona con el carácter libre de las observaciones y comentarios que conforman la base de datos. Estas características exigen una labor extensa de traducción de los comentarios libres de la ciudadanía, para interpretarlos y obtener resultados útiles para el propósito del inventario. Existen investigaciones, en el marco del litoral andaluz, que toman como referencia otras fuentes de información, cuya calidad y criterios de homogeneización es discutible (Rubio y Gutiérrez, 2020). Entre las otras aplicaciones y bases de datos de medusas disponibles en la Costa del Sol se encuentra la proporcionada por la Red de Información Ambiental de Andalucía (Rediam), que ofrece una disponibilidad temporal que abarca desde 1994 a 2017, para toda la costa andaluza. Esta base de datos podría, a priori, considerarse complementaria a InfoMedusa, ya que comienza con anterioridad a la serie 2015-2020 con la que se ha trabajado en este estudio. Un análisis de detalle de la fuente, sin embargo, descarta esta expectativa ya que Rediam, que se basa en la investigación de Prieto y Navarro (2013), no trabaja con datos en tiempo real proveniente de visitantes, lo que se traduce en un menor volumen de información. La aportación de tan sólo 12, 7 y 2 avistamientos en los años concurrentes de ambas bases de datos, esto es 2015, 2016 y 2017 respectivamente, se muestra como claramente insuficiente para la pretensión del presente estudio. Otra limitación de esta base de datos es que, además, carece de actualización, ya que está activa sólo hasta 2017. Mayor similitud en prestaciones a InfoMedusa tiene la aplicación Medusapp, que aporta datos diarios y opera en el ámbito espacial requerido en este trabajo. Uno de los aspectos negativos de esta aplicación es el intervalo de años que comprende, que comienza en 2018, lo que la limita en este aspecto, además de que los registros u observaciones que muestra sean mucho menos abundantes. Si comparamos el año 2018, que constituye el que más avistamientos posee en ambas aplicaciones, la diferencia de datos es considerable, ya que Medusapp contiene 194 comentarios en comparación a los 98.004 de InfoMedusa, en ambos casos para el conjunto del tramo occidental de la Costa del Sol. Otras aplicaciones similares como MedJelly, Grumering u Oboradar operan fuera del ámbito de análisis, por lo que se descarta en un primer momento como fuentes de trabajo. El análisis comparado de las prestaciones ofrecidas por la base de datos utilizada respecto a las otras fuentes mencionadas muestra con claridad que, a pesar de sus inconvenientes, las posibilidades que ofrecen los datos de InfoMedusa para el análisis de detalle de la frecuencia de avistamientos en la costa es incuestionablemente de mejor calidad. En este contexto, el inventario resultante de la presente investigación constituye una aportación especialmente valiosa, dada la inexistencia de una fuente de similar calidad y características en la zona de estudio. Investigaciones Geográficas, nº 77, pp. 239-258. 253 de la Fuente Roselló, A., Sortino Barrionuevo, J.F., Reyes Corredera, S. J. y Perles Roselló, M.J. Tanto InfoMedusa como Medusapp, registran los datos a nivel de playa. El desarrollo del trabajo ha mostrado que el uso de la playa como unidad de trabajo básica para la representación y cálculo estadístico de la peligrosidad presenta ventajas e inconvenientes. El principal inconveniente es la extensión variable de la misma, hecho que distorsiona la interpretación de la alta o baja frecuencia de avistamientos, dado que no se parte de un espacio potencial de frecuentación de usuarios similar. La base de datos de Rediam, por el contrario, al obtener los datos mediante recopilación de información y el procesado de imágenes de satélite, carece de este tipo de problemática. Sin embargo, como ventaja fundamental del uso de la playa como unidad de trabajo, puede señalarse que constituye un marco para la evaluación y cartografía de la susceptibilidad, coherente con la unidad fisiográfica, geomorfológica y funcional litoral básica, y facilita, por ello, la interpretación de las relaciones entre el patrón de comportamiento espacial de las medusas con las causas precisas que lo puedan estar generando. En lo que se refiere a la periodicidad de la toma de datos y la duración del periodo disponible, la fuente de datos explotada e inventariada ofrece una secuencia más sistemática que Medusapp y Rediam, con un ritmo menos frecuente y un periodo de datos menor, en el caso de Medusapp y poco actualizado con respecto a Rediam. Por último, con respecto al formato de información ofrecida por estas tres fuentes, la más operativa es, sin lugar a dudas, Rediam, que ofrece datos en formato Shape (.shp) de fácil manejo y tratamiento mediante Sistemas de Información Geográfica (SIG). Medusapp, por su parte, requiere la captura de datos procedente de la aplicación y su tratamiento para poder importarlos al SIG, tarea que se realizó para verificar su utilidad en el estudio y cuyo uso se descartó por la insuficiente suma de registros anteriormente comentada. Por último, la base de datos de InfoMedusa, si bien es la más amplia con diferencia, también es la más exigente en dedicación y necesidades de tratamiento de los datos brutos (comentarios cualitativos en formato libre). Por consiguiente, aunque la escala de trabajo sea similar en estos tres casos analizados, tanto el intervalo temporal como la disponibilidad y calidad de los datos, descarta a Rediam y Medusapp como fuente sistemáticas para comprender el comportamiento espacial de las medusas. A partir de la observación comparada de las potencialidades y limitaciones de la fuente de datos, puede afirmarse que el inventario de datos obtenido a partir de su explotación constituye, por sus características, una aportación valiosa y excepcional en el contexto de trabajo, fiable para generar indicadores de distribución espacial del fenómeno. Respecto a los indicadores calculados para evaluar la propensión de las distintas playas a recibir medusas, como se ha comentado, se ha constatado en los mismos una limitación relativa a la diferencia de tamaño de la unidad espacial de observación (la playa) y la posible diferencia asociada a su frecuentación por usuarios. Este hecho puede proporcionar una interpretación confusa de la alta o baja presencia de días con medusas, ya que el dato puede estar motivado por una alta o baja frecuentación de usuarios en la playa. Para aquilatar este problema, se ha propuesto el indicador Número de días con medusas ponderado por la superficie de la playa y Número de días sin medusas ponderado por la superficie de la playa. Mediante la ponderación por la superficie se ha intentado suavizar el hecho probable de que los valores altos de días con medusas de una playa estén motivados por la alta frecuentación de usuarios en la misma. Se ha considerado una relación directa entre la extensión de la playa y la frecuentación de usuarios. Este factor de ponderación se ha ajustado con un coeficiente de minoración (superficie de la playa* 0,5) en los casos en los que la playa esté muy alejada de los circuitos de acceso urbano. Se intenta discriminar, de esta forma, las playas de grandes dimensiones urbanas (alto potencial de frecuentación tanto por el espacio disponible como por la cercanía a la ciudad como punto emisor de usuarios), respecto a las grandes playas alejadas de la ciudad y poco frecuentadas. En cualquier caso, en avances futuros de la investigación, se están calculando indicadores empíricos de frecuentación de las playas para afinar la representación del dato estadístico. Otros estudios han intentado resolver el problema de la homogeneización de datos planteado a través de distintas soluciones. En este sentido, Rubio y Gutiérrez (2020) elaboran una cartografía de los avistamientos basada en una cuadrícula de 5x5 km sobre la que representan la cuantificación de las medusas. Este procedimiento puede ser apropiado para la escala de su investigación, ya que la unidad espacial de cálculo y representación es el municipio, pero no sería útil para el trabajo a nivel de playa. En otro estudio en la costa catalana, Benedetti-Cecchi et al. (2015) estudian los brotes de medusas por año, es decir, se analiza el patrón temporal, no el espacial, lo que hace innecesario calibrar los datos por áreas. Los resultados estadísticos y cartográficos obtenidos permiten deducir que la distribución espacial de la llegada de medusas a la costa no presenta un patrón aleatorio en las playas del tramo estudiado. Al analizar los resultados en su conjunto, se aprecia una clara tendencia al aumento de los avistamientos de medusas conforme se avanza hacia las playas más occidentales, siendo éstas por lo tanto las que presentan una mayor peligrosidad de afección por medusas. En el lado opuesto, las playas más orientales disfrutan de más registros de ausencia de medusas, siendo, por consiguiente, menos proclives a su presencia. 254 Investigaciones Geográficas, nº 77, pp. 239-258. Susceptibilidad de afección por enjambres de medusas (Pelagia noctiluca) en las playas del litoral occidental de la provincia de Málaga Algunos autores han avanzado en la búsqueda de un patrón espacial de la distribución de los enjambres. A una escala regional, de mucho menos detalle, Canepa et al. (2014) atribuyen los avistamientos costeros en el mar Adriático a la dirección y velocidad del viento y al efecto de las mareas, ya que se trata de aguas costeras poco profundas, lo que favorece grandes acumulaciones de medusas. Estos mismos autores, en un estudio del litoral de Cataluña, concluyen que, si bien las observaciones de presencia de medusas están extendidas a lo largo de toda la costa, tienen a concentrase con mayor frecuencia en las playas cercanas a los cañones submarinos, que actúan como “superautopistas” desde las aguas más profundas. A escala de la Costa del Sol, Gutiérrez-Estrada et al. (2021) identifican, al igual que Canepa et al. (2014), la velocidad y la dirección del viento como factores clave que determinan el momento y la intensidad de la llegada de las medusas a las playas. En el mismo entorno geográfico, Rubio y Gutiérrez (2020), que analizan la totalidad de la costa andaluza, relacionan una posible causa de los avistamientos con accidentes geográficos como bahías, ensenadas y costas altas con presencia de calas. Estos autores identifican la localización hacia levante, en el tramo que se extiende desde el municipio malagueño de Torrox hasta Motril en Granada como el más proclive a recibir medusas. En nuestro estudio, sin embargo, no se percibe esta tendencia, sino la contraria, en la que la peligrosidad de las playas aumenta hacia el oeste. Posiblemente motivado por el mayor nivel de detalle de nuestro estudio, puede observarse una explicación coherente a estos hechos, en principio, contradictorios. Debido a la propia incurvación del litoral mediterráneo andaluz, las playas que muestran una mayor propensión en nuestro análisis se posicionan en las franjas de costa orientadas al flujo de levante, desde dónde los estudios más generales afirman que provienen los enjambres, que se trasladan hacia la orilla desde el mar de Alborán (Bellido et al., 2020). A nivel de detalle, un análisis preliminar de la distribución espacial observada en nuestro estudio, permite asociar la distribución la mayor frecuencia de avistamientos de medusas a una serie de factores que pueden estar actuando como condicionantes. Más que la localización hacia el sector oriental de la costa, se han identificado como posibles causas de la mayor peligrosidad de algunas playas aspectos como su orientación sureste, el mayor ángulo de apertura de la playa y la ausencia de una protección lateral de la playa respecto al flujo de levante. Según se ha constatado, las playas de ángulo más abierto y orientadas hacia el este y sudeste, presentan una mayor frecuencia de enjambres. Este hecho pone de manifiesto que, cuando el análisis se realiza a nivel de detalle, la orientación de la embocadura de la playa hacia la zona de procedencia predominante de los enjambres, puede determinar la susceptibilidad, en mayor medida, que la propia localización de la playa. De las playas muestreadas en este estudio, El Cristo, más cerrada y con una orientación suroeste, que además se encuentra resguardada del flujo de levante por una barra de protección lateral en su extremo este, presenta un menor grado de peligrosidad que las de su entorno. La hipótesis que explica la distribución de la peligrosidad observada, como resultante de la orientación del tramo de costa en el que se ubica la playa y su apertura al flujo de levante, se comprueba, igualmente, al analizar el sector oriental del tramo analizado, que presenta playas menos proclives a la aparición de medusas. La protección frente al viento del este que le otorga la propia bahía de Málaga, abierta hacia el poniente, puede explicar esta menor propensión en las playas más orientales. Los valores más bajos de frecuencia de medusas en este sector costero también podrían relacionarse con la proximidad de las playas a fuentes de aporte de agua dulce, debido al río Guadalhorce. Autores como Bellido et al. (2020), citan la posible incidencia de este variable en la distribución espacial de las floraciones de medusas en el litoral y su llegada hasta las zonas de playa desde mar adentro. 5. Conclusiones La base de datos ofrecida por InfoMedusa proporciona una información esencial en lo que se refiere al conocimiento y gestión del problema de la afección por medusas. Su uso con propósitos científicos, no obstante, requiere de un tratamiento detallado y profuso de la información bruta para, después de un proceso de depuración y filtrado, obtener datos fiables. Sus potencialidades como fuente para el análisis del patrón espacial de afección de medusas en el litoral se muestran superiores a las ofrecidas por otras bases de datos de propósito similar, que resultan muy insuficientes. En este contexto, el inventario de datos aportado en la investigación adquiere una especial relevancia, dado la inexistencia de inventarios de datos de similares prestaciones en el ámbito costero andaluz. La variabilidad en los resultados de presencia o ausencia de medusas entre las distintas playas a lo largo del periodo analizado permite afirmar que existe un patrón de distribución de la peligrosidad no aleatorio. La pauta de distribución observada refleja una tendencia al aumento de la presencia de medusas conforme la playa se localiza en sectores más occidentales de la zona de estudio. Factores como la orientación abierta de las playas más proclives hacia las corrientes provenientes de levante, por la orientación general de la costa, puede relacionarse con el incremento de la susceptibilidad en las playas en el sector occidental de la zona de análisis. La orientación a sotavento respecto a los flujos de levante de las playas Investigaciones Geográficas, nº 77, pp. 239-258. 255 de la Fuente Roselló, A., Sortino Barrionuevo, J.F., Reyes Corredera, S. J. y Perles Roselló, M.J. más orientales puede explicar, de igual modo, la menor frecuencia de medusas en estas playas. Este patrón espacial no aleatorio y la coherencia general, entre el modelo de distribución espacial de la aparición de enjambres y factores explicativos asociados a la morfología y funcionamiento del litoral, permiten observar, a pesar de sus limitaciones, la potencialidad como fuente del inventario que se ha elaborado, así como la validez de los indicadores de peligrosidad. El comportamiento diferenciado de las playas objeto de estudio, que se ha constatado en este trabajo, anima a plantear futuras hipótesis, más complejas, en lo que se refiere a otros posibles factores subyacentes al patrón de susceptibilidad observado, que puedan estar causando las diferencias de afección, para avanzar hacia la generación de un modelo predictivo. La explotación de los datos sobre presencia de medusas y la metodología de análisis aplicada en la investigación, han permitido concluir aportaciones de carácter inédito y de interés para el análisis de la afección de medusas en el litoral. Los resultados ofrecen una base de partida sólida para extender el área de estudio e indagar con mayor profundidad en la identificación de factores condicionantes, al objeto de realizar cartografías predictivas de la susceptibilidad en otras zonas costeras. Agradecimientos Los autores agradecemos a J. Bellido (Aula del Mar. Málaga) el acceso a los datos que constituyen la fuente en que se fundamenta el trabajo (InfoMedusa). Referencias Aforo Costa del Sol (s.f.). Torremolinos-Planifica tu visita a la playa. Recuperado de https://aforocostadelsol. es/torremolinos/ Aula del Mar de Málaga (2021). InfoMedusa. Recuperado de https://infomedusa.es Aula del Mar (2021). Medusas en la provincia de Málaga. Recuperado de https://infomedusa.es/picadurade-medusa Ayuntamiento de Casares (s.f.). Casares. Playas. Recuperado de http://www.casares.es/turismo/playas-2/ Bellido, J., Baez, J., Souviron-Priego, L., Ferri-Yañez, F., Salas, C., López, J. A., & Real, R. (2020). Atmospheric indices allow anticipating the incidence of jellyfish coastal swarms. Mediterranean Marine Science, 21(2), 289-297. http://dx.doi.org/10.12681/mms.20983 Bellido, J., Souviron, L., Báez, J. C., Ferri-Yañez, F., & Salas, C. (2018). Seguimiento de un enjambre de Pelagia Noctiluca en la costa de la provincia de Málaga. I Foro del Mar de Alborán. Encuentro de mares, ciencias y culturas (pp. 99-102). Fuengirola. Recuperado de http://www.uicn.es/web/pdf/ XIV_foro/09%20AuladelMar_Infomedusa2.pdf Benedetti-Cecchi, L., Canepa, A., Fuentes, V., Tamburello, L., Purcell, J. E., Piraino, S., R., ... & Halpin, P. (2015). Deterministic Factors Overwhelm Stochastic Environmental Fluctuations as Drivers of Jellyfish Outbreaks. PLOS ONE, 10(10), e0141060. https://doi.org/10.1371/journal.pone.0141060 Brotz, L., Cheung, W. W. L., Kleisner, K., Pakhomov, E., & Pauly, D. (2012). Increasing jellyfish populations: trends in Large Marine Ecosystems. Hydrobiologia, 690(1), 3-20. https://doi.org/10.1007/s10750-0121039-7 Calvo, F. (2001). Sociedades y territorios en riesgo. Barcelona: Ediciones del Serbal. Canepa, A., Fuentes, V., Sabatés, Piraino, S., & Boero, F. (2014). Pelagia noctiluca in the Mediterranean Sea. En Pitt K., Lucas C. (Eds), Jellyfish Blooms (pp. 237-266). Netherlands, Dordrecht: Springer. http://dx.doi.org/10.1007/978-94-007-7015-7_11 Cohn, J. P. (2008). Citizen Science: Can Volunteers Do Real Research? BioScience, 58(3), 192-197. https:// doi.org/10.1641/B580303 Condon, R. H., Duarte, C. M., Pitt, K. A., Robinson, K. L., Lucas, C. H., Sutherland, K. R., … & Graham, W. M. (2013). Recurrent jellyfish blooms are a consequence of global oscillations. Proceedings of the National Academy of Sciences of the United States of America, 110(3), 1000-1005. https://doi. org/10.1073/pnas.1210920110 Consejería de Agricultura, Ganadería, Pesca y Desarrollo Sostenible (s.f.). Dunas de Artola o Cabopino III. 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Frontiers in Ecology and the Environment, 11(2), 91-97. https://doi.org/10.1890/110246 Enríquez, A. R., & Bujosa, A. (2020). Measuring the economic impact of climate-induced environmental changes on sun-and-beach tourism. Climatic Change, 160(2), 203-217. https://doi.org/10.1007/ s10584-020-02682-w Ghermandi, A., Galil, B., Gowdy, J., & Nunes, P. A. L. D. (2015). Jellyfish outbreak impacts on recreation in the Mediterranean Sea: welfare estimates from a socioeconomic pilot survey in Israel. Ecosystem Services, 11, 140-147. https://doi.org/10.1016/j.ecoser.2014.12.004 Goy, J., Morand, P., & Etienne, M. (1988). Long-term fluctuations of Pelagia noctiluca (Cnidaria, Scyphomedusa) in the western Mediterranean Sea. Prediction by climatic variables. Deep Sea Research, 36(2), 269-279. https://doi.org/10.1016/0198-0149(89)90138-6 Gutiérrez-Estrada, J. C., Pulido-Calvo, I., Perregrín, A., García-Gávez, A., Báez, J. C., Bellido, J. J., ... & López, J. A. (2021). Integrating local environmental data and information from non-driven citizen science to estimate jellyfish abundance in Costa del Sol (southern Spain). Estuarine, Coastal and Shelf Science, 249, 107112. https://doi.org/10.1016/j.ecss.2020.107112 Inspain. (s.f.). Playa de Sabinillas. Recuperado de https://www.inspain.org/es/malaga/manilva/playas/ sabinillas/ Irwin, A. (1995). Citizen Science: A Study of People, Expertise, and Sustainable Development. Routledge. Kogovsek, T., Bogunovic, B., & Malej, A. (2010). Recurrence of bloom-forming scyphomedusae: wavelet analysis of a 200-year time series. Hydrobiologia, 645, 81-96. https://doi.org/10.1007/s10750-0100217-8 Mills, C. (2001). Jellyfish blooms: Are populations increasing globally in response to changing ocean conditions? Hydrobiologia, 451, 55-68. https://doi.org/10.1023/A:1011888006302 MITECO (2021). Guía de Playas. Madrid: Ministerio para la Transición Ecológica y el Reto Demográfico. 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Anthropogenic causes of jellyfish blooms and their direct consequences for humans: a review. Marine Ecology Progress Series, 350, 153-174. https://doi. org/10.3354/meps07093 Purcell, J. E. (2011). Jellyfish and Ctenophore Blooms Coincide with Human Proliferations and Environmental Perturbations. Annual Review of Marine Science, 4(1), 209-235. https://doi.org/10.1146/ annurev-marine-120709-142751 Richardson, A. J., Bakun, A., Hays, G. C., & Gibbons, M. J. (2009). The jellyfish joyride: causes, consequences and management responses to a more gelatinous future. Trends in Ecology & Evolution, 24(6), 312-322. https://doi.org/10.1016/j.tree.2009.01.010 Rubio, A., & Gutiérrez, O. (2020). Impacto de las medusas y otros organismos gelatinosos en el litoral andaluz. Implicaciones para el turismo de sol y playa. Estudios Geográficos, 81(288), e038. https://doi. org/10.3989/estgeogr.202053.033 Sabatés, A., Pagès, F., Atienza, D., Fuentes, V., Purcell, J., & Gili, J.-M. (2010). Planktonic cnidarian distribution and feeding of Pelagia nociluca in the NW Mediterranean Sea. Hydrobiologia, 645, 153-165. https://doi.org/10.1007/s10750-010-0221-z Silvertown, J. (2009). A new dawn for citizen science. Trends in Ecology & Evolution, 24(9), 467-471. https://doi.org/10.1016/j.tree.2009.03.017 Theobald, E. J., Ettinger, A. K., Burgess, H. K., DeBey, L. B., Schmidt, N. R., Froehlich, H. E., ... & Parrish, J. K. (2015). Global change and local solutions: Tapping the unrealized potential of citizen science for biodiversity research. Biological Conservation, 181, 236-244. https://doi.org/10.1016/j. biocon.2014.10.021 Turismo y Planificación Costa del Sol S.L.U. (s.f.). Playa de Guadalmar. Costa del Sol Málaga. Recuperado de https://www.visitacostadelsol.com/malaga-costa-del-sol/playas/playa-de-guadalmar-p17211 Turismo y Planificación Costa del Sol S.L.U. (s.f.). Playa de El Cristo. Costa del Sol Málaga. 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The use of scenarios in climate policy planning: an assessment of actors’ experiences and lessons learned in Finland
Climate policy
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Climate Policy ISSN: (Print) (Online) Journal homepage: https://www.tandfonline.com/loi/tcpo20 Full Terms & Conditions of access and use can be found at https://www.tandfonline.com/action/journalInformation?journalCode=tcpo20 Kalle Aro, Jyrki Aakkula, Ville Lauttamäki, Vilja Varho, Pim Martens & Pasi Rikkonen To cite this article: Kalle Aro, Jyrki Aakkula, Ville Lauttamäki, Vilja Varho, Pim Martens & Pasi Rikkonen (2022): The use of scenarios in climate policy planning: an assessment of actors’ experiences and lessons learned in Finland, Climate Policy, DOI: 10.1080/14693062.2022.2123773 To link to this article: https://doi.org/10.1080/14693062.2022.2123773 © 2022 The Author(s). Published by Informa UK Limited, trading as Taylor & Francis Group Published online: 21 Sep 2022. Submit your article to this journal Article views: 257 View related articles View Crossmark data © 2022 The Author(s). Published by Informa UK Limited, trading as Taylor & Francis Group Published online: 21 Sep 2022. Submit your article to this journal Article views: 257 View related articles View Crossmark data CLIMATE POLICY https://doi.org/10.1080/14693062.2022.2123773 ABSTRACT Scenarios are often used to depict the possible outcomes of alternative future developments as part of the evaluation of climate and energy policy measures. In Finland, scenarios have become a standard practice in climate-related policy planning. However, scenario planning often results in a single cohesive narrative, which lacks transparency in why certain developments and solutions are included and others left out. This article focuses on how scenarios created during the planning of Finland’s Medium-term Plan for Climate Change Policy (KAISU) were built, and how the actors involved assessed the characteristics and quality of these scenarios. We interviewed a total of 18 participants from research, administration, and policymaking domains involved in the creation of the plan and the scenarios it contains. Semi-structured interviews provided an insight into the creation and use of scenarios as part of policy formulation, as well as points of further improvement for the process. The KAISU plan was constructed as a cross-sectoral collaborative effort between policymakers, public officials, and researchers. Despite the variety of actors involved in the process, the resulting scenarios were perceived as well- executed by the participants. However, national scenario foresight could be improved in three dimensions: 1) the process where actors collaborate to build scenarios, 2) the scenario methodology, where solutions and developments are included or excluded, and 3) use of the scenarios after their construction. KEYWORDS Scenario planning; emissions scenarios; climate change; Finland; stakeholder participation; qualitative content analysis Key policy insights: h . The scenario approach used in Finland corresponds with needs at the political level. However, the approach could be improved without compromising this goal. . The underlying scenario assumptions and the compatibility of the models used lack transparency. Many actors involved in the scenario process have a limited understanding of the models used and measures proposed outside their respective sector. p . Construction of more than one policy scenario would allow a larger variety of possible future trajectories to be evaluated in national policymaking. In this case, the aim and role of the scenario process would have to be adjusted and clarified accordingly. . Communication regarding the role and applicability of foresight methods should be improved for citizens and politicians alike. The use of scenarios in climate policy planning: an assessment of actors’ experiences and lessons learned in Finland Aakkulaa, Ville Lauttamäki b, Vilja Varhoa, Pim Martensc and Pasi Rikkonend Kalle Aro a, Jyrki Aakkulaa, Ville Lauttamäki b, Vilja Varhoa, Pim Martensc aNatural Resources Institute Finland, Helsinki, Finland; bFinland Futures Research Centre (FFRC), Turku, Finland; cMaastricht University, Campus Venlo, Venlo, Netherlands; dNatural Resources Institute Finland, Mikkeli, Finland ARTICLE HISTORY Received 22 November 2021 Accepted 7 September 2022 KEYWORDS Scenario planning; emissions scenarios; climate change; Finland; stakeholder participation; qualitative content analysis ARTICLE HISTORY Received 22 November 2021 Accepted 7 September 2022 © 2022 The Author(s). Published by Informa UK Limited, trading as Taylor & Francis Group h l d b d d h f h b h l b CONTACT Kalle Aro kalle.aro@luke.fi Natural Resources Institute Finland, Latokartanonkaari 9, 00790 Helsinki, Finland © 2022 The Author(s). Published by Informa UK Limited, trading as Taylor & Francis Group This is an Open Access article distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/by/4.0/), which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited. CONTACT Kalle Aro kalle.aro@luke.fi Natural Resources Institute Finland, Latokartanonkaari 9, 00790 Helsinki, Finland 1. Introduction Current climate action around the globe covers mitigation, adaptation, and other aspects of climate policy, and ranges in scale from individual choices and activities to globally significant actors and large cross-border initiat- ives (Hale et al., 2021). The European Union has continuously increased the ambition level of climate and energy CONTACT Kalle Aro kalle.aro@luke.fi 2 K. ARO ET AL. 2 policy in the twenty-first century. Recently, as part of the European Green Deal, the Commission proposed on September 2020 the raising of the 2030 greenhouse gas (GHG) emission reduction target, including emissions and removals, to at least 55% compared to 1990 (EU Commission, 2021). Furthermore, the EU aims to be climate-neutral by 2050. The EU has examined the actions required across all sectors, including increased energy efficiency and renewable energy, and started the process of making detailed legislative proposals in June 2021 to implement and achieve the increased ambition. This will enable the EU to move towards a climate-neutral economy and implement its commitments under the Paris Agreement. In 2020, the European Union mandated member countries to prepare and submit their national energy and climate plans. gy In Finland, climate policy is framed through a series of plans covering the various sectoral and temporal aspects of the climate domain. Government planning mostly revolves around the Energy and Climate strategy (Huttunen, 2017) and the Medium-term Climate Change Policy Plan, KAISU1 (Ministry of the Environment, 2017), which both utilize scenarios as the basis for their policy. Together, these documents form the practical basis of national climate policy in Finland by being the most regularly updated (Table 1). Of the two documents, KAISU holds a more formal position in policymaking, because its mandate comes directly from the Finnish Climate Change Act (609/2015). KAISU outlines the expected impact of existing policy measures outside the EU Emis- sions Trading System (ETS) on the projected evolution of GHG emissions, renewable energy, and energy efficiency up to 2040. The plan projects the effects of the planned policy measures on the energy system, GHG emissions and sinks, economic development, the environment, and public health. The KAISU plan also assesses the possible impact of planned and existing policy measures on investment (Ministry of Economic Affairs and Employment (MEAE) et al., 2019). 1. Introduction y The use of scenario planning in the preparation of climate actions helps evaluate the overall impacts of chosen policy packages, and at best shows the feasible pathways to stated goals. Scenarios are typically made in conjunc- tion with foresight processes that assist policymaking. The literature often differentiates between the process where scenarios are constructed, including the chosen scenario framework, actors involved and models used (e.g. Wright et al., 2019), and the use of the scenarios as a tool in policy planning (e.g. Cook et al., 2014). However, the line between scenario creation and scenario-inspired decision making becomes blurred when the two processes involve common actors with political motives and decision-making power. In other words, the political dimension can hardly be absent from the scenario-building process which sets the framework for furtherpolicyplanning,causing overlapbetweenthe two processes. Nationalemissionsscenarios can be strongly influenced by decision makers, who may approach the scenarios as a framework for applicable emissions reduction means or, as a strategic planning process (Tyler et al., 2013). Especially when the latter approach is adopted, a scenario-building process becomes inherently political, as the actors involved are negotiating and selecting the emissions reduction means to be implemented nationally. Wright and Goodwin (2009) found thatactorschosentoconductthescenario-buildingprocesscanhavea majorinfluenceonthescenario’scontents and the resulting level of predictability. Ambiguous framing of the scenario, individual biases, and erroneous per- ceptions of causality between variables contributed heavily to the low predictability of the whole scenario. National policy planning also acts as a vital mechanism for advocacy groups to legitimize, strengthen, and promote their solutions to the public agenda (Meadowcroft, 2011). Scenarios and projections can thus be under- stood as forums in which alternative low-carbon solutions compete for recognition and publicity in the national future narrative. As politicians, stakeholders and citizens have different views concerning the probable, preferred, and possible future scenarios, the processes of creating and using scenarios become intertwined. Hence, how the alternative scenarios are formed and discussed is a central question, both in environmental studies of delib- erative democracy and in the deliberative processes in the real world (Renn, 2006). Hale et al. (2021) pinpoint that policymakers need to understand which approaches are working and which are not, promoting the diffusion of best practices and creating conditions for stronger action in the future. In Finland, the scenarios created in the national energy and climate strategy have been an integral part of the policy process. The acronym was formed from the Finnish name of the plan Keskipitkän aikavälin ilmastopolitiikan suunnitelma. 1. Introduction According to the KAISU plan, the scenarios were formed by compiling sectoral estimations of possible emission reduction measures (Ministry of the Environment, 2017). The measures were evaluated based on their impact and cost-effectiveness to form a cohesive set of additional policy measures. However, the 3 CLIMATE POLICY 3 Table 1. Administrative framework for Finnish climate policy. The framework consists of multiple interlinked documents, which vary in their sectoral and temporal scope. Document Purpose Latest entry Instructive Climate Change Act (609/2015) To strengthen the governance of climate policy planning and monitoring 2015 Regulation of the Governance of the Energy Union and Climate Action (EU/ 2018/1999) To unify governance of climate policy and policy goals within the EU in accordance with the Kyoto Agreement, Paris Agreement, and the EU Climate and Energy Package 2018 Governmental Plan Includes goals in national energy and climate policy to be promoted in the current government’s term (Finnish Government, 2019)* Executive Energy and Climate Strategy To provide outlines, goals, and measures that the government deems necessary to accomplish the national goals. Updated once every parliamentary term. (Huttunen, 2017) Medium-term Plan for Climate Change Policy (KAISU) To specify policy measures outside the ETS to 2030. Updated once every parliamentary term (Ministry of the Environment, 2017) National Energy and Climate Plan To present national policy measures for energy efficiency, renewables, GHG reductions, interconnections, and research and innovation in 2021–2030 (Ministry of Economic Affairs and Employment (MEAE) et al., 2019) Adaption Plan for Climate Change To address measures and their sensitivities for climate change adaptation. Updated at least once every 10 years (Ministry of Agriculture and Forestry, 2014)** Long-term Plan for Climate Change Policy To outline national policies for climate change mitigation and adaptation to 2050. Updated at least once every 10 years n/a Monitoring Annual Climate Report Annually examines the meeting of the targets set for emission reduction obligations (Lounasheimo et al., 2021) * During the first KAISU process, the active governmental plan was that of the government of Juha Sipilä, published in 2015 ** The latest adaptation plan was created prior to the Climate Change Act, but displays clear linkages with the upcoming act. scenario-building process is poorly documented in terms of the internal choices made during the process. Bist- line et al. 1. Introduction (2021) propose that transparency should not only be understood as accessibility of data and models used, but should also include a read on the structural assumptions made during the scenario process. They suggest that efforts to increase transparency should be done especially for non-modellers, such as policymakers and stakeholders, in mind. Hence, to improve scenario work and increase the understanding of the national scenario-building processes, it is important to evaluate how the administration, policymakers, and scenario makers interact, which background assumptions guide the process and scenario models, and how scenarios influence policy decisions. This paper inspects the creation of the Finnish Medium-term Plan for Climate Change Policy (KAISU), focus- ing on the construction and use of the scenarios. It gives an in-depth portrayal of a national cross-sectoral scen- ario-building process largely absent in the current literature, and acts as a point of comparison when studying other national scenario processes. It also presents several suggestions on how to improve the scenario process, thus providing useful insights on the potential benefits and issues related to them also outside of the Finnish context. The research questions in this article are the following: RQ 1) How were the scenarios in the creation of the KAISU plan formed and used? RQ 2) How was the scenario process perceived by the KAISU process participants? 4 K. ARO ET AL. Second, 10 respondents represented public officials and other actors directly involved with the KAISU process as part of the working group. These respondents’ roles in the KAISU process were diverse, as they rep- resented multiple ministries and other organizations. Moreover, they were responsible for different actions in the working group, in which some were more involved in the KAISU plan’s coordination, management, and external communications, and others more specifically involved in scenario building by representing their cor- responding sector. Finally, four respondents represented political actors then involved in the climate policy domain. These four participants included members of parliament and their policy advisors. While neither directly involved in the scenario building nor part of the official KAISU working group, they were regularly contacted by public officials to ensure political acceptance of the measures proposed for inclusion in the KAISU plan. In certain cases, politicians also provided specific goals for inclusion in the KAISU plan, which correspondingly shaped the scenario-building process. The interviews were conducted to backtrack the KAISU process and to discover key participants’ perceptions of scenario building in the KAISU process. The interviews were semi-structured: three different interview frames were created, one for each respondent group. The interview frames contained largely similar questions, but some topics varied, based on respondent group’s expertise and involvement in the KAISU process. The inter- view frames also followed the KAISU process in chronological order, first covering the starting point and the framework for scenario building, followed by the creation of the scenarios, and finally addressing the presen- tation and further use of the finalized KAISU plan. Although the interviews’ focus was on the formulation and use of scenarios created in the KAISU process, respondents’ perceptions of the general process were also dis- cussed. Whereas interviews held with public officials followed a largely uniform pattern in line with the inter- view frames, more variance between interviews was discovered when interviewing representatives of research groups and policymakers. The interviews were held between late 2020 and early 2021 as remote video meet- ings that were recorded and transcribed. The materials were analysed using inductive content analysis (Hsieh & Shannon, 2005; Schreier, 2012) with NVivo 12 for Windows software. The purpose of the analysis was to map the KAISU process from its creation to policy use, and to discover how the participants perceived scenario formulation and use. 4 K. ARO ET AL. The interview frames were utilized as the starting point of the analysis. The transcribed interviews were first divided and segmented to correspond to different parts of the scenario process. As respondents often commented on multiple items of interest simultaneously and referred to their previous answers throughout the interviews, excerpts from other parts of the interview were added to the corresponding segment group. A more nuanced analysis was con- ducted within the segmented groups by coding the excerpts by their content. Answers with a shared meaning were coded together inductively, and new codes were created where no existing code within a segment group could accurately grasp the answer’s meaning. Some answers were coded into two or more seg- mented groups, as they provided an insight that could benefit different parts of the analysis. Finally, the result- ing codes were organized into thematic categories within the segment groups. Thematic groups were used as the main unit for the analysis, and the study’s results on the scenario process (section 3.1) and the lessons learned (section 3.2) were compiled based on a chronological and thematic reading of the resulting groups. Two rounds of coding were conducted with three months in between. The results of the two rounds were compared and evaluated against one another to validate the findings. The final thematic categories thus com- prise elements from both coding rounds. 2. Methods and materials In this study, we interviewed 18 participants involved in the creation of the Medium-term Plan for Climate Change Policy. The respondents were chosen to represent different sectors involved in KAISU planning, and their involvement in KAISU process varied as follows: First, four respondents represented research institutions and modelling groups involved mainly in scenario creation. Their role centred around utilizing modelling tools and providing scientific insights and guidance based on their knowledge of the field and the models in use. Moreover, modelling group representatives quan- tified the effect of selected policy measures. 4 K. ARO ET AL. 4 K. ARO ET AL. 3.1. Creation of medium-term plan for climate change The first Medium-term Plan for Climate Change policy was prepared and made public less than a year after the revised national Energy and Climate Strategy was published. While the two were not directly linked, it was expected that they would share a similar landscape and compatible measures across emission sectors, and many actors were involved in creating both documents. As with earlier energy and climate strategies, a quan- titative emission scenario was created by the Ministry of Economic Affairs and Employment for emission CLIMATE POLICY 5 CLIMATE POLICY 5 Figure 1. Simplified overview of the KAISU process based on the interviews. Multiple actor groups (rectangles) and documents (ellipses) influence the scenario building. (Created by authors, generated in diagrams.net). Figure 1. Simplified overview of the KAISU process based on the interviews. Multiple actor groups (rectangles) and documents (ellipses) influence the scenario building. (Created by authors, generated in diagrams.net). projection purposes. This formed the ‘with existing measures’ (WEM) scenario, against which the policy scenario was to be contrasted in the KAISU plan. According to respondents, this WEM scenario’s assumptions and pro- jections were not really discussed during the KAISU process. Instead, the WEM scenario was presented, and the group responsible for the modelling described the models’ general logic. Respondents recalled that the WEM scenario was to follow outlines provided from the EU level, which partly limited the modelling options. Additionally, as most of the modelling for the WEM scenario was conducted earlier during the creation of the Energy and Climate Strategy, the discussions took place before the KAISU process. projection purposes. This formed the ‘with existing measures’ (WEM) scenario, against which the policy scenario was to be contrasted in the KAISU plan. According to respondents, this WEM scenario’s assumptions and pro- jections were not really discussed during the KAISU process. Instead, the WEM scenario was presented, and the group responsible for the modelling described the models’ general logic. Respondents recalled that the WEM scenario was to follow outlines provided from the EU level, which partly limited the modelling options. Additionally, as most of the modelling for the WEM scenario was conducted earlier during the creation of the Energy and Climate Strategy, the discussions took place before the KAISU process. The ‘with additional measures’ (WAM) scenario’s creation differs significantly from the former. 2The Effort Sharing Regulation is part of the EU’s climate framework, which sets binding targets to national emission reductions outside of the Emissions Trading System. For Finland, the target set in the ESR was -39% compared to the emission level in 2005 (Ministry of the Environ- ment, 2022). 3.1. Creation of medium-term plan for climate change Whereas the WEM scenario was a mostly technical emission projection created during the Energy and Climate Strategy project, public officials’ and politicians’ roles were more central in the WAM scenario (Figure 1). Public officials were to draft a policy scenario including measures to meet the national emission reduction target set in the EU’s Effort Sharing Regulation (ESR).2 The discrepancy between projected emissions and the target was estimated to be 6 Mt CO2-eq. in 2030 (Ministry of the Environment, 2017). The WAM scenario’s goal was therefore to present sufficient policy measures to meet this target in the Finnish ESR sectors. The WAM scenario’s outlines were prompted by the government working group overseeing the plan alongside the con- temporary government plan. Additionally, an independent research project (KEIJU) was created to evaluate the measures in both Energy and Climate Strategy and the KAISU plan. gy gy p Four ministries were especially involved in KAISU’s creation: the Ministry of the Environment was the pro- cess’s coordinating ministry, as the preparation of KAISU plan was officially assigned to them. The Ministries of Agriculture and Forestry, Economic Affairs and Employment, and Transport and Communications partici- pated in the central working group and were responsible for measures and policy proposals in their sectors. Each administrative sector was tasked with the creation of a sectoral plan, which was constructed indepen- dently within sectors in collaboration between corresponding ministries and research institutions. The resulting sectoral plans were originally supposed to list all available and even remotely feasible policy measures accompanied by their GHG emission potential for sectors in the ESR, forming KAISU’s backbone. The Finnish 6 K. ARO ET AL. 6 K. ARO ET AL. Climate Change Panel3, as an external advisor to the process, provided a common framework in which all measures collected could be presented uniformly, thus helping evaluate different measures within and across sectors. The framework set cost-effectiveness as the foundation against which different measures were presented and evaluated. Cost-effectiveness therefore became the guiding feature in the WAM scenario’s initial creation. The common framework was generally perceived as helpful, though there were some concerns about social and larger ecological issues being somewhat side-lined due to this techno-economic emphasis. The atmosphere in the working group was highly praised by the participants. 3.1. Creation of medium-term plan for climate change Although the working group consisted of members from a variety of ministries and organizations with different practices, respondents felt that all the participants were genuinely invested in and keen to prepare KAISU to the greatest possible detail. However, the creation of KAISU was also described as rather conservative. Outlines, targets, and the practical scenario modelling work were strongly guided by the EU Climate Framework and the contemporary govern- mental plan, which limited the scenario planning. Moreover, it was felt that public officials were somewhat reluctant to include measures they deemed as lacking political support. Similarly, public officials were very cau- tious when including emerging technologies and measures with limited estimations for their effectiveness to avoid shortfalls if the WAM scenario was to be realized. This left some respondents speculating whether certain possibly effective but unproven measures were disregarded due to a conservative attitude. The scenarios’ initial formulation was largely done within ministries. As the sectors covered in the ESR are highly diverse, policy measures were generally evaluated by research institutions and public officials within administrative sectors. The identified measures were then presented to and approved by the corresponding minister’s staff. This effectively meant that the KAISU working group was more about coordinating responsibil- ities between ministries than evaluating individual policy measures in detail. Most of the reported topics for discussion within the working group were more involved in the general framing of the KAISU plan: how to account for future development of emissions sectors outside of the ESR, and how to develop a just distribution of the burden between sectors within ESR. Although the division of labour was originally designed for all sectors to collect measures to reduce a similar gross percentage of emissions, this was found to be difficult in practice. Respondents especially mentioned the agriculture and transport sectors. Whereas the public officials in the transport sector had adopted a somewhat proactive stance on climate policy by evaluating the effectiveness of different climate policy measures before the KAISU plan, agricultural sector representatives felt that not many politically feasible measures would be found in agriculture, and therefore showed more reluctance about the process. Ultimately, the transport sector was assigned to an ambitious −50% emission reduction target by 2030. The choice was above all political but relieved pressure from the other sectors, especially agriculture. This implies that political feasibility and policy goals were ultimately prioritized over cost-effectiveness. 3The Finnish Climate Change Panel is an independent advisory institution that supports and evaluates climate change policymaking. The Panel consists of 15 distinguished academic scientists, and its official position is established in the Climate Change Act (Finnish Climate Change Panel, 2021). 3.1. Creation of medium-term plan for climate change Active political lobbying was considered part of a healthy democratic process, and it could bring new ideas and perspectives to the table. The KAISU plan was accepted in late 2017 and presented to parliament. At the time of its public release, it received limited attention in the media, but it soon had a major impact on the Finnish national climate policy framework. It outlined the national climate policy’s direction, including technologies and division of labour between different emission sectors. The WAM scenario constructed in KAISU was considered the most probable and politically feasible future trajectory and has greatly influenced Finnish climate policy. Outside parliamentary policymaking, other actors in Finland take an interest in KAISU, often using the plan in their rhetoric and policy documents. Additionally, KAISU’s WAM scenario is often studied and referred to as the Finnish parliament’s official statement on the goals and means it actively works to achieve. 3.2. Lessons learned and further improvements In general, the KAISU plan’s formulation was considered well executed. Relevant ministries and research organ- izations provided sufficient high-quality estimations of the potential GHG mitigation measures and their effec- tiveness. Moreover, respondents found the process insightful and effective. Many also praised the working group’s positive and genuinely collaborative atmosphere, despite the many organizations and ministries par- ticipating. However, respondents identified multiple points where the scenario-building process, the scenarios’ content, and the eventual use of the KAISU plan could be improved (see Table 2). 3.1. Creation of medium-term plan for climate change Politically, the scenarios were discussed in a ministerial working group, which ensured the political feasibility of the overall scenario and provided a formal discussion forum for politicians and the KAISU working group. Additionally, respondents noted the importance of the political advisory level, which acted as a more pragmatic link between politicians and public officials. Political advisors thus pre-assessed the means proposed for the KAISU plan as clearing the burden from ministers who were more focused on the plan’s broader outlines. Citizens, non-governmental organizations, and other stakeholders were encouraged to participate in the KAISU process during the plan’s preparation: workshops were organized during the creation of sectoral plans, and a public hearing round was held for the KAISU draft. While public participation was deemed impor- tant by the respondents, many of them considered these public involvement efforts as largely insignificant. As actors in respective policy fields were often well acquainted with each other, respondents felt these participa- tory efforts added little to what was already known and discussed in the ministries or in the KAISU working group. In particular, the consultation round was considered ill-timed: by the time it was held at the end of the process, there was little enthusiasm for modifying the draft on the working group’s behalf. While the respondents had not encountered attempts to affect the scenario-building process, some speculated that if CLIMATE POLICY 7 Improve coordination across different processes during scenario building Provide transparent reading of models and sectoral plans used Ensure sufficient resources and time allocation Expand group of experts involved Provide transparent assessment of sensitivities and uncertainties Enhance the role of qualitative assessments Utilize a wider range of scenarios Improve communication of scenarios to wider audience Clarify role of scenarios to policymakers Utilize ex post assessments Update scenarios regularly there had been any external forces in play, those efforts would have happened outside and before the formal process. Politically, lobbying was not considered harmful to the process, but a normal two-way interaction with stakeholders. Active political lobbying was considered part of a healthy democratic process, and it could bring new ideas and perspectives to the table. there had been any external forces in play, those efforts would have happened outside and before the formal process. Politically, lobbying was not considered harmful to the process, but a normal two-way interaction with stakeholders. 3.2.1. Scenario process d b Coordination between processes was somewhat discussed by respondents. As the KAISU plan shared strong and obvious linkages with the Energy and Climate Strategy, some respondents wondered why the processes were not constructed and coordinated more closely. Similar remarks were made concerning the numerous research projects and scenario exercises conducted for the KAISU and Energy and Climate strategy. Understanding the roles, linkages, and aims of different processes were found confusing, especially by those only involved in one process. Respondents proposed that the creation of KAISU and Energy and Climate strategy should be further integrated in future to avoid obvious overlaps in the two processes and generate a more holistic stance on Finnish climate policy and the available measures across the three pillars. Of course, a strict boundary between the Effort-Sharing Sector and Emission-Trading Sector keeps emerging as problematic. It’s the same with the Effort-Sharing Sector and Land-Use Sector, as the constant motion [of emissions] across boundaries is obvious (Respondent B6). Transparency of data and sectoral plans were mentioned several times during the interviews. As every admin- istrative sector formulated its own sectoral plan mostly independently before the integrated KAISU plan, respondents from other administrative sectors often felt unable to comment on the selected measures and their rationale. Respondents also implied that they trusted their colleagues to be experts in their respective 8 K. ARO ET AL. 8 fields, thus reducing enthusiasm to challenge their assumptions and policy proposals. However, it was noted that more transparency in the selection of measures might have generated a more holistic and trans-sectoral approach to the KAISU plan, instead of treating each administrative sector independently. It’s rather hard to question representatives of other sectors over, say, whether certain cultivation methods could reduce emis- sions. In this case, the knowledge would be on the Ministry of Agriculture and Forestry’s side (Respondent B7). The compatibility and limitations of the models used were also mentioned in the interviews. As models are complex systems requiring expertise to operate and interpret, few respondents felt qualified to evaluate their functionality and effectiveness. However, the use of multiple largely disconnected models within the same scenario exercise raised some concerns about possible systematic errors resulting from differences in data and general assumptions. While this problem was somewhat addressed by maintaining the shared assumptions and communication efforts between modelling teams, it was a time-consuming process requiring significant resources. 3.2.1. Scenario process d b However, the idea of a single, more holistic, and interconnected assessment model was deemed too difficult and complex to be practical. A model this large was deemed to easily turn against its purpose, as it would require many compromises and generalizations in its internal logic to be practical, risking the loss of many nuances embedded in the current models. Notable criticism was given to the short timeframe, where scenarios had to be modelled, evaluated and compiled. Especially the modelling tasks for WAM scenario and the impact assessment (KEIJU) were conducted with short notice and given a limited timeframe. Respondents hoped that expanding the group involved in scen- ario making could help manage the problem if more people were acquainted with the scenario tools and models in both the research and administrative domains. As an added benefit, it would allow a wider range of actors to conduct scenario work more independently, thus reducing the heavy workload of current experts and generating a wider range of scenarios with different assumptions. Operating heavy-duty models and soft-linking between them is very resource-intensive, and would require multiple iteration rounds to find a stronger, broader linkage to connect them. In my opinion, a lot of questions remained regarding the coher- ence between different analyses (Respondent C4). 3.2.2. Scenario components Addressing sensitivities and uncertainties within the results lacked transparency according to several respon- dents. Respondents found that uncertainties regarding the effectiveness of particular policy measures were poorly explored and described, and done only for the final set of measures. Some respondents feared a large number of small fluctuations across different measures might cause a stark gap between the targeted GHG reduction level and the proposed measures. They felt a more careful assessment of different measures’ sensitivities could help generate a more robust climate plan, in which the risk resulting from certain estimation errors could be better nullified with a selection of measures not strongly interlinked with one another. It should be noted that some respondents recalled that sensitivities and their causes were presented during the process, and felt that confusion over them was due to limited understanding on how scenarios work in general. Many respondents also emphasized that scenarios can never present an accurate future, and the existence of sensi- tivities should therefore be understood as a default from the outset. Some sensitivity assessment was done at the end of the process. However, I found it somewhat vague to be honest (Respon- d B9) Some sensitivity assessment was done at the end of the process. However, I found it somewhat vague to be honest (Respon- dent B9). Some respondents felt the creation of a wider range of scenarios would help highlight the uncertainties of the future and overall scenario work. It was suggested that presenting only a single policy scenario was a potential cause of harmful lock-ins to certain developments and technologies, which might lead to suboptimal policy- making in an uncertain future. Moreover, some respondents suggested that multiple groups acting indepen- dently should undertake the initial scenario modelling. Comparing independently created scenarios would help to pinpoint commonly agreed trajectories across experts and possible risks regarding certain policy pro- posals, as well as to discover any new climate policy developments. Some respondents suggested the creation of multiple scenarios, portraying diverging paths to the future using different sets of policy measures. A CLIMATE POLICY CLIMATE POLICY 9 perceived benefit of multiple detached scenarios was their ability to better clarify the role of scenarios as visions of possible future pathways rather than being addressed solely as estimations of the future. I would, without a doubt, consider using a scenario spectrum, where we would try to recognize the most effective and certain measures. 3.2.2. Scenario components In a way, we would take the uncertainty of the future as a fact and try to incorporate this uncertainty into the process rather than excluding it (Respondent B7). A similar division was found in qualitative assessments in scenario work. As scenarios were built mostly on stat- istical data and estimations, qualitative assessment regarding the measures and assumptions used was found lacking by some respondents. Respondents deemed qualitative assessment helpful for discovering underlying social, cultural, and environmental conditions and trajectories, which could help analyse the preconditions and effects of the proposed policy measures. Many respondents felt that numerical estimations were easier to digest by policymakers and were thus an expected result of scenario work. Questions were, however, raised about the increased level of uncertainty when including more qualitative assessments in the scenario frame- work. Qualitative assessments, regardless of how well conducted and thorough they were, would still have to be transformed into numerical values for them to be properly included in the predominantly quantitative scenario. If this was not done, it was often the case that qualitative estimations were largely side-lined in the scenarios included in KAISU, and received only a little attention from policymakers, who were more inter- ested in the quantitative techno-economic assessments of larger trajectories. Qualitative understanding is highly important, of course, because using numbers always has a simplifying effect. So I’d say that it’s more important to understand the effects of the selected measures, what we’re changing, and what the effects of those changes are. And here it’s important to have a qualitative understanding. Yet it might be something we often don’t take seriously enough (Respondent B1). 4. Discussion Scenario planning approaches have become a widely accepted way of understanding and communicating climate change outcomes (Rogelj et al., 2012). Over the years, such scenarios as the Representative Concen- tration Pathways (RCPs) were developed to provide internally consistent sets of projections of the components of radiative forcing for use in analysis by both climate models and IAMs (van Vuuren et al., 2011). Shared socio- economic pathways (SSPs) were developed ex post to be consistent with the RCPs by integrating the descrip- tions of socioeconomic development with climate change projections and assumptions about climate mitiga- tion and adaptation policies (Ebi et al., 2014). Broader scenario planning use within climate mitigation and adaptation could be seen as supporting the evaluation of both required actions and their societal impacts. One way to improve climate policy planning is to ensure that the insight of such global scenario studies can be included more closely in policy planning to provide foresight information during the planning process. The experts who produce evidence-based research information for planning and decision making can be seen as representatives of a wider scenario setup, and thus a wider future alternative insight than just the WEM and WAM scenario approaches. In the KAISU process, the scenarios were used as a platform for integrating the understanding of the different trajectories of climate policy effects. They also serve as a medium of internal and external communication on the needed and expected additional climate policy measures. Other potential benefits of scenario work, such as the ability to prepare and manage uncertainties, identify discontinuities (Volkery & Ribeiro, 2009) or weak signals, did not appear as priorities in this process. Wright et al. (2020) identify the challenge of understanding risks and opportunities following emerging tech- nological development in policy formulation. In particular, the impact of emerging technologies on different domains in society can be difficult to grasp by policymakers, and this difficulty may lead to disregarding emer- ging issues when designing policies. In a similar fashion, Pye et al. (2021) propose that current modelling approaches often lack radicalism in the solutions included, as many potentially effective, yet politically unfea- sible, solutions are left unaccounted for in the models or side-lined in the scenario process. 3.2.3. Use of scenarios ARO ET AL. 10 10 multiple scenarios with different assumptions, some respondents felt that generating multiple policy scenarios would cause only more confusion and would easily reduce the role of scenarios from a strategic tool to a narra- tive battleground. A single, well-considered, and carefully assessed policy scenario was therefore found more appealing, as it streamlined the policy process and made the KAISU plan more approachable overall. multiple scenarios with different assumptions, some respondents felt that generating multiple policy scenarios would cause only more confusion and would easily reduce the role of scenarios from a strategic tool to a narra- tive battleground. A single, well-considered, and carefully assessed policy scenario was therefore found more appealing, as it streamlined the policy process and made the KAISU plan more approachable overall. 3.2.3. Use of scenarios Several respondents suggested more frequent revisiting and ex post assessment of previously created scenarios. Respondents found that scenario work would greatly benefit from regular revisions where the scenarios could be re-evaluated against contemporary developments. This might enable regular subtle adjustments instead of generating new scenarios every four years. Moreover, some respondents found scenario work in its current form inadequate in providing significant help to policymakers. KAISU plan scenarios were only done once in a parliamentary term. As the level of uncertainty in scenario projections increased significantly with every year, scenarios might lose their practicality quite quickly. To accumulate further understanding from the pre- vious modelling efforts, scenarios should be exposed to ex post analysis. Comparing previous scenarios with contemporary developments would highlight discrepancies between the two. This in turn could help under- stand limitations within the models and data used, and create more robust future scenario modelling. If they hit us once every four years with this vast project, where we then rush to formulate WEM and WAM scenarios, it’s bound to be challenging (Respondent C1). Finally, external communication regarding the scenarios was found lacking, especially for wider audiences such as citizens and the media. While respondents regarded the participation efforts during the KAISU process and external relations after its publication as better than average, they also felt more resources should be allocated to external briefing in future. It was deemed quite difficult for an ordinary citizen to holistically understand the policy framework in climate- and energy-related affairs, and accurately situate KAISU scenarios in this framework. From a communication perspective, it is key that citizens understand the outlines [of climate policy]. Currently, it’s quite hard to grasp, as the pieces are in silos all over the place (Respondent B8). From a communication perspective, it is key that citizens understand the outlines [of climate policy]. Currently, it’s quite hard to grasp, as the pieces are in silos all over the place (Respondent B8). Moreover, the respondents found it hard to explain the applicability and limitations of scenarios for policy- makers, who were not familiar with the methodology. Policymakers were prone to treat the WAM scenario as a deterministic narrative, which downplayed the uncertainty embedded in the foresight method. Additionally, policymakers sometimes pinpointed a certain estimation or causality as incorrect, thus generating a reserved posture toward the rest of the scenario. While this phenomenon could be somewhat addressed by utilizing K. 4. Discussion In our findings, lack of radicalism can be found in the strict modelling boundaries, which gave hardly any leeway to imagine vastly different economic or energy systems, and the precautionary tendencies of the participants to avoid measures with higher levels of uncertainty. As a solution, Wright et al. (2020) suggest forming scenarios accompanied by comparative analyses with regard to different policy aspects and policy recommendations from stakeholders. Hanna and Gross (2021) point to several instances where energy scenario modelling has failed to anticipate changes that have taken place within the energy domain. Similar shortfalls were apparent in the KAISU process as, especially where the WAM scenario is concerned, the pressure to quantify GHG impacts may lead to distor- tion when selecting policy measures. If the models available for the scenario work cannot be used for a quan- titative impact analysis of a certain type of policy measure, there is an obvious temptation to replace such policy measures with measures that can be modelled quantitatively more easily. This underlines the potential disparity in the applicability of qualitative and quantitative scenario approaches, when goals steering the scenario design are set in quantitative terms. Another difficulty with scenarios arises from the modelling and quantification of the impact of proposed measures. In this case, many of the interviewees felt that in the scenario work various models which were used to quantify the impacts of applied policy measures or GHG emission reduction techniques were difficult to perceive and not very transparent in terms of their embedded causality. Of course, this is to some extent an inevitable outcome, because modelling becomes very complex when all the sectors in the economy and their GHG emissions and sinks are included. Not even the researchers and experts responsible for the use of models can master all the models simultaneously, but rather the ones in which they happen to be specialized. This means actors in all the different preparation spheres (research, administration, policymaking) must trust each other. This was the case in the KAISU process, which was indicated in several interview responses. CLIMATE POLICY 11 The idea of having a reference scenario based on currently applied policy measures (WEM) and a policy scenario including policy measures required to achieve the new policy goals set (WAM) is doubtless a practical way to prepare an action plan. 5. Conclusions The complexity of combining cross-sectoral climate policy, the uncertainty inherently embedded in scenario studies, and value-based policymaking demand a high level of planning, cooperation, and context-driven approaches to scenario building. Despite this complexity, the KAISU process appears quite well executed, based on this study’s empirical evidence. The researchers, public officials and political actors involved described the KAISU working group as highly motivated and genuinely engaged with the scenario work, thus mediating the complexity of an intersectoral process. The 2017 KAISU plan generated the framework for Finnish climate policy for several years, creating a more predictable policy landscape for citizens, investors, and other stake- holders. Despite the success of the initial process, many issues were mentioned for potential future improve- ment. Practical scenario building could benefit from better overall integration, transparency, and additional allocated resources. In terms of the scenarios themselves, more variety in scenarios and scenario-specific assess- ments might prepare better for uncertainties. Concerning scenario use, approaching scenarios as a continuous learning and communication process might strengthen their role and practicality as policy planning tools. However, it should be remembered that it is vital to maintain the practicality of scenarios created for policy- making. Holding high academic ideals in a policy process may prompt new ideas for imagining the future, but it may also result in the scenarios being unusable in political decision making and evaluation processes. The scen- ario approach selected for KAISU appeared quite straightforward with its emphasis on quantitative assessment and a baseline - policy scenario dynamic. While the approach may seem low in creativity and leaves room for further improvement, the selected approach and the fundamental goals set for its creation have given policy- makers a cohesive and consistent roadmap to follow. Improving the use of scenarios as part of a policy process thus requires balancing of scientifically considered ideals with the practical realities of representative politics. Finding a middle-road between the two calls for unambiguous goal setting and transparent methodology, as well as understanding on how the scenarios will be used in the context of policy planning. Acknowledgments We thank the interviewees for sharing their time and expertise. We also wish to thank the three anonymous reviewers for their con- structive feedback. 4. Discussion However, with only one alternative WAM scenario, the choice of policy options and instru- ments becomes limited and may lead to an emphasis on already existing, and thus evident, policy measures. According to Koskimaa et al. (2021) the central role of semi-autonomous public officials strengthens the long- term perspective in environmental policy planning, as it balances out the more short-term perspective stemming from representative and party politics. However, our results suggest that this emphasis on long-term develop- ments can also act as a restraint on new emerging climate solutions. Constructing a single alternative scenario is likely to amplify this restraining effect when public officials are more inclined to apply a stricter principle of pre- caution. Keeping this in mind, we can conclude that, although most interviewees agreed that the WEM-WAM scen- ario approach was beneficial in the KAISU process as such, a more extensive array of policy scenarios would be desirable when the long- and medium-term climate policy goals and related measures are to be determined. Disclosure statement No potential conflict of interest was reported by the author(s). We thank the interviewees for sharing their time and expertise. We also wish to thank the three anonymous reviewers for their con- structive feedback. References Bistline, J., Budolfson, M., & Francis, B. (2021). Deepening transparency about value-laden assumptions in energy and environmental modelling: Improving best practices for both modellers and Non-modellers. Climate Policy, 21(1), 1–15. https://doi.org/10.1080/ 14693062.2020.1781048 Bistline, J., Budolfson, M., & Francis, B. (2021). Deepening transparency about value-laden assumptions in energy and environmental modelling: Improving best practices for both modellers and Non-modellers. Climate Policy, 21(1), 1–15. https://doi.org/10.1080/ 14693062.2020.1781048 Climate Change Act (609/2015). (2015). https://www.finlex.fi/fi/laki/alkup/2015/20150609 Unofficial English translation available at: https://www.finlex.fi/fi/laki/kaannokset/2015/en20150609.pdfAnon 2015. Climate Change Act (609/2015). Climate Change Act (609/2015). (2015). https://www.finlex.fi/fi/laki/alkup/2015/20150609 Unofficial English translation available at: https://www.finlex.fi/fi/laki/kaannokset/2015/en20150609.pdfAnon 2015. Climate Change Act (609/2015). Cook, C. N., Inayatullah, S., Burgman, M. A., Sutherland, W. J., & Wintle, B. A. (2014). Strategic foresight: How planning for the unpre- dictable Can improve environmental decision-making. Trends in Ecology & Evolution, 29(9), 531–41. https://doi.org/10.1016/j.tree. 2014.07.005 Cook, C. N., Inayatullah, S., Burgman, M. A., Sutherland, W. J., & Wintle, B. A. (2014). Strategic foresight: How planning for the unpre- dictable Can improve environmental decision-making. Trends in Ecology & Evolution, 29(9), 531–41. https://doi.org/10.1016/j.tree. 2014.07.005 Cook, C. N., Inayatullah, S., Burgman, M. A., Sutherland, W. J., & Wintle, B. A. (2014). Strategic foresight: How planning for the unpre- dictable Can improve environmental decision-making. Trends in Ecology & Evolution, 29(9), 531–41. https://doi.org/10.1016/j.tree. 2014.07.005 Ebi, K. L., Hallegatte, S., Kram, T., Arnell, N. W., Carter, T. R., Edmonds, J., Kriegler, E., Mathur, R., O’Neill, B. C., Riahi, K., Winkler, H., Vuuren, D. P., & Zwickel, T. (2014). A New scenario framework for climate change research: Background, process, and future direc- tions. Climatic Change, 122(122), 363–72. https://doi.org/10.1007/s10584-013-0912-3 EU Comission. (2021). A European Green Deal.” Https://Ec.Europa.Eu/Info/Strategy/Priorities-2019-202 Finnish Climate Change Panel. (2021). Finnish climate change panel. Retrieved October 5, 2021, from https://www.ilmastopaneeli.fi/ en/ Finnish Government. (2019). Programme of Prime Minister Sanna Marin’s Government 10 December 2019. Inclusive and Competent Finland—A Socially, Economically and Ecologically Sustainable Society. Publications of the Finnish Government 2019:33. http:// urn.fi/URN:ISBN:978-952-287-811-3. Hale, T. N., Chan, S., Hsu, A., Clapper, A., Elliott, C., Faria, P., Kuramochi, T., McDaniel, S., Morgado, M., Roelfsema, M., Santaella, M., Singh, N., Tout, I., Weber, C., Weinfurter, A., & Widerberg, O. (2021). Sub- and Non-state climate action: A framework to assess progress, implementation and impact. Climate Policy, 21(3), 406–20. https://doi.org/10.1080/14693062.2020.1828796 Hanna, R., & Gross, R. (2021). How Do energy systems model and scenario studies explicitly represent socio-economic, political and technological disruption and discontinuity? Implications for policy and practitioners. References Finland’s National Climate Change Adaptation Plan 2022. https://mmm.fi/documents/ 1410837/5120838/MMM-_193086-v1-Finland_s_National_climate_Change_Adaptation_Plan_2022.pdf/582041ee-3518-4a63- bf60-7133aed95a9c/MMM-_193086-v1-Finland_s_National_climate_Change_Adaptation_Plan_2022.pdf?t=1507187377000 Ministry of Economic Affairs and Employment (MEAE), Ministry of the Environment, Ministry of Agriculture and Forestry, Ministry of Transport and Communications, and Ministry of Finance. . (2019). Finland’s Integrated Energy and Climate Plan. 2019:66. Ministry of Economic Affairs and Employment. http://urn.fi/URN:ISBN:978-952-327-478-5 Ministry of the Environment. (2017). 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Government Report on Medium-Term Climate Change Plan for 2030 – Towards Climate-Smart Day-to-Day Living. Ministry of the Environment. http://urn.fi/URN:ISBN:978-952-11-4752-4 Ministry of the Environment. (2017). Government Report on Medium Term Climate Change Plan for 2030 Towards Climate Smart Day-to-Day Living. Ministry of the Environment. http://urn.fi/URN:ISBN:978-952-11-4752-4 Pye, S., Broad, O., Bataille, C., Brockway, P., Daly, H. E., Freeman, R., Gambhir, A., Geden, O., Rogan, F., Sanghvi, S., Tomei, J., Vorushylo, I., & Watson, J. (2021). Modelling Net-zero emissions energy systems requires a change in approach. Climate Policy, 21(2), 222–31. https://doi.org/10.1080/14693062.2020.1824891 Pye, S., Broad, O., Bataille, C., Brockway, P., Daly, H. E., Freeman, R., Gambhir, A., Geden, O., Rogan, F., Sanghvi, S., Tomei, J., Vorushylo, I., & Watson, J. (2021). Modelling Net-zero emissions energy systems requires a change in approach. Climate Policy, 21(2), 222–31. https://doi.org/10.1080/14693062.2020.1824891 Renn, O. (2006). Participatory processes for designing environmental policies. Land Use Policy, 23(23), 34–43. https://doi.org/10.1016/ j.landusepol.2004.08.005 Renn, O. (2006). Participatory processes for designing environmental policies. Land Use Policy, 23(23), 34–43. https://doi.org/10.1016/ j.landusepol.2004.08.005 Rogelj, J., Meinshausen, M., & Knutti, R. (2012). Global warming under Old and New scenarios using IPCC climate sensitivity range estimates. Nature Climate Change, 2(2), 248–53. https://doi.org/10.1038/nclimate1385 Rogelj, J., Meinshausen, M., & Knutti, R. (2012). Global warming under Old and New scenario estimates. Nature Climate Change, 2(2), 248–53. https://doi.org/10.1038/nclimate1385 Rogelj, J., Meinshausen, M., & Knutti, R. (2012). ORCID Kalle Aro http://orcid.org/0000-0002-4171-4304 Ville Lauttamäki http://orcid.org/0000-0002-0688-5618 Funding This study was funded by the Academy of Finland in projects Towards deliberative climate and energy foresight (DECENT) under grant numbers 325209 and 325207, and Determinants of successful foresight system in preventing food crisis (DEFORFO) under grant number 335648. 12 K. ARO ET AL. 12 K. ARO ET AL. References Energy Policy, 149, 111984. https://doi.org/ 10.1016/j.enpol.2020.111984 j p Hsieh, H.-F., & Shannon, S. E. (2005). Three approaches to qualitative content analysis. Qualitative Health Research, 15(9), 1277–88. https://doi.org/10.1177/1049732305276687 Huttunen, R. (2017). Government report on the national energy and climate strategy for 2030 (unofficial translation). Ministry of Economic Affairs and Employment. http://urn.fi/URN:ISBN:978-952-327-199-9 Koskimaa, V., Rapeli, L., & Hiedanpää, J. (2021). Governing through strategies: How does Finland sustain a future-oriented environ- mental policy for the long term? Futures, 125, 102667. https://doi.org/10.1016/j.futures.2020.102667 Koskimaa, V., Rapeli, L., & Hiedanpää, J. (2021). Governing through strategies: How does Finland sustain a future-oriented environ- mental policy for the long term? Futures, 125, 102667. https://doi.org/10.1016/j.futures.2020.102667 Meadowcroft, J. (2011). Engaging with the politics of sustainability transitions. Environmental Innovation and Societal Transitions, 1(1), 70–75. https://doi.org/10.1016/j.eist.2011.02.003 Meadowcroft, J. (2011). Engaging with the politics of sustainability transitions. Environmental Innovation and Societal Transitions, 1(1), 70–75. https://doi.org/10.1016/j.eist.2011.02.003 p g j Ministry of Agriculture and Forestry. (2014). Finland’s National Climate Change Adaptation Plan 2022. https://mmm.fi/documents/ 1410837/5120838/MMM- 193086-v1-Finland s National climate Change Adaptation Plan 2022.pdf/582041ee-3518-4a63- p g j Ministry of Agriculture and Forestry. (2014). Finland’s National Climate Change Adaptation Plan 2022. https://mmm.fi/documents/ 1410837/5120838/MMM-_193086-v1-Finland_s_National_climate_Change_Adaptation_Plan_2022.pdf/582041ee-3518-4a63- bf60-7133aed95a9c/MMM-_193086-v1-Finland_s_National_climate_Change_Adaptation_Plan_2022.pdf?t=1507187377000 Ministry of Economic Affairs and Employment (MEAE), Ministry of the Environment, Ministry of Agriculture and Forestry, Ministry of Transport and Communications, and Ministry of Finance. . (2019). Finland’s Integrated Energy and Climate Plan. 2019:66. Ministry of Economic Affairs and Employment. http://urn.fi/URN:ISBN:978-952-327-478-5 Mi i t f th E i t (2017) G t R t M di T Cli t Ch Pl f 2030 T d Cli t S t Ministry of Agriculture and Forestry. (2014). 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Pye, S., Broad, O., Bataille, C., Brockway, P., Daly, H. E., Freeman, R., Gambhir, A., Geden, O., Rogan, F., Sanghvi, S., Tomei, J., Vorushylo, I., & Watson, J. (2021). Modelling Net-zero emissions energy systems requires a change in approach. Climate Policy, 21(2), 222–31. https://doi.org/10.1080/14693062.2020.1824891 Ministry of Agriculture and Forestry. (2014). References Global warming under Old and New scenarios using IPCC climate sensitivity range estimates. Nature Climate Change, 2(2), 248–53. https://doi.org/10.1038/nclimate1385 Schreier, M. (2012). Qualitative content analysis in practice. Sage. Schreier, M. (2012). Qualitative content analysis in practice. Sage. Schreier, M. (2012). Qualitative content analysis in practice. Sage. Schreier, M. (2012). Qualitative content analysis in practice. Sage. Tyler E Boyd A Coetzee K Gunfaus M T & Winkler H (2013) Developing country perspectives on ‘mitigation actions’ ‘NAMAs’ Schreier, M. (2012). Qualitative content analysis in practice. Sage. Tyler, E., Boyd, A., Coetzee, K., Gunfaus, M. T., & Winkler, H. (2013). Developing country perspectives on ‘mitigation actions’, ‘NAMAs’, and ‘LCDS.’. Climate Policy, 13(6), 770–76. https://doi.org/10.1080/14693062.2013.823334 Tyler, E., Boyd, A., Coetzee, K., Gunfaus, M. T., & Winkler, H. (2013). Developing country perspectives on and ‘LCDS.’. Climate Policy, 13(6), 770–76. https://doi.org/10.1080/14693062.2013.823334 13 van Vuuren, D. P., Edmonds, J., Kainuma, M., Riahi, K., Thomson, A., Hibbard, K., Hurtt, G. C., Kram, T., Krey, V., Lamarque, J.-F., Masui, T., Meinshausen, M., Nakicenovic, N., Smith, S. J., & Rose, S. K. (2011). The representative concentration pathways: An overview. Climatic Change, 109(5), 5–31. https://doi.org/10.1007/s10584-011-0148-z g p g Volkery, A., & Ribeiro, T. (2009). Scenario planning in public policy: Understanding Use, impacts and the role of institutional context factors. 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Neurodevelopmental Changes in Excitatory Synaptic Structure and Function in the Cerebral Cortex of Sanfilippo Syndrome IIIA Mice
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Neurodevelopmental Changes in Excitatory Synaptic Structure and Function in the Cerebral Cortex of Sanfilippo Syndrome IIIA Mice Chrissa A. Dwyer1, Samantha L. Scudder2, Ying Lin1, Lara E. Dozier2, Dustin Phan1, Nicola J Allen3 Gentry N Patrick2 & Jeffrey D Esko1 received: 06 December 2016 accepted: 17 March 2017 Published: 18 April 2017 Sanfilippo syndrome, MPS IIIA-D, results from deficits in lysosomal enzymes that specifically degrade heparan sulfate, a sulfated glycosaminoglycan. The accumulation of heparan sulfate results in neurological symptoms, culminating in extensive neurodegeneration and early death. To study the impact of storage in postnatal neurodevelopment, we examined murine models of MPS IIIA, which lack the enzyme sulfamidase. We show that changes occur in excitatory postsynaptic structure and function in the somatosensory cortex prior to signs of neurodegeneration. These changes coincide with accumulation of heparan sulfate with characteristic non-reducing ends, which is present at birth in the mutant mice. Accumulation of heparan sulfate was also detected in primary cultures of cortical neural cells, especially astrocytes. Accumulation of heparan sulfate in cultured astrocytes corresponded with augmented extracellular heparan sulfate and glypican 4 levels. Heparan sulfate from the cerebral cortex of MPS IIIA mice showed enhanced ability to increase glutamate AMPA receptor subunits at the cell surface of wild type neurons. These data support the idea that abnormalities in heparan sulfate content and distribution contribute to alterations in postsynaptic function. Our findings identify a disease- induced developmental phenotype that temporally overlaps with the onset of behavioral changes in a mouse model of MPS IIIA. All cell types in vertebrates and invertebrates, including cells in the central nervous system (CNS), produce plasma membrane and extracellular matrix heparan sulfate proteoglycans (HSPGs). HSPGs consist of a pro- tein core with one or more covalently attached heparan sulfate (HS) chains. HS is comprised of alternating glu- cosamine and uronic acids (d-glucuronic and l-iduronic) variously modified by sulfate groups1. Studies of animal models deficient in HS biosynthesis show that HS has a role in nearly every developmental process in the brain, including neural progenitor cell proliferation and differentiation2–4, axon guidance5,6 and synapse formation7,8. HS in the adult brain also plays a role in maintaining synaptic function9. These processes depend on the binding of numerous growth factors, receptors and guidance proteins to sulfated domains within the chains1. HS turnover occurs through several mechanisms, including proteolytic shedding of proteoglycans from the cell surface, limited endolytic cleavage of the chains by heparanase, and endocytosis, which culminates in lysosomal degradation. Degradation of HS in the lysosome occurs in a sequential manner beginning at the non-reducing end of the chain. www.nature.com/scientificreports www.nature.com/scientificreports www.nature.com/scientificreports SCIeNTIfIC REPortS | 7:46576 | DOI: 10.1038/srep46576 Results 1I), showing that excitatory postsynaptic strength is reduced in Sgshh/h mice. No change in mEPSC inter-event inter- val (Fig. 1J) was observed, consistent with the absence of change in the number of dendritic spines. These find- ings reflect reduced glutamatergic excitatory synaptic strength in layer II/III pyramidal cortical neurons without alterations in synapse number or presynaptic neurotransmitter vesicle release. Evaluation of α​-amino-3-hydroxy- 5-methyl-4-isoxazolepropionic acid (AMPA) receptor subunit levels, GluA1 and GluA2, showed no change in Sgshh/h mice by Western blotting (Fig. S1D,E) and surface expression by biotinylation (Fig. S1F,G). Thus, reduced glutamatergic synaptic strength does not reflect a loss of GluA1 or GluA2 receptors. g g y p gl p To determine whether changes in synaptic function occurred in other brain regions, we also assessed a form of short-term synaptic plasticity in the hippocampus as measured by excitatory synaptic basal transmission and paired pulse facilitation. Basal synaptic transmission in the Schaffer collateral pathway in acute hippocampal slices had normal field excitatory postsynaptic potentials (fEPSPs) in response to increasing stimulation intensi- ties (Fig. S2A). Paired-pulse facilitation in these hippocampi was intact, although a small reduction in facilitation with 50 msec separation of pulses was observed (Fig. S2B). Thus, MPS IIIA mice exhibit specific deficits in excita- tory postsynaptic structure and function in the developing postnatal primary somatosensory cortex. Lysosomal expansion, but not astrocytosis and ganglioside accumulation, are present in the developing postnatal somatosensory cortex. To determine whether changes in postsynaptic structure and function might be due to lysosomal expansion in mutant animals, we analyzed the lysosomal marker, LAMP- 1, in 14, 21 and 61 day old mice. Enhanced LAMP-1 immunostaining was detected throughout the cortical layers of the primary somatosensory cortex at all ages in Sgshh/h mice relative to littermate Sgsh+/h controls (Fig. 2A,B). We also evaluated astrogliosis and secondary accumulation of GM3 ganglioside, traits associated with neuro- degenerative stages of disease progression in the aged MPS IIIA brain. Staining tissue sections with antibodies against glial fibrillary protein (GFAP) showed reactive astrocytes along the pial basement membrane (P), a subset of blood vessels (BV) and white matter (WM) tracks in control mice (Fig. 2C). Staining in the vicinity of the blood vessels and white matter tracts was elevated in Sgshh/h mice (Fig. 2C), culminating at 61 days of age in widespread distribution of GFAP-positive astrocytes throughout the somatosensory cortex (Fig. 2C,D). Results Alterations in excitatory synaptic structure and function occurs in the developing brain of Sgsh mutant mice. To determine whether synaptic development was altered in MPS IIIA mice, we used a nat- urally occurring hypomorphic mutant (Sgshh/h)16,17. This line contains a missense mutation in the sulfamidase gene, Sgsh, reducing enzyme activity by >​95%. Sgshh/h mice exhibit hyperactivity and signs of reduced anxiety as early as 3 weeks of age, which is exaggerated by repeated behavioral testing18,19. Early behavioral abnormalities in human patients and mouse models are consistent with manifestation of disease pathology in the somatosensory cortex, a region of the cerebral cortex involved in the integration of sensory input. Formation and refinement of the neural circuitry in this region of the brain occurs from postnatal day 7 to 28 in mice. Staining of sections of postnatal day 21 somatosensory cortex with the excitatory postsynaptic marker postsynaptic density 95 (PSD-95) showed enhanced puncta number in cortical layers I, II/III and V in Sgshh/h mice compared to heterozygous mice (Sgsh+/h), which do not store HS or present any disease symptoms (Fig. 1A,B; Fig. S1A,B). Western blot analysis on whole tissue homogenates and synaptosomes prepared from the cerebral cortex confirmed the accumulation of PSD-95 in Sgshh/h mice (Fig. 1C). The increase in PSD-95 was not observed in 14-day old mice (Fig. S1C). In the healthy brain, the postsynaptic apparatus containing PSD-95 localizes to the head of membranous protrusions called dendritic spines. Golgi silver-impregnation of layer II/III pyramidal neurons in the primary somatosensory cortex of Sgshh/h mice at day 21 showed that the number of dendritic spines was not altered (Sgsh+/h 0.84 ±​ 0.12, Sgshh/h 0.79 ±​ 0.11 protrusions/μ​m, p >​ 0.05), but a modest increase in spine head width (Fig. 1D,E) and the per- centage of mushroom-shaped spines was observed (Fig. 1F). These findings suggest that synapse architecture is altered by the end of postnatal neurodevelopment in Sgshh/h mice. y p p g Synapse strength closely correlates with dendritic spine morphology (reviewed in ref. 20). To determine whether excitatory synaptic function was altered, electrophysiological recordings were performed in layer II/III pyramidal neurons in the primary somatosensory cortex of acutely isolated brain slices from 21-day old Sgshh/h mice (Fig. 1G). Evaluation of total miniature excitatory postsynaptic current (mEPSC) events revealed fewer large events in the mutant (Fig. 1H). Average mEPSCs amplitude was also significantly reduced in the mutant (Fig. Neurodevelopmental Changes in Excitatory Synaptic Structure and Function in the Cerebral Cortex of Sanfilippo Syndrome IIIA Mice Chrissa A. Dwyer1, Samantha L. Scudder2, Ying Lin1, Lara E. Dozier2, Dustin Phan1, Nicola J Allen3 Gentry N Patrick2 & Jeffrey D Esko1 Thus, inactivating mutations in any of the degradative enzymes arrests the pro- cess, causing accumulation of HS fragments in the lysosome. In humans and animal models, storage results in mucopolysaccharidosis (MPS), a lysosomal storage disorder10. Sanfilippo syndromes (MPS IIIA-D) arise from deficits in enzymes that hydrolyze or modify the non-reducing terminal glucosamine unit. MPS IIIA (OMIM 252900) is a homozygous recessive, congenital disorder caused by the lack of the enzyme sulfamidase (SGSH), which removes the N-sulfate group from terminal N-sulfoglucosamine units. Failure to degrade the chains and/ or accumulation of chains with terminal N-sulfoglucosamine units results in cell death and neurodegeneration. 1Department of Cellular and Molecular Medicine, Glycobiology Research and Training Center, University of California, San Diego, La Jolla, CA, 92093-0687, USA. 2Section of Neurobiology, Division of Biological Sciences, University of California, San Diego, La Jolla, CA 92093-0347, USA. 3Salk Institute for Biological Studies, Molecular Neurobiology Laboratory, La Jolla, CA 92037, USA. Correspondence and requests for materials should be addressed to C.A.D. (email: cdwyer@ucsd.edu) or J.D.E. (email: jesko@ucsd.edu) SCIeNTIfIC REPortS | 7:46576 | DOI: 10.1038/srep46576 1 www.nature.com/scientificreports/ The syndrome also manifests behavioral changes during childhood11,12. MPS IIIB (OMIM 252920), MPS IIIC (OMIM 252930) and MPS IIID (OMIM 252940), caused by deficiencies in other enzymes involved in lysosomal processing of the non-reducing terminal glucosamine residue in HS, have similar clinical consequences. Recently, mutation of a fifth enzyme involved in processing of terminal glucosamine residues (3-sulfoglucosamine sulfa- tase) was shown to result in lysosomal storage and neuropathology in mice13.if y g p gy Sanfilippo syndrome affects the central nervous system and is considered a childhood neurodegenerative disorder. MPS III children present typically with developmental delay that can be accompanied by severe hyper- activity, autistic-like social behaviors and insomnia. In animal models of MPS III, the appearance of abnormal neurological behaviors precedes neuronal cell loss and nervous system atrophy14,15. This observation suggests that changes in neuronal circuit function may give rise to neurological dysfunction at disease onset. Here we show that HS accumulation occurs at birth in the developing MPS IIIA brain and impacts neuronal function at early time points in disease pathogenesis. The results demonstrate that MPS IIIA mice exhibit deficits in synaptic function in the developing somatosensory cortex, which may explain early behavioral abnormalities in MPS III patients. Results (B) Quantification of PSD-95 puncta in cortical layers I, II/III, and V of the primary somatosensory cortex of 21d unaffected Sgsh+/h and Sgshh/h mice. Mean ±​ SEM. N =​ 5–6 animals per genotype. (C) Western blot for PSD-95 levels in cortical homogenate and synaptosome preparations from 21d unaffected and hypomorphic MPS IIIA mice. Band pixel intensities were measured in ImageJ and normalized to β​-actin. Mean ±​ SEM. N =​ 3 animals per genotype, spanning 2 litters. (D) Representative maximum intensity projection z-stacks of dendritic segments from pyramidal neurons in layer II/III of the somatosensory cortex of 21d unaffected and hypomorphic MPS IIIA mice. (E) Quantification of dendritic spine head width and shaft length of pyramidal neurons in layer II/III of the somatosensory cortex of 21d unaffected and hypomorphic MPS IIIA mice. Mean ±​ SEM. N =​ 3 animals per genotype. (F) Quantification of dendritic spine type. Mean ±​ SEM. (G) Representative mEPSC recordings from pyramidal neurons in layer II/III of the somatosensory cortex of 21–22d unaffected and hypomorphic MPS IIIA mice. Scale bar depicts 20 pA, 200 msec. (H) Cumulative probability distributions of all mEPSC event amplitudes recorded from pyramidal neurons in layer II/III of the somatosensory cortex of 21–22d unaffected and hypomorphic MPS IIIA (mice, depicting reduced amplitude in mutant animals. N =​ 1883, 2362 events. p <​ 0.001, Kolmogorov-Smirnov test. (I) Mean mEPSC amplitude. Mean ±​ SEM. (J) Mean mEPSC inter-event interval. N =​ 5 mice per genotype, 16–19 cells total. p =​ 0.015, Student’s t test. Heparan sulfate storage occurs at birth and increases coincident with postnatal brain growth. Accumulation of LAMP-1 stained lysosomes is consistent with storage of HS fragments. HS that accumulates in MPS IIIA has diagnostic terminal non-reducing end (NRE) glycan structures that serve as bio- markers of the disease21. Digestion of the chains with bacterial lyases releases these NRE glycans, which can be readily quantified by liquid chromatography/mass spectrometry (LC/MS)22. Accumulation of NRE gly- cans in MPS disorders is usually quite dramatic in comparison to overall HS or glycosaminoglycan (GAG) accumulation because the biomarker is typically present at very low levels in normal tissues, whereas all cells make HS. Results GM3 ganglioside also accumulated in cortical layers II/III and V of the somatosensory cortex of 61-day-old adult mutant mice (Fig. 2C,E), but not in younger or unaffected mice. Thus, lysosomal expansion and post-synaptic dysfunction precedes widespread astrocytosis and secondary storage of GM3 ganglioside. SCIeNTIfIC REPortS | 7:46576 | DOI: 10.1038/srep46576 2 www.nature.com/scientificreports/ Figure 1. Excitatory postsynaptic structure and function of layer II/III pyramidal neurons from the primary somatosensory cortex of postnatal MPS IIIA mice is abnormal. (A) Representative confocal images show an increase in PSD-95 puncta (red) in layer II/III of the primary somatosensory cortex in 21d (day-old) hypomorphic MPS IIIA (Sgshh/h) mice. Scale, 50 μ​m. Nuclei are stained with DAPI (blue). (B) Quantification of PSD-95 puncta in cortical layers I, II/III, and V of the primary somatosensory cortex of 21d unaffected Sgsh+/h and Sgshh/h mice. Mean ±​ SEM. N =​ 5–6 animals per genotype. (C) Western blot for PSD-95 levels in cortical homogenate and synaptosome preparations from 21d unaffected and hypomorphic MPS IIIA mice. Band pixel intensities were measured in ImageJ and normalized to β​-actin. Mean ±​ SEM. N =​ 3 animals per genotype, spanning 2 litters. (D) Representative maximum intensity projection z-stacks of dendritic segments from pyramidal neurons in layer II/III of the somatosensory cortex of 21d unaffected and hypomorphic MPS IIIA mice. (E) Quantification of dendritic spine head width and shaft length of pyramidal neurons in layer II/III of the somatosensory cortex of 21d unaffected and hypomorphic MPS IIIA mice. Mean ±​ SEM. N =​ 3 animals per genotype. (F) Quantification of dendritic spine type. Mean ±​ SEM. (G) Representative mEPSC recordings from pyramidal neurons in layer II/III of the somatosensory cortex of 21–22d unaffected and hypomorphic MPS IIIA mice. Scale bar depicts 20 pA, 200 msec. (H) Cumulative probability distributions of all mEPSC event amplitudes recorded from pyramidal neurons in layer II/III of the somatosensory cortex of 21 22d unaffected Figure 1. Excitatory postsynaptic structure and function of layer II/III pyramidal neurons from the Figure 1. Excitatory postsynaptic structure and function of layer II/III pyramidal neurons from the primary somatosensory cortex of postnatal MPS IIIA mice is abnormal. (A) Representative confocal images show an increase in PSD-95 puncta (red) in layer II/III of the primary somatosensory cortex in 21d (day-old) hypomorphic MPS IIIA (Sgshh/h) mice. Scale, 50 μ​m. Nuclei are stained with DAPI (blue). Results Quantification of total GAG (as uronic acid) and the NRE biomarker characteristic of MPS IIIA (N-sulfoglucosamine) from whole brains of wild type, Sgsh+/h and Sgshh/h mice at birth and postnatal days 8, 10, 14, 37, and 58–60 showed that GAG accumulation in the brain was apparent at birth (Fig. 3A), with striking accu- mulation of the diagnostic NRE biomarker at all time points (Fig. 3B). In mice, brain mass increases as cells are added during the first several postnatal weeks, a process that did not differ significantly between genotypes at any SCIeNTIfIC REPortS | 7:46576 | DOI: 10.1038/srep46576 3 www.nature.com/scientificreports/ Figure 2. Lysosomal expansion but not astrocytosis and ganglioside accumulation are present in the developing postnatal primary somatosensory cortex. (A) Representative confocal images from unaffected (Sgsh+/h*) and hypomorphic MPS IIIA (Sgshh/h) mouse primary somatosensory cortex stained with LAMP-1 (green). Left panels show low magnification images, scale bar 100 μ​m. Right panels show high magnification of layer II/III (boxed region), scale bar 50 μ​m. Insets in lower right corner show single cells. Nuclei were stained with DAPI (blue). (B) Quantification of LAMP-1 stained vesicle area from high magnification images. (C) Representative confocal images stained with a marker for reactive cortical astrocytes, GFAP (red) and ganglioside GM3 (green), as described in (A). Arrows in low magnification images point to cells with intracellular staining of GM3, denoted by asterisks in high magnification images. P, pial basement membrane; WM, white matter; BV, blood vessel. (D) Quantification of GFAP immunoreactivity in the parenchyma of the somatosensory cortex. (E) Quantification of GM3 immunoreactivity in the parenchyma of the somatosensory cortex. N =​ 3 animals per genotype per age, 2 sections per animal, 2 regions of interest per section were quantified. All brains were collected, stained, and analyzed at the same time. Findings were confirmed on a separate cohort of N =​ 3 animals per genotype per age. Mean ±​ SD. Figure 2. Lysosomal expansion but not astrocytosis and ganglioside accumulation are present in the developing postnatal primary somatosensory cortex. (A) Representative confocal images from unaffected (Sgsh+/h*) and hypomorphic MPS IIIA (Sgshh/h) mouse primary somatosensory cortex stained with LAMP-1 (green). Left panels show low magnification images, scale bar 100 μ​m. Right panels show high magnification of layer II/III (boxed region), scale bar 50 μ​m. Insets in lower right corner show single cells. Nuclei were stained with DAPI (blue). Results (B) Quantification of LAMP-1 stained vesicle area from high magnification images. (C) Representative confocal images stained with a marker for reactive cortical astrocytes, GFAP (red) and ganglioside GM3 (green), as described in (A). Arrows in low magnification images point to cells with intracellular staining of GM3, denoted by asterisks in high magnification images. P, pial basement membrane; WM, white matter; BV, blood vessel. (D) Quantification of GFAP immunoreactivity in the parenchyma of the somatosensory cortex. (E) Quantification of GM3 immunoreactivity in the parenchyma of the somatosensory cortex. N =​ 3 animals per genotype per age, 2 sections per animal, 2 regions of interest per section were quantified. All brains were collected, stained, and analyzed at the same time. Findings were confirmed on a separate cohort of N =​ 3 animals per genotype per age. Mean ±​ SD. age (Fig. S3A). The extent of accumulation of NRE biomarker, when normalized to brain weight, did not change significantly over the 8-week observation period. gi y p Disaccharide analysis also was performed using LC/MS, which provides compositional data as well as an independent means for assessing total HS content23. HS was significantly elevated in cerebral cortical homoge- nates prepared from Sgshh/h mice and increased modestly from 14 days to 58–60 days (Fig. 3C), as did NRE levels (Fig. 3D). NRE levels were elevated to a greater extent in cortical samples compared to whole brain (11 ±​ 2 (14d) and 16 ±​ 4 (58d) nmol NRE/g tissue wet weight in cortical samples versus 5 ±​ 1 (14d) and 7 ±​ 1 (58d) nmol NRE/g tissue wet weight in whole brain). The NRE biomarker was elevated to a similar extent at 14 days of age in the cerebral cortex of a novel MPS IIIA mouse strain bearing a null allele for Sgsh (Sgsh−/−) (see methods; compare Fig. 3E and B). Based on disaccharide analysis, the degree of 2-O-sulfation increased over time in Sgshh/h mice (Fig. S3B), with corresponding changes in 2-O-sulfated disaccharides at 58 days (Fig. S3C). In contrast, no changes in chondroitin/dermatan sulfate (CS/DS) levels or disaccharide composition were observed between genotypes (Fig. S3D–F). These results show that the developing postnatal cerebral cortex is heavily burdened with a pathological form of HS having an altered composition and characteristic NRE, which we refer to as “patholog- ical HS”. Importantly, HS accumulates in the absence of secondary pathology and precedes synaptic dysfunction. Results (C) Cerebral cortex heparan sulfate levels measured by LC/MS. (D) Cerebral cortex NRE levels by LC/MS. (E) Sgsh null (Sgsh−/−) cerebral cortex NRE levels by LC/MS. Values normalized to wet tissue mass. Unaffected wild type and heterozygous (Sgsh+/h* open bars) and hypomorphic MPS IIIA (S hh/h l d b ) l th i p ifi d D f bi th 0 d N 3 i l p t p p Figure 3. Accumulation of undegraded heparan sulfate occurs in the developing postnatal MPS IIIA mouse brain and cerebral cortex. (A) Whole brain glycosaminoglycan uronic acid levels by carbazole assay. (B) Whole brain NRE levels (N-sulfoglucosamine) by LC/MS. (A,B) values are normalized to brain wet weight, which is presented in Fig. S3A. (C) Cerebral cortex heparan sulfate levels measured by LC/MS. (D) Cerebral cortex NRE levels by LC/MS. (E) Sgsh null (Sgsh−/−) cerebral cortex NRE levels by LC/MS. Values normalized to wet tissue mass. Unaffected wild type and heterozygous (Sgsh+/h* open bars) and hypomorphic MPS IIIA (Sgshh/h closed bars), unless otherwise specified. Day of birth =​ 0 day. N =​ 3 animals per genotype per age. Transcardial perfusion with PBS was performed on all animals to eliminate contamination from residual blood in tissue. In all cases graphs show mean ±​ SD. Figure 3. Accumulation of undegraded heparan sulfate occurs in the developing postnatal MPS IIIA mouse brain and cerebral cortex. (A) Whole brain glycosaminoglycan uronic acid levels by carbazole assay. (B) Whole brain NRE levels (N-sulfoglucosamine) by LC/MS. (A,B) values are normalized to brain wet weight which is presented in Fig. S3A. (C) Cerebral cortex heparan sulfate levels measured by LC/MS. (D) Cerebral cortex NRE levels by LC/MS. (E) Sgsh null (Sgsh−/−) cerebral cortex NRE levels by LC/MS. Values normalized to wet tissue mass. Unaffected wild type and heterozygous (Sgsh+/h* open bars) and hypomorphic MPS IIIA (S hh/h l d b ) l h fi d f b h d l Figure 3. Accumulation of undegraded heparan sulfate occurs in the developing postnatal MPS IIIA mouse brain and cerebral cortex. (A) Whole brain glycosaminoglycan uronic acid levels by carbazole assay. (B) Whole brain NRE levels (N-sulfoglucosamine) by LC/MS. (A,B) values are normalized to brain wet weight, which is presented in Fig. S3A. (C) Cerebral cortex heparan sulfate levels measured by LC/MS. (D) Cerebral cortex NRE levels by LC/MS. (E) Sgsh null (Sgsh−/−) cerebral cortex NRE levels by LC/MS. Results Values normalized to wet tissue mass. Unaffected wild type and heterozygous (Sgsh+/h* open bars) and hypomorphic MPS IIIA (Sgshh/h closed bars), unless otherwise specified. Day of birth =​ 0 day. N =​ 3 animals per genotype per age. Transcardial perfusion with PBS was performed on all animals to eliminate contamination from residual blood in tissue. In all cases graphs show mean ±​ SD. Sgshf/f mice to strains carrying different cell-specific Cre drivers (Fig. 4D) resulted in extensive recombination of the floxed Sgsh allele based on PCR analysis of DNA derived from cerebral cortex (Fig. S4A,B). However, inactivation of Sgsh in neurons (Sgshf/fSyn1-cre+), astrocytes (Sgshf/fGFAP-cre+) or endothelia/myeloid cells (Sgshf/fTie2-cre+) did not result in NRE biomarker accumulation in the cerebral cortex (Fig. 4E), most likely due to cross correction by secreted sulfamidase25. Consistent with this hypothesis, Sgshf/fNes-cre+ animals, which results in Sgsh inactivation in the majority of cells in the brain, accumulated NRE biomarker (Fig. 4E) and LAMP-1 immunoreactive lysosomes, albeit to a lesser extent than Sgshh/h (Fig. 2A) and Sgsh−/− mice (Fig. S4C). y g g g g Although we were unable to assess the contribution of astrocyte accumulation to the neurodevelopmental phenotype in vivo, we demonstrated that pathological HS that accumulates in the cerebral cortex affects the sur- face expression of AMPA receptors in the postsynaptic membrane of rat cortical neurons cultured in vitro. HS chains from the cerebral cortex of Sgsh+/h or Sgshh/h mice, which mostly derives from resident astrocytes, were purified and added to cultures of mature wild type rat cortical neurons in equal “brain equivalents,” which mirrors the two-fold increase in overall brain HS in the mutant. Plasma membrane AMPA receptor subunit levels were assessed by cell surface biotinylation followed by precipitation with streptavidin beads and immunoblotting for AMPA receptor GluA2 or GluA1 subunits. The addition of HS from either the mutant or the unaffected animals did not affect the total level of GluA2 subunits (Fig. 5A), but plasma membrane associated GluA2 subunits were increased when HS from the mutant was added or when higher concentrations of HS were added (Fig. 5B). Note that the horizontal line with grey shading shows control levels of GluA receptor subunits in untreated samples. The addition of HS chains did not alter GluA1 levels, suggesting the elevated HS expressed in the mutant has a specific effect on AMPA receptor GluA2 subunits (Fig. Results Pathological heparan sulfate alters cell surface AMPA GluA2 subunits. To determine the cellular origin of HS bearing the NRE biomarker, primary cultures of highly enriched neurons or astrocytes (>​95% purity) were prepared. Dramatic accumulation of NRE biomarker was present in cell lysates prepared from mixed primary cultures containing neurons and glia isolated from the cerebral cortex of newborn Sgshh/h mice (Fig. 4A). Most of this material derives from astrocytes based on 60-fold more NRE in enriched astrocyte preparations compared to isolated neurons (Fig. 4B). Evaluation of Sgsh transcript levels in available RNAseq datasets showed significantly higher expression of Sgsh transcripts in astrocytes than neurons, an observation that we confirmed in the astrocyte and neuronal cell cultures by qPCR (Fig. 4C)24. Several HS-degrading lysosomal hydrolases and genes that regulate lysosomal biogenesis, including transcription factor EB (TCFEB) also were enriched in astro- cytes compared to neurons (Table S1). y p Synapse formation has both a cell autonomous neuronal component and non-cell autonomous glial compo- nent. We attempted to measure the contribution of different cell types to disease pathogenesis in vivo by selective deletion of a “floxed” allele of Sgsh (Sgshf/f) in astrocytes and other cell types found in the brain. Crossbreeding SCIeNTIfIC REPortS | 7:46576 | DOI: 10.1038/srep46576 4 www.nature.com/scientificreports/ Figure 3. Accumulation of undegraded heparan sulfate occurs in the developing postnatal MPS IIIA mouse brain and cerebral cortex. (A) Whole brain glycosaminoglycan uronic acid levels by carbazole assay. (B) Whole brain NRE levels (N-sulfoglucosamine) by LC/MS. (A,B) values are normalized to brain wet weight, which is presented in Fig. S3A. (C) Cerebral cortex heparan sulfate levels measured by LC/MS. (D) Cerebral cortex NRE levels by LC/MS. (E) Sgsh null (Sgsh−/−) cerebral cortex NRE levels by LC/MS. Values normalized to wet tissue mass. Unaffected wild type and heterozygous (Sgsh+/h* open bars) and hypomorphic MPS IIIA (Sgshh/h closed bars), unless otherwise specified. Day of birth =​ 0 day. N =​ 3 animals per genotype per age. Transcardial perfusion with PBS was performed on all animals to eliminate contamination from residual blood in tissue. In all cases graphs show mean ±​ SD. Figure 3. Accumulation of undegraded heparan sulfate occurs in the developing postnatal MPS IIIA mouse brain and cerebral cortex. (A) Whole brain glycosaminoglycan uronic acid levels by carbazole assay. (B) Whole brain NRE levels (N-sulfoglucosamine) by LC/MS. (A,B) values are normalized to brain wet weight, which is presented in Fig. S3A. Results (B) NRE levels in intracellular preparations from mature primary cortical cultures of enriched neurons or astrocytes from unaffected and hypomorphic MPS IIIA mice. Note that differences are presented using a logarithmic scale. N =​ 3 individual animals per genotype spanning two litters. Cell preparations of neurons and astrocytes were obtained from different animals. (C) Relative Sgsh transcript expression by quantitative RT-PCR in wild type primary cultures enriched for neurons or astrocytes. N =​ 3–4 individual animals spanning two litters. (D) Genomic arrangement of conditional Sgsh allele and Cre-recombinase drivers. Table shows the affected cell types and developmental stage when cre recombinase is expressed for each strain. (E) NRE levels in the cerebral cortex from mice at 182 days of age. N =​ 3 animals per genotype. Figure 4. Contributions of neurons and astrocytes to HS accumulation in the cerebral cortex. (A) NRE Figure 4. Contributions of neurons and astrocytes to HS accumulation in the cerebral cortex. (A) NRE levels in cell lysates prepared from primary mature cortical neural cultures containing a mixture of neurons and glia isolated from unaffected (Sgsh+/h*) and hypomorphic MPS IIIA (Sgshh/h) mice. N =​ 3 individual animals per genotype spanning two litters. (B) NRE levels in intracellular preparations from mature primary cortical cultures of enriched neurons or astrocytes from unaffected and hypomorphic MPS IIIA mice. Note that differences are presented using a logarithmic scale. N =​ 3 individual animals per genotype spanning two litters. Cell preparations of neurons and astrocytes were obtained from different animals. (C) Relative Sgsh transcript expression by quantitative RT-PCR in wild type primary cultures enriched for neurons or astrocytes. N =​ 3–4 individual animals spanning two litters. (D) Genomic arrangement of conditional Sgsh allele and Cre-recombinase drivers. Table shows the affected cell types and developmental stage when cre recombinase is expressed for each strain. (E) NRE levels in the cerebral cortex from mice at 182 days of age. N =​ 3 animals per genotype. is sufficient to increase GluA2 levels and shows a dependence on concentration and that astrocytes may be the primary source of HS that contributes to alterations in post-synaptic function. is sufficient to increase GluA2 levels and shows a dependence on concentration and that astrocytes may be the primary source of HS that contributes to alterations in post-synaptic function. Results 5C).h pif p ( g ) The high level of HS accumulation in astrocytes and the predominance of astrocytes in the brain suggest that these cells are the primary source of HS. Analysis of HS distribution in astrocytes by radiolabeling with 35SO4 showed that cell surface and secreted GAG levels did not differ in unaffected and mutant cells, but accumula- tion of intracellular GAG was obvious in the mutant (Fig. 5D). In a label-chase experiment, which tracks the turnover of metabolically labeled HS, both intracellular and cell surface GAG was increased in Sgshh/h astrocytes (Fig. 5E). Purification of HS from these samples confirmed elevation of HS at the cell surface (Fig. 5F). Glypican 4 levels were also modestly elevated in astrocyte lysates derived from Sgshh/h mice, although the difference did not reach significance (Sgsh+/h 1.0 ±​ 0.37, Sgshh/h 1.36 ±​ 0.56) (Fig. 5G). These findings show HS supplementation SCIeNTIfIC REPortS | 7:46576 | DOI: 10.1038/srep46576 5 www.nature.com/scientificreports/ p Figure 4. Contributions of neurons and astrocytes to HS accumulation in the cerebral cortex. (A) NRE levels in cell lysates prepared from primary mature cortical neural cultures containing a mixture of neurons and glia isolated from unaffected (Sgsh+/h*) and hypomorphic MPS IIIA (Sgshh/h) mice. N =​ 3 individual animals per genotype spanning two litters. (B) NRE levels in intracellular preparations from mature primary cortical cultures of enriched neurons or astrocytes from unaffected and hypomorphic MPS IIIA mice. Note that differences are presented using a logarithmic scale. N =​ 3 individual animals per genotype spanning two litters. Cell preparations of neurons and astrocytes were obtained from different animals. (C) Relative Sgsh transcript expression by quantitative RT-PCR in wild type primary cultures enriched for neurons or astrocytes. N =​ 3–4 individual animals spanning two litters. (D) Genomic arrangement of conditional Sgsh allele and Cre-recombinase drivers. Table shows the affected cell types and developmental stage when cre recombinase is expressed for each strain. (E) NRE levels in the cerebral cortex from mice at 182 days of age. N =​ 3 animals per genotype. Figure 4. Contributions of neurons and astrocytes to HS accumulation in the cerebral cortex. (A) NRE levels in cell lysates prepared from primary mature cortical neural cultures containing a mixture of neurons and glia isolated from unaffected (Sgsh+/h*) and hypomorphic MPS IIIA (Sgshh/h) mice. N =​ 3 individual animals per genotype spanning two litters. Discussion In this report, we show that excitatory postsynaptic structure and function is abnormal in the developing somatosensory cortex in MPS IIIA mice, that HS accumulation is present at birth and remains at an elevated steady-state level in the developing postnatal brain, and that HS accumulation occurs prior to astrocytosis and secondary storage of ganglioside GM3. Furthermore, we show that HS accumulation mostly derives from astro- cytes and that exogenous HS is sufficient to alter the level of AMPA receptor, GluA2, on the cell surface. The reduced excitatory synaptic strength observed in 21 day-old MPS IIIA mice coincides with the appearance of behavioral changes that have been published by other research groups18, suggesting a potential causal relation- ship. Together these findings provide the first evidence that neurodevelopmental abnormalities contribute to disease pathogenesis in MPS III. To our knowledge, previous studies have not addressed whether pathological HS accumulates early in brain development and prior to histological hallmarks of neurodegeneration. In part, this lack of information reflects a technological limitation for assessing HS (and other GAGs) in small samples, a problem solved using LC/MS assessment of the MPS IIIA NRE biomarker, N-sulfoglucosamine. This method has a wide dynamic range, can detect storage in as little as 104 cells or ≤​1 mg wet weight of tissue, and has a signal (mutant) to noise (wildtype) ratio that cannot be achieved using methods that simply measure total GAG by dye binding or chemical methods. Using LC/MS NRE analysis, we show that storage of pathological HS occurs in the brain at birth and did not change over the first 8 postnatal weeks when normalized to brain weight, potentially explaining why the expected increase in NRE is not observed over this period. We also show that HS accumulates differentially in isolated SCIeNTIfIC REPortS | 7:46576 | DOI: 10.1038/srep46576 6 www.nature.com/scientificreports/ Figure 5. HS alters AMPA receptor subunit GluA2 recruitment to the cell surface. (A–C) Mature wildtype rat cortical neurons were treated for 72 hours with different brain equivalents of HS purified from the cerebral cortex of unaffected and hypomorphic MPS IIIA mice. Note that the mutant brain contains twice the amount of HS as the unaffected mice. (A) Western blots for GluA2 AMPA receptor subunit shows cell surface GluA2 levels are enhanced following addition of HS, while total GluA2 levels are unchanged. (B) Quantification of surface GluA2 levels normalized to total GluA2 from panel A. Mean ±​ SEM. Discussion (C) Quantification of surface GluA1 levels normalized to total GluA1 for the same samples in (B). Mean ±​ SEM. N =​ 3–6 wells per concentration from N =​ 3 experiments. (B,C) Horizontal line with grey shading shows control levels in untreated samples as mean ±​ SEM used for statistical comparison. (D) Purified GAGs from intracellular, cell surface, and media fractions from primary astrocyte cultures from unaffected (Sgsh+/h) and hypomorphic MPS IIIA (Sgshh/h) mice following a 48-hour radiolabeling with 35SO4. (E) Purified GAGs from corresponding fractions following a 48- hour chase. (F) Purified HS from the fractions shown in panel E. Mean ±​ SD. N =​ 2 animals per genotype, 2 wells per animal. Results were confirmed on a separate cohort of N =​ 2 animals per genotype. (G) Western blot for glypican 4 in cell lysates of cultured primary astrocytes. Buffer control (−​), heparin lyase treated (+​). re 5. HS alters AMPA receptor subunit GluA2 recruitment to the cell surface. (A–C) Mature wildtype fi Figure 5. HS alters AMPA receptor subunit GluA2 recruitment to the cell surface. (A–C) Mature wildtype rat cortical neurons were treated for 72 hours with different brain equivalents of HS purified from the cerebral cortex of unaffected and hypomorphic MPS IIIA mice. Note that the mutant brain contains twice the amount of HS as the unaffected mice. (A) Western blots for GluA2 AMPA receptor subunit shows cell surface GluA2 levels are enhanced following addition of HS, while total GluA2 levels are unchanged. (B) Quantification of surface GluA2 levels normalized to total GluA2 from panel A. Mean ±​ SEM. (C) Quantification of surface GluA1 levels normalized to total GluA1 for the same samples in (B). Mean ±​ SEM. N =​ 3–6 wells per concentration from N =​ 3 experiments. (B,C) Horizontal line with grey shading shows control levels in untreated samples as mean ±​ SEM used for statistical comparison. (D) Purified GAGs from intracellular, cell surface, and media fractions from primary astrocyte cultures from unaffected (Sgsh+/h) and hypomorphic MPS IIIA (Sgshh/h) mice following a 48-hour radiolabeling with 35SO4. (E) Purified GAGs from corresponding fractions following a 48- hour chase. (F) Purified HS from the fractions shown in panel E. Mean ±​ SD. N =​ 2 animals per genotype, 2 wells per animal. Results were confirmed on a separate cohort of N =​ 2 animals per genotype. www.nature.com/scientificreports/ www.nature.com/scientificreports/ considered as hallmarks of the later neurodegenerative phase of the disease. The delay in primary and secondary pathology likely reflects the progressive nature of neurological deterioration in the aging MPS IIIA brain. p gy yl p g g g g Studies to evaluate alterations in synaptic function were performed primarily in the somatosensory cortex, which is a region of the brain involved in processing sensory information. A striking increase in the number of PSD-95 positive puncta was observed at 21 days of age prior to astrocytosis and ganglioside accumulation. In the normal brain, PSD-95 localizes to dendritic spine heads. However, the number of dendritic spines on pyramidal neurons in layer II/III did not differ from normal littermates and no change in mEPSC inter-event interval was observed. Together these data indicate that the enhancement in PSD-95 puncta does not reflect an increase in synapse number. Given the degree of HS accumulation in the 21-day old mouse brain, the absence of substantial alterations in synapse number is surprising and suggests that the establishment of the appropriate number of synaptic connections can occur even in the presence of lysosomal storage. y p p y g We observed a modest but consistent reduction in mEPSC amplitude. Reduced mEPSC amplitude was unex- pectedly accompanied by enhanced dendritic spine width, suggesting that AMPA receptor function was reduced. Free HS chains are sufficient to alter AMPA receptor subunit levels at the cell surface based on the impact of add- ing HS isolated from the cerebral cortex to rat cortical neurons. The elevated level of glypican 4 and cell surface HS in MPS IIIA astrocytes support that astrocyte HS may be the primary source of HS that drives changes in post- synaptic function in MPS IIIA. Allen et al. recently showed that the addition of HS proteoglycans, glypicans 4 or 6, to retinal ganglion cells increases mEPSC amplitude and promotes the formation of excitatory synapses, whereas deletion of glypican 4 reduces mEPSC amplitude7. Irie et al. reported that deletion of the HS co-polymerase Ext1 in post-mitotic neurons led to a reduction in plasma membrane levels of glutamate AMPA receptors, specifically GluA2, and a modest but significant reduction in mEPSC amplitude9. The sensitivity of this receptor system to both elevated and reduced HS levels is consistent with the bell shaped response curves reported by Allen and colleagues7. www.nature.com/scientificreports/ Glypican 4 may be just one of several HSPGs that accumulate in MPS III, based on the observation that MPS IIIB mice have elevated levels of glypican 1 in the medial entorhinal cortex28. g yp Sambri and colleagues recently reported lysosomal impairment in 10-month-old MPS IIIA mice causes a loss of presynaptic function in the hippocampus by inhibiting synaptic vesicle trafficking29. Thus, we suggest that different forms of synaptic impairment likely cause changes in neurological function in the developing and aged MPSIIIA brain, consistent with progressive neurological deterioration associated with this disorder. Future studies to develop therapeutic treatments will benefit from this enhanced understanding of the disease process.f p pi g p Only limited information is available on the contribution of different neural cell types to disease pathology in MPS III30,31. Towards this goal, we examined a new mouse model carrying a conditional allele of Sgsh, driving deletion of Sgsh in specific cell types by intercrossing Sgshf/f mice to animals expressing Cre recombinase in differ- ent neural cell types, including neurons, endothelial cells, and astrocytes. The lack of NRE biomarker accumula- tion in conditional knockout mice shows that cross-correction by secreted sulfamidase apparently occurs across different cell types in the brain. Lower, but significant levels of biomarker were observed in Sgshf/fNestin-Cre mice, suggesting that a non-neuroepithelial cell type (microglia, meningeal fibroblasts, and ependymal cells) may also participate in cross-correction in the brain. p p In summary, we show for the first time that MPS IIIA has a neurodevelopmental component most likely induced by accumulation of pathological forms of HS. Further studies are warranted to explore the relationship of HS content and composition in synaptic biology not only in MPS disorders, but also in other neurodevelopmental and neurodegenerative disorders that have shared characteristics with MPS III32. Discussion (G) Western blot for glypican 4 in cell lysates of cultured primary astrocytes. Buffer control (−​), heparin lyase treated (+​). Figure 5. HS alters AMPA receptor subunit GluA2 recruitment to the cell surface. (A–C) Mat Figure 5. HS alters AMPA receptor subunit GluA2 recruitment to the cell surface. (A–C) Mature wildtype rat cortical neurons were treated for 72 hours with different brain equivalents of HS purified from the cerebral cortex of unaffected and hypomorphic MPS IIIA mice. Note that the mutant brain contains twice the amount of HS as the unaffected mice. (A) Western blots for GluA2 AMPA receptor subunit shows cell surface GluA2 levels are enhanced following addition of HS, while total GluA2 levels are unchanged. (B) Quantification of surface GluA2 levels normalized to total GluA2 from panel A. Mean ±​ SEM. (C) Quantification of surface GluA1 levels normalized to total GluA1 for the same samples in (B). Mean ±​ SEM. N =​ 3–6 wells per concentration from N =​ 3 experiments. (B,C) Horizontal line with grey shading shows control levels in untreated samples as mean ±​ SEM used for statistical comparison. (D) Purified GAGs from intracellular, cell surface, and media fractions from primary astrocyte cultures from unaffected (Sgsh+/h) and hypomorphic MPS IIIA (Sgshh/h) mice following a 48-hour radiolabeling with 35SO4. (E) Purified GAGs from corresponding fractions following a 48- hour chase. (F) Purified HS from the fractions shown in panel E. Mean ±​ SD. N =​ 2 animals per genotype, 2 wells per animal. Results were confirmed on a separate cohort of N =​ 2 animals per genotype. (G) Western blot for glypican 4 in cell lysates of cultured primary astrocytes. Buffer control (−​), heparin lyase treated (+​). cortical neurons and astrocytes. Other secondary markers of disease, including GM326, CS/DS27, 2-O-sulfated HS derived disaccharides15, and astrocytosis are not present in the developing MPS IIIA brain, and should be SCIeNTIfIC REPortS | 7:46576 | DOI: 10.1038/srep46576 7 www.nature.com/scientificreports/ i For staining of PSD-95 in tissue sections, PBS perfused whole brains were dissected and cut down the mid- line. The right hemisphere was flash frozen in OCT in a bath of isopentane that had been pre-cooled in liquid nitrogen. Tissue was transferred to dry ice and frozen at −​80 °C for a minimum of one day. Sagittal sections (12 μ​ m) were cut using a cryostat and mounted directly onto glass slides. Glass slides were fixed for 8 min at −​20 °C in methanol, and then washed and fixed for 4 min in 4% PB-PFA at room temperature. Sections were stained with an anti-PSD95 antibody (LifeTech 51-6900) as previously described34.i Species-specific Alexa Fluor-conjugated secondary antibodies were used to visualize primary antibody stain- ing. Confocal images were collected on a Nikon Ti microscope equipped with the Nikon A1R confocal system using a high NA 63X oil objective. The ND acquisition tool was used to acquire Z stacks in three channels. Quantification of PSD95 was performed using the puncta analyzer ImageJ plugin on 5 μ​m max projection confo- cal images constructed from z-stacks. Data from Sgshh/h sections were normalized to unaffected littermates that were sectioned and stained at the same time. N =​ 5–6 animals per genotype, 2 sections per animal, 2 images per section, 2 regions of interest per image. Golgi impregnation and dendritic spine analysis and quantification were performed exactly as described35. Western blot analysis. SDS-PAGE gels were cast manually. After electrophoresis of samples, bands were transferred to either nitrocellulose or PVDF membrane. Western blotting was performed as previously described36. Glycosaminoglycan Purification. Mice were heavily anesthetized using a ketamine/xylazine mixture, and transcardial perfusion with Dulbecco’s PBS (dPBS) was performed. Whole brains were removed and dissected into anatomical sub regions, when specified. Organs were homogenized in ice-cold buffer containing 50 mM sodium acetate (pH 6.0) and 0.2 M sodium chloride. The tissue was dissociated with a Polytron homogenizer, and the resulting homogenates were digested overnight at 37 °C with 0.1 mg/ml Pronase (type XIV from Streptomyces griseus, Sigma Aldrich) containing 0.1% Triton X-100. Tissue homogenates were filtered through 0.45 μ​m syringe filters or centrifuged at 10,000×​ g for 10 min to remove insoluble debris. Glycosaminoglycans were purified from the supernates by anion exchange chromatography as described previously37. Non-reducing end and glycosaminoglycan analysis. The disaccharides resulting from enzymatic depolymerization were dried and tagged with [12C6] aniline as described23. www.nature.com/scientificreports/ prior to decapitation and brains were incubated at 4 °C in modified artificial cerebrospinal fluid (ACSF) contain- ing sucrose (83 mM NaCl, 2.5 mM KCl, 1 mM NaH2PO4, 26.2 mM NaHCO3, 22 mM glucose, 72 mM sucrose, 0.5 mM CaCl2, and 3.3 mM MgSO4). Coronal slices were prepared (350 μ​m) using a Leica VT1200 vibratome and recovered in standard ACSF (119 mM NaCl, 5 mM KCl, 1 mM NaH2PO4, 26 mM NaHCO3, 11 mM glucose, 2 mM CaCl2, and 1 mM MgSO4). The slices were incubated at 34 °C for 30 min and at room temperature prior to recordings. Slices were transferred to a submerged recording chamber and perfused with 30 °C oxygenated ACSF (containing 0.1 mM picrotoxin). For miniature excitatory postsynaptic current (mEPSC) recordings, 1 μ​M tetro- dotoxin (TTX) was included in the circulating ACSF. g For mEPSC recordings, neurons displaying pyramidal-like morphology in layer II/III of primary somatosen- sory cortex were whole-cell voltage-clamped at −​70 mV with glass pipettes of 2.5–3.5 MΩ​ resistance filled with a cesium-based internal solution (10 mM CsCl, 105 mM CsCH3SO3, 0.5 mM ATP, 0.3 mM GTP, 10 mM HEPES, 5 mM glucose, 2 mM MgCl2, and 1 mM EGTA, pH 7.2). mEPSC amplitude and inter-event interval were ana- lyzed from 80–150 events per cell using Clampfit 10.3 and differences were determined using unpaired t-test in GraphPad Prism. Immunohistochemistry. Mice of various ages were heavily anesthetized using a mixture of ketamine/xyla- zine and transcardially perfused dPBS followed by 4% phosphate-buffered paraformaldehyde (PB-PFA). Tissues were post-fixed overnight at 4 °C and cryo-protected by sinking in 30% PB-sucrose at 4 °C and cut on a cry- ostat. Coronal sections (40 μ​m) were collected and stained as free-floating sections. Primary antibodies used were LAMP-1 (1D4B, Developmental Studies Hybridoma Bank, University of Iowa), glial fibrillary acidic protein (GFAP) (Sigma, G3893), and ganglioside GM3 (Cosmo Bio Co., NBT-M101). Confocal images were collected on a Nikon Ti microscope equipped with the Nikon A1R confocal system using a high NA 63X oil, 10X, or 20X objectives. The ND acquisition tool was used to acquire Z stacks in three channels. Quantification of LAMP-1 ves- icle size was performed on high magnification max projection Z-stacks that span 4 μ​m in depth using the puncta analyzer Image J plugin. Quantification of area stained by GFAP and GM3 was performed on images set to the same threshold and area quantification was performed using Image J. Materials and Methods Mouse models. Hypomorphic MPS IIIA mice were purchased from Jackson Laboratory (B6. Cg-Sgshmps3a/PstJ)17. These mice carry a homozygous missense mutation in the Sgsh gene and exhibit <​5% residual activity of sulfamidase. A mouse model of MPS IIIA bearing a conditional “floxed” allele of Sgsh was obtained from the Knockout Mouse Project Repository (Sgshtm1a(KOMP)Wtsi). The Sgsh “knockout first” allele was converted to a conditional allele via flippase-mediated recombination, which exposed two lox-P sequences flank- ing exons 3 through 6 of Sgsh. Subsequent crossing to animals bearing germ-line expression of bacterial Cre recombinase eliminated these 4 exons and induced a frame-shift mutation at the junction between exons 2 and 7 and a premature stop codon, thus abolishing enzyme function and/or synthesis of the enzyme. Sgshf/f mice were crossed to Nestin-cre mice (Jackson Laboratory, Tg(Nes-cre)2Wme), Synapsin1-cre (Jackson Laboratory, B6. Cg-Tg(Syn1-cre)671Jxm/J), and a GFAP-cre line (generated by the Messing Laboratory and provided by Don Cleveland, UCSD33. To generate a mouse line bearing a null allele of Sgsh, Sgshf/+ mice were crossed to EIIA- cre mice purchased from Jackson Laboratory (Bg.FVB-Tg(EIIa-cre)C5379Lmgd/L). Female offspring of genotype Sgshf/+EIIA-cre+​ were crossed to wildtype C57BL/6 h mice to obtain germline transmission of the null Sgsh allele and to remove the cre transgene. Heterozygous offspring were bred to produce wild type, Sgsh+/− and Sgsh−/− off- spring. Supplemental Table S2 contains primer sequences for genotyping.i g g y g Plug discovery was counted as embryonic day 0 (E0) and the date of birth was defined as postnatal day 0 (P0). Male and female mutant mice were analyzed and gender was carefully monitored in all experiments. Unaffected littermate animals of the same gender were analyzed to control for inter-litter and male/female variation. In all experiments gender did not systematically vary. Data collected in experiments employing different experimental techniques was generated using separate cohorts of mice. Animals were housed and bred in vivaria approved by the Association for Assessment and Accreditation of Laboratory Animal Care located in the School of Medicine, UCSD. All experiments were performed in accordance with relevant guidelines and regulations following stand- ards and procedures approved by the UCSD Institutional Animal Care and Use Committee. Slice Electrophysiology. Acute hippocampal and cortical slices were prepared from 21–22 day old Sgsh+/h and Sgshh/h mice with the experimenter blinded to genotype. Mice were deeply anesthetized with isofluorane 8 SCIeNTIfIC REPortS | 7:46576 | DOI: 10.1038/srep46576 www.nature.com/scientificreports/ www.nature.com/scientificreports/ Each sample was mixed with a known amount of [13C6] aniline tagged standards, including N-sulfoglucosamine, the primary saturated non-reducing end biomarker for MPS IIIA (Carbosynth). Samples were analyzed by liquid chromatography-mass spectrometry (LC/MS) using an LTQ Orbitrap Discovery electrospray ionization mass spectrometer (Thermo Scientific) as described22. Internal disaccharides and the non-reducing end monosaccharide were quantified manually based on their unique mass signatures relative to internal standards and normalized by either tissue wet weight or pro- tein concentration, determined by BCA assay (Pierce). Data is presented using a disaccharide structural code38. Primary Neuron and Astrocyte Isolation. Mixed cortical cultures were prepared from single newborn MPS IIIA pups as described39. Cells were cultured for 21 days prior to NRE and morphological analyses. Enriched cultures of primary cortical neurons were prepared from single mouse embryos (E15.5) as described40. To select for post-mitotic neurons, 5 μ​M cytosine arabinoside (AraC) was added at 1 day and removed with a medium change at 3 days. cultures of primary cortical neurons were prepared from single mouse embryos (E15.5) as described . To select for post-mitotic neurons, 5 μ​M cytosine arabinoside (AraC) was added at 1 day and removed with a medium change at 3 days. Astrocyte cultures were obtained from the cortex of single newborn mice as described41. For NRE analysis, cells cultured to maturation (18–21 days) were treated with 0.05% trypsin-EDTA to remove cell surface HSPGs. g y Astrocyte cultures were obtained from the cortex of single newborn mice as described41. For NRE anal cells cultured to maturation (18–21 days) were treated with 0.05% trypsin-EDTA to remove cell surface HSP SCIeNTIfIC REPortS | 7:46576 | DOI: 10.1038/srep46576 9 www.nature.com/scientificreports/ Intact cells containing intracellular storage material were then collected by centrifugation. RNA was isolated from enriched cultures of wild type or heterozygous neurons and astrocytes and Sgsh expression was assessed by qRT-PCR (primer sequences, Table S2). Intact cells containing intracellular storage material were then collected by centrifugation. RNA was isolated from enriched cultures of wild type or heterozygous neurons and astrocytes and Sgsh expression was assessed by qRT-PCR (primer sequences, Table S2). Data quantification and statistical analyses. In all cases a researcher blinded to genotype performed image quantification. www.nature.com/scientificreports/ Statistical analyses were performed using GraphPad Prism software Synaptic analyses are presented as average values ±​ SEM to take into account the large number of samples and size of datasets, whereas other data is presented as average values ±​ SD. *p <​ 0.05, **p <​ 0.01, ***p <​ 0.001. p g p p p Additional details for experimental procedures including mouse breeding scheme, immunohistochemistry, glycosaminoglycan purifications, primary cultures for neuron and astrocyte enrichment, slice electrophysiology for field recordings, Western blot analysis, evaluation of surface AMPA receptor levels, and metabolic label/chase experiments can be found in Supplementary Information under Materials and Methods. References 1. Xu, D. & Esko, J. D. Demystifying heparan sulfate-protein interactions. Annu Rev Biochem 83, 129–157 (2014). 1. Xu, D. & Esko, J. D. Demystifying heparan sulfate-protein interactions. Annu Rev Biochem 83, 129–157 (2014). 1. Xu, D. & Esko, J. D. Demystifying heparan sulfate-protein interactions. Annu Rev Biochem 83, 129–157 (2014). b l b l h l d f l d f l k h lf d f l 2. Grobe, K. et al. Cerebral hypoplasia and craniofacial defects in mice lacking heparan sulfate Ndst1 gene function. Development 132 3777–3786 (2005). 3. Inatani, M., Irie, F., Plump, A. S., Tessier-Lavigne, M. & Yamaguchi, Y. Mammalian brain morphogenesis and midline axon guidance require heparan sulfate. Science 302, 1044–1046 (2003).ii q p 4. McLaughlin, D. et al. Specific modification of heparan sulphate is required for normal cerebral cortical development. Mech Dev 120, 1481–1488 (2003). 5. Matsumoto, Y., Irie, F., Inatani, M., Tessier-Lavigne, M. & Yamaguchi, Y. Netrin-1/DCC signaling in commissural axon guidance requires cell-autonomous expression of heparan sulfate. J Neurosci 27, 4342–4350 (2007). 6. Pratt, T., Conway, C. D., Tian, N. M., Price, D. J. & Mason, J. O. Heparan sulphation patterns generated by specific heparan sulfotransferase enzymes direct distinct aspects of retinal axon guidance at the optic chiasm. J Neurosci 26, 6911–6923 (2006). 7 All N J t l A t t l pi 4 d 6 p t f ti f it t p i Gl A1 AMPA pt N t 486 6. Pratt, T., Conway, C. D., Tian, N. M., Price, D. J. & Mason, J. O. Heparan sulphation patterns generated by specific heparan sulfotransferase enzymes direct distinct aspects of retinal axon guidance at the optic chiasm. J Neurosci 26, 6911–6923 (2006). 7. Allen, N. J. et al. Astrocyte glypicans 4 and 6 promote formation of excitatory synapses via GluA1 AMPA receptors. Nature 486, 410–414 (2012). sulfotransferase enzymes direct distinct aspects of retinal axon guidance at the optic chiasm. J Neurosci 26, 6911 6923 (2006). 7. Allen, N. J. et al. Astrocyte glypicans 4 and 6 promote formation of excitatory synapses via GluA1 AMPA receptors. Nature 486, 410–414 (2012). 8. Ko, J. S. et al. PTPsigma functions as a presynaptic receptor for the glypican-4/LRRTM4 complex and is essential for excitatory synaptic transmission. Proc Natl Acad Sci USA 112, 1874–1879 (2015).i 9. Irie, F., Badie-Mahdavi, H. & Yamaguchi, Y. Autism-like socio-communicative deficits and stereotypies in mice lacking heparan sulfate. References Proc Natl Acad Sci USA 109, 5052–5056 (2012). 10. Neufeld, E. F. & Muenzer, J. In Metabolic and Molecular Basis of Inherited Disease Vol. 3 (eds Scriver, C. R. et al.) Ch. 136, 3421–345 (MacGraw-Hill, 2001). 1. Shapiro, E. G. et al. A prospective natural history study of mucopolysaccharidosis Type IIIA. J Pediatr 170, 278–287 (2016).i p p p y y p y yp 12. Wijburg, F. A., Wegrzyn, G., Burton, B. K. & Tylki-Szymanska, A. Mucopolysaccharidosis type III (Sanfilippo syndrome) and misdiagnosis of idiopathic developmental delay, attention deficit/hyperactivity disorder or autism spectrum disorder. Acta Paediatr 102, 462–470 (2013). 13. Kowalewski, B. et al. Arylsulfatase G inactivation causes loss of heparan sulfate 3-O-sulfatase activity and mucopolysaccharido mice. Proc Natl Acad Sci USA 109, 10310–10315 (2012). 14. Vitry, S. et al. Enhanced degradation of synaptophysin by the proteasome in mucopolysaccharidosis type IIIB. Mol Cell Neuros 8–18 (2009). ( ) 15. Wilkinson, F. L. et al. Neuropathology in mouse models of Mucopolysaccharidosis Type I, IIIA and IIIB. PLoS ONE 7, e3 (2012). 6. Bhattacharyya, R., Gliddon, B., Beccari, T., Hopwood, J. J. & Stanley, P. A novel missense mutation in lysosomal sulfamidase is the basis of MPS III A in a spontaneous mouse mutant. Glycobiology 11, 99–103 (2001).i p y gy 17. Bhaumik, M. et al. A mouse model for mucopolysaccharidosis type III A (Sanfilippo syndrome). Glycobiology 9, 1389–1396 (1999). p y gy 7. Bhaumik, M. et al. A mouse model for mucopolysaccharidosis type III A (Sanfilippo syndrome). Glycobiology 9, 1389–1396 (1999) i 8. Hemsley, K. M. & Hopwood, J. J. Development of motor deficits in a murine model of mucopolysaccharidosis type IIIA (MPS-IIIA) Behav Brain Res 158, 191–199 (2005). 19. Lau, A. A., Crawley, A. C., Hopwood, J. J. & Hemsley, K. M. Open field locomotor activity and anxiety-related behaviors in mucopolysaccharidosis type IIIA mice. Behav Brain Res 191, 130–136 (2008). 20. Adrian, M. et al. Barriers in the brain: resolving dendritic spine morphology and compartmentalization. Front Neuroanat 8 (2014). ( ) 21. Lawrence, R. et al. Glycan-based biomarkers for mucopolysaccharidoses. Mol Genet Metab 111, 73–83 (2014).i y p y 22. Lawrence, R. et al. Disease-specific non-reducing end carbohydrate biomarkers for mucopolysaccharidoses. Nat Chem Biol 8, 197–204 (2012).fi 3. Lawrence, R. et al. Evolutionary differences in glycosaminoglycan fine structure detected by quantitative glycan reductive isotope labeling. J Biol Chem 283, 33674–33684 (2008). 24. Zhang, Y. et al. 5. Risher, W. C., Ustunkaya, T., Singh Alvarado, J. & Eroglu, C. Rapid Golgi analysis method for efficient and unbiased classification o dendritic spines. PLoS One 9, e107591 (2014). Acknowledgementsh g This work was supported in part by T32 training grant, NIH-T32 HL086344-08 (to C.A.D.), a grant from the National MPS Society (to J.D.E.) and a grant from the Cure Sanfilippo Syndrome Foundation (to J.D.E.), NIH- NINDS R01 NS089791 (to N.J.A.). We thank Elizabeth Neufeld for many helpful comments. References & Schuman, E. M. Ubiquitin-mediated proteasome activity is required for agonist-induced endocytosis of GluRs. Curr Biol 13, 2073–2081 (2003). y 0. Dwyer, C. A., Katoh, T., Tiemeyer, M. & Matthews, R. T. Neurons and glia modify receptor protein-tyrosine phosphatase zeta (RPTPzeta)/phosphacan with cell-specific O-mannosyl glycans in the developing brain. J Biol Chem 290, 10256–10273 (2015). ( ) p p pi y g y p g J , ( ) 1. Mecha, M. et al. An easy and fast way to obtain a high number of glial cells from rat cerebral tissue: A beginners approach. Protoco Exchange, 10.1038/protex.2011.218 (2011). Author Contributions Designed and performed research, analyzed data, wrote the paper (C.A.D.), performed electrophysiology experiments, analyzed data, wrote respective sections of the paper (S.L.S.), performed research and analysis of data (Y.L.; D.P; L.E.D.), guidance with research design regarding synaptic analysis, technical advice (N.J.A.), design of research, analyzed data electrophysiology experiments (G.N.P.). Designed research, analyzed data, wrote the paper (J.D.E.). References An RNA-sequencing transcriptome and splicing database of glia, neurons, and vascular cells of the cerebral cortex. J Neurosci 34, 11929–11947 (2014). 25. Del Monte, M. A., Maumenee, I. H. & Edwards, R. B. Glycosaminoglycan degradation by cultured retinal pigment epithelium from patients with retinitis pigmentosa. Curr Eye Res 10, 241–248 (1991).ti p p g y 6. Dawson, G., Fuller, M., Helmsley, K. M. & Hopwood, J. J. Abnormal gangliosides are localized in lipid rafts in Sanfilippo (MPS3a mouse brain. Neurochem Res 37, 1372–1380 (2012).i 27. Lamanna, W. C., Lawrence, R., Sarrazin, S. & Esko, J. D. Secondary storage of dermatan sulfate in Sanfilippo disease. J Biol Chem 286, 6955–6962 (2011).i 28. Ohmi, K., Zhao, H. Z. & Neufeld, E. F. Defects in the medial entorhinal cortex and dentate gyrus in the mouse model of Sanfilippo syndrome type B. PLoS One 6, e27461 (2011). 9. Sambri, I. et al. Lysosomal dysfunction disrupts presynaptic maintenance and restoration of presynaptic function prevent neurodegeneration in lysosomal storage diseases. EMBO Mol Med (2016). 30. Bruyere, J. et al. Heparan sulfate saccharides modify focal adhesions: implication in mucopolysaccharidosis neuropathophysiology. J Mol Biol 427, 775–791 (2015). 31. Garbuzova-Davis, S. et al. Blood-brain barrier impairment in an animal model of MPS III B. PLoS One 6, e16601 (2011). p 32. Dwyer, C. A. & Esko, J. D. Glycan susceptibility factors in autism spectrum disorders. Mol Aspects Med (2016). . Dwyer, C. A. & Esko, J. D. Glycan susceptibility factors in autism y , , J y p y p p ( ) 33. Zhuo, L. et al. hGFAP-cre transgenic mice for manipulation of glial and neuronal function in vivo. Genesis 31, 85–94 (2001). 34. Ippolito, D. M. & Eroglu, C. Quantifying synapses: an immunocytochemistry-based assay to quantify synapse number. J Vis Exp (2010).fii SCIeNTIfIC REPortS | 7:46576 | DOI: 10.1038/srep46576 10 www.nature.com/scientificreports/ 36. Dwyer, C. A., Baker, E., Hu, H. & Matthews, R. T. RPTPzeta/phosphacan is abnormally glycosylated in a model of muscle-eye-b disease lacking functional POMGnT1. Neuroscience 220, 47–61 (2012). 7. Esko, J. D. In Current protocols in molecular biology (eds Ausubel, F. et al.) 17.12.11–17.12.19 (Greene Publishing and Wiley Interscience, 1993). 8. Lawrence, R., Lu, H., Rosenberg, R. D., Esko, J. D. & Zhang, L. Disaccharide structure code for the easy representation of constituen oligosaccharides from glycosaminoglycans. Nat Methods 5, 291–292 (2008). g g y g y 9. Patrick, G. N., Bingol, B., Weld, H. A. SCIeNTIfIC REPortS | 7:46576 | DOI: 10.1038/srep46576 © The Author(s) 2017 Additional Information Supplementary information accompanies this paper at http://www.nature.com/srep Supplementary information accompanies this paper at http://www.nature.com/srep Competing Interests: The authors declare no competing financial interests. Competing Interests: The authors declare no competing financial interests. How to cite this article: Dwyer, C. A. et al. Neurodevelopmental Changes in Excitatory Synaptic Structure and Function in the Cerebral Cortex of Sanfilippo Syndrome IIIA Mice. Sci. Rep. 7, 46576; doi: 10.1038/srep46576 (2017). How to cite this article: Dwyer, C. A. et al. Neurodevelopmental Changes in Excitatory Synaptic Structure and Function in the Cerebral Cortex of Sanfilippo Syndrome IIIA Mice. Sci. Rep. 7, 46576; doi: 10.1038/srep46576 (2017). 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. This work is licensed under a Creative Commons Attribution 4.0 International License. The images or other third party material in this article are included in the article’s Creative Commons license, unless indicated otherwise in the credit line; if the material is not included under the Creative Commons license, users will need to obtain permission from the license holder to reproduce the material. To view a copy of this license, visit http://creativecommons.org/licenses/by/4.0/ SCIeNTIfIC REPortS | 7:46576 | DOI: 10.1038/srep46576 11
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Analytic Pragmatism and Universal LX Vocabulary
Philosophia
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1 Department of Philosophy, The Ohio State University, Columbus, OH, USA 2 Department of Philosophy; Arché Philosophical Research Centre; Centre for Exoplanet Science, University of Saint Andrews, St Andrews, Fife, UK Philosophia (2017) 45:1803–1827 DOI 10.1007/s11406-017-9866-4 Analytic Pragmatism and Universal LX Vocabulary Richard Samuels1 & Kevin Scharp2 Received: 6 June 2017 /Accepted: 16 June 2017 / Published online: 24 August 2017 # The Author(s) 2017. This article is an open access publication Received: 6 June 2017 /Accepted: 16 June 2017 / Published online: 24 August 2017 # The Author(s) 2017. This article is an open access publication # The Author(s) 2017. This article is an open access publication Abstract In his recent John Locke Lectures – published as Between Saying and Doing – Brandom extends and refines his views on the nature of language and philosophy by developing a position that he calls Analytic Pragmatism. Although Brandom’s project bears on an extraordinarily rich array of different philosophical issues, we focus here on the contention that certain vocabularies have a privileged status within our linguistic practices, and that when adequately understood, the practices in which these vocabu- laries figure can help furnish us with an account of semantic intentionality. Brandom’s claim is that such vocabularies are privileged because they are a species of what he calls universal LX vocabulary –roughly, vocabulary whose mastery is implicit in any linguistic practice whatsoever. We show that, contrary to Brandom’s claim, logical vocabulary per se fails to satisfy the conditions that must be met for something to count as universal LX vocabulary. Further, we show that exactly analogous considerations undermine his claim that modal vocabulary is universal LX. If our arguments are sound, then, contrary to what Brandom maintains, intentionality cannot be explicated as a Bpragmatically mediated semantic phenomenon^, at any rate not of the sort that he proposes. Keywords Pragmatism.Conceptualanalysis.Logic.Conditionals.LogicalConstants. Intentionality * Kevin Scharp kscharp@gmail.com 1 Introduction Over the past three decades or so, Robert Brandom has exerted an enormous influence on debates regarding the nature of language and philosophy. In his recent John Locke * Kevin Scharp kscharp@gmail.com * Kevin Scharp kscharp@gmail.com 1804 Philosophia (2017) 45:1803–1827 Lectures – published as Between Saying and Doing – Brandom further extends and refines his views on these matters by seeking to develop and defend a position that he calls Analytic Pragmatism. As we will see, this position combines two commitments that are exceedingly widely endorsed amongst analytic philosophers; and a careful discussion of the resulting view is of importance if for no other reason than, to date, Brandom’s position constitutes the most detailed and systematic endeavor to develop such a view. Although Brandom’s project bears on an extraordinarily rich array of different philo- sophical issues, we focus here on a single central strand that runs throughout, and without which the project as a whole fails. In brief, the strand in question concerns the contention that certain vocabularies have a privileged status within our linguistic practices, and that when adequately understood, the practices in which these vocabularies figure can help furnish us with an account of semantic intentionality. More specifically, we focus on Brandom’s discussion of two vocabularies that have long figured prominently in analytic philosophy: logical vocabulary – especially the conditional – and modal vocabulary, such as that used to express necessity and possibility.1 Brandom’s claim is that such vocabularies are privileged because they are a species of what he calls universal LX vocabulary –roughly, vocabulary whose mastery is implicit in any linguistic practice whatsoever. On such a view, then, logical and modal vocabularies are privileged because they possess a kind of transcendental status. Further, according to Brandom, this transcendental status, at least in the case of modal vocabulary, is important to characterizing the sort of intentionality essential to thought and language. This paper has three main aims. First, we show that logical vocabulary per se fails to satisfy the conditions that must be met for something to count as universal LX vocabulary. We focus in particular on the case of the conditional, which Brandom takes to be a paradigmatic example of logical vocabulary. If our argument goes though, then the conditional is not universal LX, and Brandom’s efforts to defend the privileged status of logic per se fail. 1 Following Brandom, we are interested here in aleithic modality as opposed to epistemic modality. 2 Brandom (2008: 189). 2 Brandom (2008: 189). 1 Introduction Second, we show that exactly analogous considerations undermine the claim that modal vocabulary is universal LX. If this is so, then another of Brandom’s central claims is undermined – what he calls the modal Kant-Sellars thesis – the thesis that the use of ordinary, empirical vocabulary presupposes those capacities required for the introduction and deployment of modal vocabulary. Finally, we argue briefly that given our criticism of the modal Kant-Sellars thesis, Brandom’s conception of intentionality, which he articulates in the final pages of his Locke Lectures, is also untenable. If our arguments are sound, then, contrary to what Brandom maintains, intentionality cannot be explicated as a Bpragmatically mediated semantic phenomenon^,2 at any rate not of the sort that he proposes. Here is how we proceed. In Section 1, we provide an overview of Brandom’s Analytic Pragmatism. In Section 2, we focus on the notion of universal LX vocabulary, and both explain what it is and why is it so important to Brandom’s overarching project. In Section 3, we set out Brandom’s case for the conditional being universal LX. Then in Section 4, we present our criticisms of this case; and in Section 5, we turn our attention to modal vocabulary and argue that considerations exactly analogous to those discussed in Section 4 also undermine the modal Kant-Sellars thesis. Finally, in Section 6, we 1805 Philosophia (2017) 45:1803–1827 draw out the implications of the forgoing discussion for Brandom’s account of intentionality. draw out the implications of the forgoing discussion for Brandom’s account of intentionality. 3 Brandom (2008: 1). 2 Analytic Pragmatism Brandom’s overall project – what he calls Analytic Pragmatism – can be thought of as a consequence of endorsing two commitments. The first is a methodological commit- ment, which he takes to be central to traditional analytic philosophy: The Analytic Project: A central goal of philosophy ought to be to establish semantic relations between vocabularies – kinds of linguistic expressions – in order to Bmake sense of the meanings expressed by one kind of locution in terms of the meanings expressed by another kind of locution^.3 Paradigmatically, these semantic relations include translation, paraphrase, a priori entailment, and truth- making. The history of analytic philosophy is, of course, littered with attempts to produce such analyses —including, the reduction of arithmetic to logic, the translation of psychological vocabulary into physical vocabulary, and the analysis of moral vocabu- lary in non-moral terms. By endorsing this commitment, then, Brandom locates his position as continuous with the project of traditional analytic philosophy. Brandom’s second central commitment is a general thesis about the metaphysics of meaning: Semantic Pragmatism: The meaning of a linguistic expression is determined by its use in a linguistic practice. 4 Terminological note: Brandom tends variously to use the expressions ‘practice’, ‘activity’, ‘ability’, ‘exercise of a practice’, and ‘practice-or-ability’ more-or-less interchangeably; see Brandom (2008: 9). For simplicity and ease of exposition we just use the term ‘practice’. Semantic Pragmatism: The meaning of a linguistic expression is determined by its use in a linguistic practice. This sort of meaning pragmatism has also been prominent in the history of analytic philosophy. Though we suspect that many proponents of the analytic project have also endorsed Semantic Pragmatism, far from being a natural complement to the Analytic Project, such pragmatism poses, as Brandom notes, a serious challenge to it. The challenge takes a variety of forms, but the rough idea is this: If our practices determine the meanings of linguistic expressions, then it is highly implausible to suppose that expressions comprising different vocabularies – and, hence, having different meaning- determining practices – will tend to bear systematic semantic relations to each other, such as translation, reduction and paraphrase. Thus by endorsing both the Analytic Project and Semantic Pragmatism, Brandom seeks to combine a pair of commitments that seem to be in tension with each other. As a consequence, a central challenge for Brandom – and one that exerts an enormous influence on the overall shape of his Analytic Pragmatism – is to reconcile this apparent tension: to show that, contrary to appearances, one can pursue the goal of philosophical analysis at the same time as cleaving to Semantic Pragmatism. 3 Brandom (2008: 1). 1806 Philosophia (2017) 45:1803–1827 How is this to be done? Brandom’s solution is to propose that Semantic Pragmatism requires that we rethink the fundamental semantic relations between the analyzed and analyzing vocabularies in philosophical analyses. Specifically, he proposes that if linguistic meaning is determined by use, then the relationship between distinct vocab- ularies should be mediated by linguistic practices.4 Thus, for Brandom, the way to be a pragmatist and to pursue the Analytic Project is to take the analyzing vocabulary to describe those practices that are sufficient to deploy the target vocabulary. If one could make good on this suggestion, then semantic pragmatists could have their analytic cake, and eat it too. 3 Universal LX Vocabulary So far we have sketched the overarching shape of Brandom’s project. But why does the notion of universal LX vocabulary come to play such a central role? 3.1 Why Universal LX Vocabulary Matters 3.1 Why Universal LX Vocabulary Matters 3.1 Why Universal LX Vocabulary Matters There are two quite different roles that the notion of universal LX vocabulary plays in Brandom’s project. The first, exemplified by its application to logic, is to justify parts of the analyzing base that everyone must presuppose in order to pursue the Analytic Project. The second, exemplified by Brandom’s discussion of modality, is to justify or vindicate the use of philosophically contested notions. In both sorts of case, however, Brandom maintains that the response should be the same. Roughly, where some vocabulary – contested or otherwise – is universal LX, we get it for free since the capacity to master that vocabulary is a necessary condition for the possibility of any language-use whatsoever. Let us consider the cases of logical vocabulary and modality in a bit more detail. Logic One way to make vivid the potential significance of the notion of universal LX vocabulary for analytic philosophy is to note, as Brandom does, that within any project of analysis – classical or pragmatist – logical vocabulary has a privileged status. Roughly put, no matter what the analysandum, it is taken for granted that logical vocabulary must be part of the analyzing base. If, for example, one seeks to translate psychological vocabulary into physical vocabulary, it is not merely that one uses the substantive terms of physics; one also uses – and indeed must use – such logical resources as conjunction, disjunction, quantifiers, and of course the conditional. But if this is so, then as Brandom notes, some justification or explanation is required for why logical vocabulary should have this privileged status. The notion of universal LX vocabulary is central to Brandom’s effort to address this challenge. Logical vocabulary is supposed to be privileged because it is a species of universal LX vocabulary, and universal LX vocabulary is supposed to be privileged. 1807 Philosophia (2017) 45:1803–1827 Modality At least since Locke, modal vocabulary has been a point of contention within philosophy, especially amongst those of an empiricist bent.5 Suspicion of alethic modality traditionally comes in at least three forms. First, there are commonplace epistemic concerns regarding how we can come to know – or justifiably believe – modal claims. Second, there are familiar metaphysical concerns about the Bweirdness^ of an ontology that allows for modal truths that are not merely truths of language. 3.1 Why Universal LX Vocabulary Matters Finally, empiricists have long questioned the intelligibility of modal locutions —what claims to possibility or necessity might even mean. Despite these familiar concerns there has been, as Brandom notes, something of a sea change in philosophical attitudes towards modality in recent decades, in large measure due to developments in the formal semantics of modal logics. Nonetheless, Brandom argues – and we are inclined to agree – that such developments leave largely unaddressed the original philosophical challenges to modality. It is here that the notion of universal LX vocabulary is supposed to enter the picture. For if modal locutions are universal LX, then no matter what philosophical puzzle- ment accompanies them, they ought to be construed as holding a privileged and legitimate status. Modal vocabulary is supposed to be privileged because it is a species of universal LX vocabulary, and universal LX vocabulary is supposed to be privileged. 5 The same is, of course, true of normative vocabulary —a topic in which Brandom also invests much time in his Locke lectures. 3.2 What Universal LX Vocabulary is and Why It Matters We now need to say more about Brandom’s notion of universal LX vocabulary. Here is a first pass. Universal LX vocabulary is supposed to be a privileged class of linguistic expressions in that they satisfy the following two conditions: a) The vocabulary allows one to describe practices that are necessary for having mastery for any vocabulary whatsoever, and a) The vocabulary allows one to describe practices that are necessary for having mastery for any vocabulary whatsoever, and b) Once one masters the described practice, one can in principle deploy the very vocabulary used to describe it. b) Once one masters the described practice, one can in principle deploy the very vocabulary used to describe it. Crudely put, Universal LX vocabulary describes the necessary conditions for mastery of any linguistic expression whatsoever, including universal LX vocabulary itself. Crudely put, Universal LX vocabulary describes the necessary conditions for mastery of any linguistic expression whatsoever, including universal LX vocabulary itself. Even this rough formulation is a bit of a mouthful. But to understand clearly Brandom’s notion of universal LX vocabulary we need to explain several additional notions. First, we need to explain two notions of autonomy. The first is the notion of an autonomous vocabulary. To a first approximation: A vocabulary is autonomous if and only if one could use it even though one could use no other vocabulary whatsoever. Suppose, for example, that it was possible to master the language of arithmetic and nothing else. Then the language of arithmetic would be an autonomous vocabulary in the relevant sense. The second notion of autonomy – an autonomous practice – can be defined in terms of the previous notion. To a first approximation: an autonomous practice is any practice that is sufficient for mastery of an autonomous vocabulary. 1808 Philosophia (2017) 45:1803–1827 As we will soon see, the paired notions of an autonomous vocabulary and an autonomous practice are important because they are required in order to specify what is it for a LX vocabulary to be universal. But we are getting ahead of ourselves. Before explaining what makes an LX vocabulary universal, we first need to explain what makes a vocabulary LX, and this in turn requires that we characterize a range of what Brandom calls meaning-use relations. 3.2 What Universal LX Vocabulary is and Why It Matters Given the project of Analytic Pragmatism – to specify relations between vocabularies that are mediated by practices – Brandom appeals to a number of such relations: 1) PV-Sufficiency: Where one has mastery of any vocabulary – autonomous or otherwise – Brandom supposes that there must be some set of practices that suffices for mastery of that vocabulary. He calls this relation PV-sufficien- cy. Roughly: a practice is PV-sufficient for a vocabulary just in case one has mastery of the vocabulary, if one engages in that practice. 2) PV-Necessity: Now suppose that for some vocabulary there is a practice that one must engage in to count as having mastery of the vocabulary. Suppose, for example, that the capacity to identify the successor of any natural number is required for mastery of the language of arithmetic. Then this practice – identifying successors – would be PV-necessary for mastery of arithmetic vocabulary. 2) PV-Necessity: Now suppose that for some vocabulary there is a practice that one must engage in to count as having mastery of the vocabulary. Suppose, for example, that the capacity to identify the successor of any natural number is required for mastery of the language of arithmetic. Then this practice – identifying successors – would be PV-necessary for mastery of arithmetic vocabulary. 3) VP-Sufficiency: This relation between vocabularies and practices obtains when a practice can be specified in a particular vocabulary. That is, a vocabulary is VP- sufficient for a practice just in case that practice can be described using that vocabulary.6 4) PP-Sufficiency: The fourth relation that we consider here obtains between prac- tices. If, when one engages in a particular practice, one can in principle do everything needed to engage in some other practice, then the former is PP- sufficient for the latter. For example, perhaps being able to add any three numbers between 0 and 9 is PP-sufficient for being able to find the sum of any two numbers by the usual method of addition. In characterizing PP- sufficiency, Brandom tends to express a preference for using the framework of computational theory, but there is no reason to think that this is essential to the notion.7 5) PP-Necessity: The final relation we consider is also a relation between practices. One practice is PP-necessary for another if it is impossible to engage in the second without engaging in the first. 6 We discuss this relation in far greater detail in Section 4.1. 7 7 See Brandom (2008: ch. 2). (a) P2 is PV-necessary for V2, (b) P2 is PP-sufficient for P1, 7 See Brandom (2008: ch. 2). (d) V1 is VP-sufficient for P2. In effect, what this says is that V1 specifies a practice P2 that one must be able to master in order to be a competent user of another vocabulary V2 and, moreover, the practice P2 incorporates everything that is required, in principle, in order to engage in a practice that the use of V1 depends upon –i.e., P1. This set of conditions can be captured, perhaps more intuitively, in what Brandom calls a meaning-use diagram (see Fig. 1). Finally, we are in position to characterize the notion of universal LX vocabulary. A vocabulary is universal LX iff it is an LX vocabulary for any autonomous vocabulary. We can capture this in terms of an additional meaning- use relation: (e) P2 is PP-necessary for any autonomous practice – i.e. a practice that is PV- sufficient for an autonomous vocabulary. 3.2 What Universal LX Vocabulary is and Why It Matters For example, the practice of asserting is PP-necessary for the practice of asserting conditionals. 5) PP-Necessity: The final relation we consider is also a relation between practices. One practice is PP-necessary for another if it is impossible to engage in the second without engaging in the first. For example, the practice of asserting is PP-necessary for the practice of asserting conditionals. With this terminology in hand, we can now provide a more explicit formu- lation of LX vocabulary. A vocabulary V1 is LX for a vocabulary V2 iff there exists practices P1 and P2, such that: (a) P2 is PV-necessary for V2, (b) P2 is PP-sufficient for P1, 1809 Philosophia (2017) 45:1803–1827 (c) P1 is PV-sufficient for V1, (d) V1 is VP-sufficient for P2. (c) P1 is PV-sufficient for V1, (d) V1 is VP-sufficient for P2. (c) P1 is PV-sufficient for V1, (d) V1 is VP-sufficient for P2. 4 Logic as Universal LX: The Case of the Conditional It is time to consider how the notion of universal LX vocabulary is supposed to apply to Brandom’s paradigmatic example of a logical expression: the condi- tional. In order for the conditional to be universal LX, it must be LX for any autonomous vocabulary whatsoever. So, applying the definition from Section 2.2, let us assume that V2 is any arbitrary autonomous vocabulary, and V1 is the conditional. Moreover, let us follow Brandom as characterizing P1 as the practice of using the conditional, and P2 as the practice of inferring one claim from another. In which case, again following Brandom, there are five conditions—represented by the meaning-use diagram in Fig. 2—that need to be satisfied. Condition 1. According to the first condition: P2, the practice of inferring one claim from another, is PV-necessary for the autonomous vocabulary, V2. In other words, one must be able to infer one claim from another in order to count as using any vocabulary whatsoever. This is a central tenet of Brandom’s inferentialism on which the capacity to draw inferences is a metaphysically necessary condition on being a concept-user – hence language-user – of any sort.9 As Brandom puts it: Assertions are essentially, and not just accidentally, speech acts that can play the role both of premises and as conclusions of inferences … According to this way of thinking, inferential practices are PP-necessary components of every autono- mous discursive practice, hence PV-necessary for the deployment of every autonomous vocabulary, hence PV-necessary for the deployment of every vocab- ulary whatsoever.10 Though there are those who might wish to take issue with this claim, in what follows we grant this assumption, at least for the sake of argument. Though there are those who might wish to take issue with this claim, in what follows we grant this assumption, at least for the sake of argument. Condition 2. The second condition concerns a relationship between practices. According to Brandom: P2, the practice of inferring, is PP-sufficient for P1, the practice of using the conditional. In other words, if a person can infer one claim from another, then she can, at least in principle, do everything that she needs to be able to do to count as having mastery of the conditional. Again, Brandom’s inferentialism, and the connection it assumes between asserting and inferring, is central here. 8 Brandom (2008: 52-53). 9 See Brandom (1994). 10 Brandom (2008: 42-43). (e) P2 is PP-necessary for any autonomous practice – i.e. a practice that is PV- sufficient for an autonomous vocabulary. (e) P2 is PP-necessary for any autonomous practice – i.e. a practice that is PV- sufficient for an autonomous vocabulary. Thus if a vocabulary satisfies the conditions (i.e., (a)-(e)) for being universal LX, then not only are the practices it describes necessary for being a language user of any sort, but the practices so described also suffice for mastery of the universal LX vocabulary itself. As Brandom puts it for the case of logic, Banyone who can talk at all, hence can deploy any base vocabulary, can already do everything one needs to be Fig. 1 General meaning-use diagram for LX Vocabulary Fig. 1 General meaning-use diagram for LX Vocabulary Fig. 1 General meaning-use diagram for LX Vocabulary 1810 Philosophia (2017) 45:1803–1827 able to do in order in principle to be able to say what logical vocabulary lets one say.^8 We might add that if Brandom is correct, then the same is true for modal (and normative) vocabulary as well. 4 Logic as Universal LX: The Case of the Conditional According to this view, if we begin with an agent that can assert and infer, then: …we can teach it also to produce-assertively tokenings of the new form Bif p then q.^…The system must respond to its assertion of the conditional Bif p then q^ by …we can teach it also to produce-assertively tokenings of the new form Bif p then …we can teach it also to produce-assertively tokenings of the new form if p the q.^…The system must respond to its assertion of the conditional Bif p then q^ b Philosophia (2017) 45:1803–1827 1811 Fig. 2 Our meaning-use diagrams differ from Brandom’s in that we list the PP-necessity relation as an arrow instead of as inclusion. See Fig. 1.9 on Brandom (2008: 28) Fig. 2 Our meaning-use diagrams differ from Brandom’s in that we list the PP-necessity relation as an arrow instead of as inclusion. See Fig. 1.9 on Brandom (2008: 28) treating the inference from p to q as a good one—for instance, by being disposed to endorse q assertionally if it is disposed to endorse p assertionally… In a clear sense, then, the capacity to distinguish good from bad inferences involving non- logical sentences is (PP-) sufficient for the ability to deploy conditionals involving those sentences.11 Condition 3. According to the third condition that needs to be satisfied for ‘If__, then__’ to be universal LX: P1, the practice of using conditionals, is PV-sufficient for mastery of V1, conditional vocabulary. This is true given the definitions of P1 and V1. then__’ to be universal LX: P1, the practice of using conditionals, is PV-sufficient for mastery of V1, conditional vocabulary. This is true given the definitions of P1 and V1. Condition 4. The fourth condition on ‘If__, then__’ being universal LX is that the vocabulary of conditionals permits us to specify adequately the practice of inferring. In other words: V1, the vocabulary of conditionals, is VP-sufficient for P2, the practice of inferring. This condition is captured by the diagonal arrow in Fig. 2. Notice that the existence of such VP-relations is crucial to the overall success of Brandom’s Analytic Pragmatism. For precisely what VP-sufficiency does is establish practice-mediated rela- tions between pairs of vocabularies. In other words, without VP-sufficiency we would be unable to provide a practice-mediated analysis of one vocabulary in terms of another. 11 Brandom (2008: 45). 4 Logic as Universal LX: The Case of the Conditional In the present case, conditional vocabulary is taken to provide a means of specifying the practice of inference making, and thereby Bmake explicit^ what was only implicit in the practice of drawing inferences. As Brandom puts it: BConditionals make explicit 1812 Philosophia (2017) 45:1803–1827 something that otherwise was implicit in the practical sorting of non-logical inferences into good and bad.^12 Condition 5: If a vocabulary meets conditions 1–4, then it is LX. The final condition, which renders it universal LX, is that the practice of inferring is PP- necessary for any autonomous practice. In other words, every language-user must be proficient at inferring. Again, this is controversial; but we grant it here, at least for the sake of argument. To summarize: Brandom maintains that conditionals are universal LX because they satisfy all of the above conditions. The burden of the next section is to show that he is mistaken. 5 Conditionals are Not Universal LX Our complaint against Brandom’s treatment of the conditional is threefold. First, we maintain that there are two quite different readings of VP-sufficiency and that there are serious problems with the claim that conditionals are, in the sense relevant to the Analytic Project, VP-sufficient for the practice of inferring. Second, even if the first objection is waived, we maintain that PP-sufficiency (i.e., Condition 2) fails. Third, even if one supplements the account so that Condition 2 holds, Condition 4 fails; and moreover it fails no matter which of the two readings of VP-sufficiency one adopts. Thus, we maintain that even if conditionals are VP-sufficient for the practice of inferring, it still follows that conditionals are not Universal LX. 12 Brandom (2008: 46). 5.1 Descriptive and Expressive Notions of VP-Sufficiency The first problem arises from the fact that Brandom’s discussion incorporates two rather different construals of VP-sufficiency: what we call the descriptive and expressive readings. The problem for Brandom is that, on the one hand, only the descriptive reading will do the work required by Analytic Pragmatism, whilst on other the other hand, conditionals can, at most, provide the basis for the expressive form of VP- sufficiency. Let us explain this in more detail. As characterized by Brandom, the objective of Analytic Pragmatism is to provide pragmatic analyses —i.e., to specify, in one vocabulary, the practices that determine the content of another vocabulary. This is precisely the role that VP-sufficiency is supposed to play within the broader project. But talk of Bspecifying^ appears, given the context, to involve describing a set of practices. Indeed, his paradigm cases of VP-sufficiency confirm this impression. For example, Brandom glosses what he calls the Kaplan- Stalnaker semantics for indexicals by saying the following: [It provides a] purely non-indexical vocabulary [that] can serve as an adequate pragmatic metavocabulary for indexical vocabulary. That is, one can say (that is, describe), in wholly non-indexical terms, everything one needs to do in order to 1813 Philosophia (2017) 45:1803–1827 use indexical vocabulary. Non-indexical vocabulary is VP-sufficient to specify practices-or-abilities PV-sufficient to deploy indexical vocabulary.13 In short: VP-sufficiency, on this descriptive construal, involves the description of linguistic practices. Let us turn to the second, expressive construal of VP-sufficiency. On this view, VP- sufficiency need not involve the description of practices. Instead it consists in one vocabulary making explicit what is only implicit in a linguistic practice. Very roughly, one makes explicit a linguistic practice or ability when one introduces an expression into the language that allows us to formulate claims about – and rationally assess – performances of the practice.14 To take a relatively simple example, Brandom claims in some earlier work that the negation sign renders explicit what would otherwise only be implicit in our linguistic practices — namely, treating two claims as incompatible. As he puts it: Negation, as a logical connective supporting formally valid inferences, plays the same explicitating role with respect to material incompatability relations among judgeable (that is propositional) contents that the conditional plays with respect to the material inferential relations among such contents.15 n this expressive construal of VP-sufficiency, then, no description need occur. 5.1 Descriptive and Expressive Notions of VP-Sufficiency p y, , p The problem is that a vocabulary that only makes explicit some feature of a practice need not describe that practice. Yet precisely what pragmatic analyses seem to require is a description of the practices. One way to see the point is to consider the pragmatic Banalysis^ that would result from merely making explicit the practice of treating two claims as incompatible. If all that were required for VP-sufficiency was that this be made explicit, then the Banalysis^ would consist in one word: not! By any lights, such a result would not be an analysis worthy of the name. But it should be clear that conditionals can no more describe the practice of inference than the negation sign can describe the practice of treating-as- incompatible. Instead, what it more plausibly does, is make some element(s) or aspect(s) of the practice explicit. And indeed, this is what Brandom ends up emphasizing when he argues for the claim that conditionals are universal LX.16 Our complaint, in short, is that Brandom slides from a descriptive notion of VP-sufficiency that cannot be met by conditional vocabulary to an expressive conception of VP-sufficiency that can be played by the conditional but which is not fit for the purposes of Analytic Pragmatism. 13 Brandom (2008: 56). 14 Brandom’s notion of making explicit is a notoriously hard one to articulate precisely. The present way of formulating the idea might sound as if making a practice explicit consists in producing a name for that practice. For reasons that we need not go into here, however, it is quite clear that this is not Brandom’s intention. Moreover, it is clearly false that logical expressions, such as negation, are names for practices. 15 Brandom (1994: 115). 15 Brandom (1994: 115). ( ) 16 Brandom (2008: 44-47). 17 Brandom (2008: 42). 5.2 Inferring, Asserting and Inferring-and-Asserting Let us waive the previous objection for the sake of argument. Still, there is another – and in our view more serious – problem for the claim that conditionals are VP- 1814 Philosophia (2017) 45:1803–1827 sufficient for the practice of inferring. Here the problem concerns how to understand the bottom right-hand interior box in Fig. 2. According to Brandom’s characterization of conditionals as universal LX, the practice of inferring is supposed to be PP-sufficient for the practice of using conditionals. But this cannot be right. For, in order to have mastery of conditionals, one must also be able to engage in the practice of asserting. This is a point that Brandom would himself readily endorse and for a general and rather obvious reason: a language user who is incapable of assertion would be no language user at all. But if this is so, then the practice of inferring is not PP-sufficient for the practice of using conditionals, and so conditionals (on this construal of the diagram) are not universal LX. Now, it should be clear what is going on here. Brandom is assuming that the practices of asserting and inferring go together. As he puts it: Bthe beginning of wisdom^ in understanding the nature of assertion Bis to see that asserting and inferring are internally related practices, in the sense that each is PP-necessary for the other.^17 So, in order to accurately capture the meaning-use relations for conditionals, we need to augment the lower right-hand interior box in the diagram in Fig. 2 by having the conjunction of inferring and asserting as PP-sufficient for the practice of using condi- tionals. But now the problem crops up elsewhere. Even if we grant that the conjoint practice of inferring-and-asserting is PP-sufficient for the practice of using conditionals and that this practice is, in turn, PV-sufficient for the mastery of the conditional, it is simply false that the conditional locution is VP-sufficient for the practice of inferring- and-asserting. Moreover, this is so irrespective of whether one adopts a descriptive or an expressive conception of the VP-sufficiency relation. Now, it should be clear what is going on here. Brandom is assuming that the practices of asserting and inferring go together. 5.2 Inferring, Asserting and Inferring-and-Asserting As he puts it: Bthe beginning of wisdom^ in understanding the nature of assertion Bis to see that asserting and inferring are internally related practices, in the sense that each is PP-necessary for the other.^17 Consider the options. On the descriptive reading, the ‘if__then__’ locution would need to describe the practice of inferring-and-asserting, which it clearly does not: any more than ‘not’ describes the practice of treating two claims as incompatible. On the alternative expressive reading, the ‘if__then__’ locution would need to make explicit the practice of inferring-and-asserting. But even if we grant that the conditional makes the practice of inferring explicit, it surely does not make inferring-and-asserting explicit. To see this, we need only reflect upon the vocabulary that would make asserting explicit. According to one familiar proposal, for example, the Fregean assertion stroke (├) can be used to express assertoric force. But on the face of it, the conditional, by itself, does not obviate the need for the assertion sign, any more than, say, disjunction does. When one asserts a conditional one does not thereby assert either the antecedent or the consequent. Moreover, in expressing conditionals, one does not thereby resolve the force of the utterance since conditionals can, like most other expressions, figure in assertions, suppositions, queries, commands, and so on. Thus, however we construe VP-sufficiency (whether descriptively or expressively) condi- tionals are not VP-sufficient for the practice of inferring-and-asserting. In sum: The practice of inferring, by itself, is not PP-sufficient for the practice of using conditionals. Perhaps the practice of inferring-and-asserting is PP-sufficient for the practice of using conditionals. But even if this is true, conditionals are not VP- sufficient for the practice of inferring-and-asserting. Thus, either Condition 2 fails or Condition 4 fails. Either way, conditionals turn out not to be LX, much less universal LX. Philosophia (2017) 45:1803–1827 1815 6 Modal Vocabulary is Not Universal LX (or Why the Modal Kant-Sellars Thesis is False) 6 Modal Vocabulary is Not Universal LX (or Why the Modal Kant-Sellars Thesis is False) In the preceding sections we argued at some length that Brandom’s primary example of logical vocabulary – the conditional – is not universal LX. & It is PV-necessary for using any empirical vocabulary; 5.2 Inferring, Asserting and Inferring-and-Asserting Since the claim that logical vocabulary is universal LX is amongst the central claims of Between Saying and Doing, and since the conditional is the only piece of logical vocabulary that he considers at any length, we take this to pose a serious problem for his project. We now propose to challenge another central thesis of the book – what Brandom calls the modal Kant- Sellars thesis —roughly, the claim that modal vocabulary (e.g., ‘possible’, ‘necessary’, and ‘contingent’) is universal LX. More specifically, we maintain that it is subject to exactly analogous problems to those set out earlier. 18 Brandom (2008: 103). 19 Brandom illustrates the point with the following example: BOne grasps the claim ‘the lioness is hungry’ only insofar as one takes it to have various consequences (which would be true if it were true) and to rule out some others (which would not be true if it were true). And it is not intelligible that one should endorse as materially good an inference involving it, such as the inference from ‘the lioness is hungry’ to ‘nearby prey animals visible to and accessible by the lioness are in danger of being eaten,’ but be disposed to make no distinction at all between collateral premises that would, and those that would not, if true infirm the inference.^ (p. 105) 18 Brandom (2008: 103). 19 6.1 The Modal Kant-Sellars Thesis and Its Philosophical Significance As noted in Section 2.1, a central goal of Brandom’s project in Between Saying and Doing is to argue for the thesis, which he calls the modal Kant-Sellars thesis, on which modal vocabulary has a privileged status akin to logical vocabulary. Via an extensive discussion that focuses primarily on Sellars – but takes in Ryle, Kripke, and Quine along the way – Brandom fashions a characterization of the modal Kant-Sellars thesis that conforms to the pragmatic conception of meaning so central to Brandom’s overarching project. In particular, the version of the modal Kant-Sellars thesis that he endorses is formulated as the conjunction of two claims: 1. In using ordinary empirical vocabulary, one already knows how to do everything one needs to know how to do in order to introduce and deploy modal vocabulary. 1. In using ordinary empirical vocabulary, one already knows how to do everything one needs to know how to do in order to introduce and deploy modal vocabulary. 2. The expressive role characteristic of alethic modal vocabulary is to make explicit semantic, conceptual connections and commitments that are already implicit in the use of ordinary empirical vocabulary. As Brandom observes, this formulation of the modal Kant-Sellars thesis is amena- ble, with minimal modification, to the sort of meaning-use analysis that we have already encountered. Claim 1 maintains that some set of practices that are PV- necessary for the use of any empirical vocabulary are PP-sufficient for practices that are PV-sufficient to deploy modal vocabulary. Claim 2 maintains that modal vocabulary makes explicit – is VP-sufficient for – those aspects of practices that are implicit in – i.e., PV-necessary for – the use of any empirical vocabulary whatsoever. Thus, accord- ing to Brandom’s modal Kant-Sellars thesis, modal vocabulary is universal LX. This is represented diagrammatically in Fig. 3 (‘CRI’ stands for ‘counterfactually robust inference’, to be explained below). There is a crucial component of Brandom’s discussion of the modal Kant-Sellars thesis that we have yet to touch upon. From inspection of Fig. 3, it is clear that the thesis requires that there be some set of practices – corresponding to the bottom-right interior box – that satisfies three conditions: Philosophia (2017) 45:1803–1827 1816 Fig. 3 A meaning-use diagram for modal vocabulary Fig. 6.2 Why Brandom’s Modal Kant-Sellars Thesis is False Although we do not deny the general thesis that modal notions are in some way implicit in ordinary descriptive, linguistic practices, we do reject the claim that modal vocab- ulary is universal LX, and with it Brandom’s specific formulation of the modal Kant- Sellars thesis. Moreover, our line of criticism is exactly analogous to the objection we leveled against the claim that the conditional is universal LX in Section 4.2. In order to have mastery of modal locutions, one must also be able to engage in the practice of asserting —i.e. PModal in Fig. 3 must involve the ability to make assertions. This is a point that Brandom himself would readily endorse and for precisely the same reason mentioned in the case of the conditional. But if this is so, then the practice of counterfactually robust inferring (hereon CRI) is not PP-sufficient for the practice of using modal notions, and so modal vocabulary (on this construal of the diagram) is not universal LX. Now, as with the case of the conditional, one might respond to this problem by maintaining that the capacity for asserting is PP-necessary for the capacity for CRI and vice versa. In which case, to capture the meaning-use relations for modals, we need to augment the lower interior right-hand box in the diagram by having the conjunction of CRI and asserting as PP-sufficient for the practice of using modal locutions. But just as before, the problem now crops up elsewhere. Even if we grant that the conjoint practice of CRI-and-asserting is PP-sufficient for the practice of using conditionals, and that this practice is, in turn, PV-sufficient for the mastery of modals, it is simply false that modal locutions are VP-sufficient for the practice of CRI-and-asserting. Moreover, this is so irrespective of whether one adopts a descriptive or an expressive conception of the VP- sufficiency relation. Consider the options. On the descriptive reading, modal locutions would need to describe the practice of CRI-and-asserting, which they clearly do not: any more than ‘not’ describes the practice of treating two claims as incompatible. On the alternative expressive reading, modal locutions would need to make explicit the practice of CRI- and-asserting. But even if we grant that modals make the practice of CRI explicit, they surely do not make CRI-and-asserting explicit. To see this, we need only reflect again upon the vocabulary that would make asserting explicit. 6.1 The Modal Kant-Sellars Thesis and Its Philosophical Significance 3 A meaning-use diagram for modal vocabulary & It is PP-sufficient for those practices PV-sufficient for deploying modal vocabulary and & It is PP-sufficient for those practices PV-sufficient for deploying modal vocabulary; and & It is PP-sufficient for those practices PV-sufficient for deploying modal vocabulary; and & Modal vocabulary is VP-sufficient for these practices. & Modal vocabulary is VP-sufficient for these practices. Yet as formulated no commitment is made regarding what these practices are. This is a gap that Brandom fills in the course of arguing for the modal Kant-Sellars thesis. In brief, his argumentative strategy is to justify the thesis by showing that there is a practice that satisfies the above conditions. What might it be? His answer is that it is the capacity for what he calls Bcounterfactually robust inference^: the capacity to associate a range of Bcounterfactual robustness^ with each materially good inference. 18 In other words, he maintains that mastery of any empirical vocabulary requires that one be able to distinguish the counterfactual conditions in which an inference would fail from those in which it would go through. For otherwise one would not count as possessing the relevant empirical concepts.19 Philosophia (2017) 45:1803–1827 1817 6.2 Why Brandom’s Modal Kant-Sellars Thesis is False According to one familiar proposal, for example, the Fregean assertion stroke can be used to express assertoric force. But on the face of it, modal vocabulary, by itself does not obviate the need for the assertion sign, any more than, say, the conditional does. Thus, whichever way we construe VP-sufficiency – whether it be descriptively or expressively – modals are not VP-sufficient for the practice of CRI-and-asserting. In sum: The practice of CRI, by itself, is not PP-sufficient for the practice of using modals. Perhaps the practice of CRI-and-asserting is PP-sufficient for the practice of using modals. But even if this is true, modals are not VP-sufficient for the practice of CRI-and-asserting. Thus, either Condition 2 fails or Condition 4 fails. Either way modals turn out not to be LX, much less universal LX. 6.3 The Normative Kant-Sellars Thesis We have argued that the modal Kant-Sellars thesis fails. But those familiar with Between Saying and Doing might wonder whether any of this applies to what Brandom 1818 Philosophia (2017) 45:1803–1827 calls the normative Kant-Sellars thesis: the claim that in order to apply ordinary, empirical, descriptive vocabulary – and hence to deploy any autonomous vocabulary whatsoever – one must already be able to do everything needed to introduce normative vocabulary.20 This thesis is defended in tandem with the modal version of the Kant- Sellars thesis. Moreover, Brandom appears to suppose that the two theses are strongly analogous, being answers to two variants of a single issue: Kant read Hume's theoretical and practical philosophies as raising variants of a single question. On the side of theoretical reasoning, Hume asks what our warrant is for moving from descriptions of what in fact happens to characteriza- tions of what must happen, and what could not happen. How, he wants to know, can we rationally justify the move from descriptions of matter-of- factual regularities to formulations of necessary laws? On the side of practical reasoning, Hume asks what our warrant is for moving from descriptions of how things are to prescriptions of how they ought to be. How, he wants to know, can we rationally justify the move from ‘is’ to ‘ought’? … As we have seen, on the modal side, Kant's response is that Hume's predicament is not a real one. One cannot in fact fully understand the descriptive, empirical employment of ordinary determinate concepts such as cat without at least im- plicitly understanding also what is made explicit by the modal concepts that articulate laws. Kant mounts a corresponding line of thought on the side of normative or practical necessity. Normative concepts make explicit commitments that are implicit in any use of concepts, whether theoretically in judgment or practically in acting intentionally.21 In view of this apparently tight connection between the two Kant-Sellars theses one might wonder whether the normative version is subject to the same problems we level against the modal version. We suspect that the normative Kant-Sellars thesis is unscathed by the sorts of considerations raised above. In order to see this, consider the meaning-use diagram that Brandom provides for the normative Kant-Sellars thesis (‘GAR’ stands for ‘the game of giving and asking for reasons’) Fig. 4. 20 Anyone familiar with Brandom’s earlier work, Making It Explicit, should recognize that such a thesis about normative vocabulary is central to Brandom’s philosophical position. 21 Brandom (2008: 109-110). 22 This is not, of course, to deny that some other objection might apply. But we put such issues to aside here. 6.3 The Normative Kant-Sellars Thesis In order for normative vocabulary to be universal LX, it must be VP-sufficient for some practice that is, in turn, PP-sufficient for the practice of using normative vocabulary. According to Brandom the practice in question is giving and asking for reasons. But this practice is – and this is the crucial point – one that incorporates the practice of assertion. Giving a reason just is a kind of asserting. But if this is so, then we have no reason to doubt that it is PP- sufficient for the practice of using normative vocabulary. Thus no analog of our earlier objections can be run for the present case.22 1819 Philosophia (2017) 45:1803–1827 Fig. 4 A meaning-use diagram for normative vocabulary Fig. 4 A meaning-use diagram for normative vocabulary 7 Intentionality as a Pragmatically Mediated Relation The final central thesis of Brandom’s Between Saying and Doing concerns the nature of intentionality – the world-directed character of language and thought – that we call the pragmatically mediated conception of intentionality. In our view, this proposal is of profound importance to the overarching project of Analytic Pragmatism. Yet Brandom’s discussion is underdeveloped and, at times, frustratingly obscure. With this in mind, we propose to start with a careful and charitable reconstruction of the proposal. Having done so, we show that the arguments of the foregoing sections provide reason to reject the proposal. 23 See Rosen (2001) for example. As David Lewis once wrote in this spirit, Bsemantics with no treatment of truth conditions is not semantics^ (Lewis 1983: 190). 24 Brandom labels this principle P3; Brandom (2008: 178). 7.1 Motivation: A Persistent Criticism of Semantic Pragmatism One way to appreciate the central motivation for – and significance of – Brandom’s pragmatically mediated conception of intentionality is to start by focusing on a persistent criticism of the semantic assumptions of Analytic Pragmatism. More or less by definition, Analytic Pragmatism is committed to Semantic Pragmatism: the thesis that linguistic meaning is determined by use. Moreover, the sorts of analyses that Brandom seeks to provide consist in specifying the linguistic practices on which our mastery of various vocabularies are thought to depend. But as Brandom himself readily acknowledges, there is a worry that such analyses are, in fact, not semantic at all; and not for any reason that has anything specific to do with the particular analyses that he advocates. Rather, the worry is quite general. Such analyses fail to be semantic – and hence to do the work required of them by Analytic Pragmatism – because they conform 1820 Philosophia (2017) 45:1803–1827 to the general precepts of Semantic Pragmatism, and Semantic Pragmatism is unsatis- factory as an account of semantic properties. On such a view, meaning is ultimately to be understood in terms of the linguistic practices in which we engage, not the relations that terms bear to aspects of the world. But according to many, semantics, if it is anything, is concerned with precisely such word-world relations – e.g., with represen- tation, truth, and reference.23 In which case, it may seem that to the extent that Analytic Pragmatism seeks to provide semantic analyses, it is doomed to fail. Thus whatever else the value of pragmatic analysis, it cannot be a satisfactory extension of the project initiated by Frege, Russell and Moore. 7.2 The Pragmatist Responds One obvious response to the above concern is simply to deny that semantics is centrally concerned with word-world relations. Yet clearly this is not Brandom’s response. Rather, he thinks that there is something importantly right about the idea that semantic properties involve word-world relations. The challenge, then, is to explain why this is so in a manner consistent with Semantic Pragmatism. This is precisely the role that his account of intentionality is supposed to fill. Step 1: The Nature of Practices Brandom’s response can be viewed as having two main steps. The first is driven by what he takes to be a very general fact about the nature of practices, which he views as a central claim of the pragmatist tradition. Of course language use qua practice involves relations between subjects and the world because in general practices involve aspects of the world: they are thick.24 To take some simple examples, the practice of cooking cannot be adequately characterized absent such things as food items and kitchen utensils; and the practice of drinking tea cannot be adequately characterized without reference to such things as cups, saucers, and, of course, tea leaves. But such components of the practices are also aspects of the world, and thus the existence of these practices carry a commitment to subject-world relations. And what goes for cooking and tea drinking is also true of language use. Qua practice, it too ineliminably involves subject-world relations. So there is a sense in which, according to Brandom, the worry with which we started is no objection at all. That is, if the concern is that Semantic Pragmatism severs the connection between word and world, then the concern is misplaced. At best it is an artifact of assuming that the relevant practices ought to be characterized Bthinly^, in such a way as to avoid reference to aspects of the world. But as Brandom clearly recognizes, this does not settle the matter. The real problem for the semantic pragmatist is not that there are no relations between language-using subjects and the world. On the contrary, there are a great many such relations. Such relations are cheap. The real problem for semantic pragmatists is specifying the right relation(s) – those relevant for the purposes of characterizing the intentional character of language use; and this, according to Brandom, requires a more detailed, pragmatist account of intentionality. 7.2 The Pragmatist Responds 1821 Philosophia (2017) 45:1803–1827 Step 2: Combining the Accounts of Modal and Normative Vocabulary To appre- ciate Brandom’s account of intentionality one needs first to make three general points clear. The first, already hinted at above, is that he views intentionality of the sort manifested by the practice of language use as a specific instance of a broader phenom- enon, manifested by all human practices, which he calls practical intentionality. Practices quite generally, you may recall, involve relations between subjects and aspects of the world. Further, as Brandom notes, practices invariably involve what he calls practical intentionality: feedback-governed relations in which the results of earlier actions are used as guides to future, goal-directed behavior. This is true, for example, of cooking and tea drinking, but also football kicking and everything else for that matter, including language use. According to Brandom, then, the semantic intentionality characteristic of language use is best thought of as a specific instance of practical intentionality.25 The second point concerns the order of explanation one ought to adopt in explaining semantic intentionality. According to Brandom, some traditions26 purport to start with independent conceptions of the states of the subject that represent and of the entities so represented, and then seek to characterize the nature of the intentional relation that holds between them —to somehow Bbolt^ them together.27 Brandom maintains that such an approach is wrong-headed in that it gets the proper order of explanation exactly backwards, and thereby ignores precisely that which allows for intentionality in the first place: Bthe thick, essentially world-involving practices^ in which we engage.28 In its place, Brandom proposes that should start, as traditional pragmatists have advocated, with the world-involving practices themselves; and only characterize intentionality and its relata as a kind of abstraction from these practices.29 Thus it is only by taking our rich, thickly characterized linguistic practices as primary and subjecting them to careful enquiry that, according to Brandom, we have any hope of understanding the general phenomenon of semantic intentionality.30 The final general point concerns the specific practices most central to explaining intentionality. According to Brandom, the most central are those concerned with our use of normative and modal vocabularies. Indeed his strategy is, in effect, to explain intentionality by combining the two Kant-Sellars Theses discussed in Section 5, and seeking to draw out certain consequences of their joint endorsement. 25 Brandom labels this claim P4; Brandom (2008: 179). 26 Though he does not explicitly mention any philosophical theories that cleave this methodology, we suspect that he has in mind, amongst other things, causal theories of content, such as those advocated by Fodor and Dretske; see Dretske 1981) and Fodor (1992). 27 Brandom (2008: 179). 28 Brandom (2008: 180). 29 Historical note: This sort of view was characteristic of the views of pragmatists, such as Dewey and James, but similar views can also be found in Russell (1945: 812) among others. 30 Superficial appearances not withstanding, Brandom is not claiming that there could be no objective world without the existence of our linguistic practices. His purpose is not to advocate some kind of linguistic idealism, but instead to defend an approach to the study of intentionality. 26 Though he does not explicitly mention any philosophical theories that cleave this methodology, we suspe that he has in mind, amongst other things, causal theories of content, such as those advocated by Fodor an Dretske; see Dretske 1981) and Fodor (1992). 27 d 25 Brandom labels this claim P4; Brandom (2008: 179). 26 ( ) 29 Historical note: This sort of view was characteristic of the views of pragmatists, such as Dewey and James, but similar views can also be found in Russell (1945: 812) among others. 30 Superficial appearances not withstanding, Brandom is not claiming that there could be no objective world without the existence of our linguistic practices. His purpose is not to advocate some kind of linguistic idealism, but instead to defend an approach to the study of intentionality. 28 Brandom (2008: 180). 29 25 Brandom labels this claim P4; Brandom (2008: 179). 26 7.2 The Pragmatist Responds In Section 6.3 we argue that this strategy does not work. But first some further unpacking is in order. Recall: for Brandom, though an explanation of semantic intentionality requires an account of the relevant subject-world relations, such an account is to be provided by abstraction from the relevant linguistic practices. The primary task, then, is to supply an adequate account of the relevant practices; and for Brandom the two central practices 1822 Philosophia (2017) 45:1803–1827 are those concerning modal vocabulary and normative vocabulary, since they inform us about the relata of semantic intentional relations: the world and the rational subject, respectively. The practices made explicit by modal vocabulary (CRI) impose con- straints on the modal structure of reality. Roughly, they reflect constraints, implicit in our thought and language, on ways that the world could be and must be. In contrast, the practices made explicit by normative vocabulary (GAR) impose constraints on rational agency. Roughly, they tell us what is it to be a rational subject – a producer and consumer of reasons. Thus for Brandom these linguistic practices, though not them- selves the poles of intentional relations, are crucial to explicating the nature of intentionality, because it is by providing appropriately perspicuous characterizations of them that we are able, by abstraction, to reconstruct the nature of the relata. So far so good. But an account of intentionality must do more than merely explicate those practices that concern the relata. Rather, one needs to explain how each of these practices are related to each other so as to yield the larger practice on which semantic intentionality depends. In effect, Brandom must address an analog of the problem that confronts more traditional approaches to intentionality of the sort that he rejects. For as we have already noted, on some approaches to the explanation of intentionality, the challenge is to specify the relevant relation between the relata: i.e., subject and world. For Brandom, the challenge is to specify the relationship between two practices: the world-oriented practices made explicit by modal vocabulary and the subject-oriented practices made explicit by normative vocabulary. How is this to be done? Brandom’s answer is that the two practices intersect in what he calls rational rectification, which is the practice by which new commitments are integrated into one’s cognitive economy – one’s system of beliefs, desires, goals, and other intentional states. 7.2 The Pragmatist Responds Such a practice involves the modification of one’s beliefs – often in the light of new information – so as to eliminate inconsis- tencies among one’s current commitments. But the capacity to do so presup- poses a capacity to recognize inconsistencies, which in turn presupposes a capacity to engage in inferences that draw out the consequences of one’s current beliefs. According to Brandom, all of these cognitive capacities are aspects of the practice made explicit by use of modal vocabulary; and thus are implicit in the practice of rational rectification. But there is more to rational rectification than this. Rational rectification requires more of us than the mere capacity to recognize inconsistency – and whatever inferential capacities this, in turn, presupposes. Among other things, this alone would be compat- ible with continuing to endorse inconsistent commitments. In addition to this, rational rectification requires that one take oneself to have an obligation to eliminate such inconsistencies – to structure one’s commitments in such a way as not to violate facts about the modal structure of the world. And according to Brandom, the practice of having one’s commitments conform to such constraints are amongst those made explicit by our use of normative vocabulary. It is time to bring all the pieces together. What we have said so far on the topic intentionality is quite impressionistic; and indeed most of Brandom’s own discussion is similarly gestural. Nevertheless, in accord with his practice elsewhere in the book, when Brandom seeks to spell out his views precisely, he presents a meaning-use diagram. In the case of intentionality, the diagram that he uses to distill his views is reproduced as Fig. 5. Philosophia (2017) 45:1803–1827 1823 Fig. 5 This figure has three arrows numbered for ease of reference below. See Fig. 6.1 on Brandom (2008: 183) Fig. 5 This figure has three arrows numbered for ease of reference below. See Fig. 6.1 on Brandom (200 Fig. 5 This figure has three arrows numbered for ease of reference below. See Fig. 6.1 on Brandom (2008: 183) Although there is a lot going on here, we restrict ourselves to the following observations. First, the diagram aims to depict a kind of practice – indeed any practice of using an autonomous vocabulary – as an object for which intentional vocabulary is VP-sufficient – i.e., either made explicit by or describable by intentional vocabulary. 31 Brandom uses the term ‘discursive’ as a general term for any activity in which only rational entities can engage. 7.3 An Objection In view of our earlier discussion, one problem with Brandom’s proposed account of intentionality should be clear. As we have already seen, modal vocabulary is not universal LX. So, there might be a language user that cannot already do everything needed to be able to use modal vocabulary; and if that is right, then the account of semantic intentionality, as represented in Fig. 5, obviously cannot be right. How deep does this objection go? To see the significance of the objection, we need to rehearse some aspects of our earlier discussion, and introduce some features of Brandom’s theory of content. First, recall that we showed earlier that the practice of counterfactually robust inference is not PP-sufficient for the practice of using modal vocabulary. But this is just to deny that arrow 1 in Fig. 5 holds. In other words, the practice labeled Pobj-modal in Fig. 5 is not PP-sufficient for the practice of using modal vocabulary; and this means that Brandom has failed to characterize adequately the nature of the practice that, on his view, mediates the intentionality relation. In particular, if our arguments from Section 5 are correct, then the aspect of the practice that is supposed to explicate the Bworld pole^ of the intentionality relation has not have been accurately characterized. Of course, this is not the end of the matter. Following the dialectic set out in Section 5, Brandom might seek to preserve the connection between Pobj-modal and the practices sufficient for the use of modal vocabulary by enriching Pobj-modal. Specifically, he might add the assumption that Pobj-modal is constituted, in part, by the ability to make assertions. If such a maneuver is pursued, then our earlier objection to arrow 1 fails. But now – just as before – the problem crops up elsewhere. Though arrow 2 holds more or less by definition, arrow 3 fails. That is, as argued earlier, we no longer have any reason to suppose that modal vocabulary is VP-sufficient for Pobj-modal. In which case, modal vocabulary still fails to be universal LX, and the modal Kant-Sellars Thesis is false. As a consequence, even if one pursues this escape route, Brandom’s pragmatic theory of intentionality, at least as represented by Fig. 5, is false. 7.2 The Pragmatist Responds Second, the diagram represents any autonomous discursive practice as incorporating two essential components: a practice made explicit by modal vocabulary and a practice made explicit by normative vocabulary.31 Thus the diagram represents mastery of such practices as necessary for being a language-user at all. Finally, the diagram represents anyone who can engage in these two practices as being capable, in principle, of deploying the vocabularies that make these practices explicit – i.e., modal and normative vocabulary. In other words, the diagram represents the fact that Brandom’s account of intentionality – in conjunction with the earlier discussed claims regarding modal and normative vocabulary – entails the modal and normative Kant-Sellars theses. This is important for present purposes, because by tying his account of intentionality to his previous claims about modality and normativity, Brandom provides a partial explication of the nature of the practices on which intentionality depends. As we noted earlier, the real challenge for a pragmatic account of intentionality of the sort advocated by Brandom is to explicate the word-world relations that is relevant to intentionality; and with respect to this task, the primary significance of the Kant-Sellars theses is that they permit Brandom is specify central 1824 Philosophia (2017) 45:1803–1827 aspects of the relevant practices. Brandom is thus relying on his earlier discussion of modality and normativity in order to flesh out his account of intentionality. aspects of the relevant practices. Brandom is thus relying on his earlier discussion of modality and normativity in order to flesh out his account of intentionality. 7.3 An Objection One might, however, accept our criticism and yet insist that the objection is easily met so as to preserve the spirit, though not the letter, of Brandom’s account of intentionality. Specifically, it seems eminently reasonable to ask why the account of intentionality as such should require that modal vocabulary be VP-sufficient for Pobj- modal. Clearly, on anything like Brandom’s view, Pobj-modal should be PP-sufficient for Pmodal. Absent this connection Brandom would, by his own lights, have failed to explicate the Bworld pole^ of the intentionality relation. Further, on anything like Brandom’s account, a relation of PV-sufficiency must hold between Pmodal and Vmodal. After all, on his view, the existence of such a connection obtains more or less by definition. In contrast, it is utterly unclear why the account of intentionality requires that Vmodal be VP-sufficient for Pobj-modal and, hence, that modal vocabulary be universal LX. To the extent that we concerned only with the account of intentionality, then, such a commitment seems entirely optional. 1825 Philosophia (2017) 45:1803–1827 There is a sense in which we think that this response is entirely correct. Brandom’s approach to intentionality, need not – and indeed should not – demand that Vmodal be VP-sufficient for Pobj-modal. But we think that the assumption that it should is deeply engrained in Brandom’s views about semantics and, hence, that preserving the Brandonian approach to intentionality requires that one give up a deep-seated commit- ment of Brandom’s philosophical worldview, sometimes called expressive equilibrium. In our experience, the issue is not easily appreciated; and so we propose to creep up on the relevant assumption by first considering another assumption – expressive complete- ness – also deep-seated in Brandom’s philosophy, that does not suffice to mandate the commitment to arrow 3, but that does motivate the endorsement of expressive equilibrium Expressive Completeness Brandom is, of course, a semantic pragmatist, and so his theory of content takes the form of a theory of those discursive practices that are adequate to confer semantic content on the expressions involved. Expressive complete- ness is invoked as a condition of adequacy on such a theory. To a first approximation, expressive completeness requires that a theory of discursive practices should, in principle, be available to its practitioners. 32 Wittgenstein (1922). 7.3 An Objection To see the significance of this notion for Brandom’s semantic project we need to rehearse briefly the overall structure of the approach to semantic properties that Brandom adopts. Roughly put, he advocates a two-part approach: 1) The basic theory: This describes what might be called basic discursive practices – i.e. those involving no logical vocabulary. This theory will, of course, use logical vocabulary, but it will not explain how such expressions get their meanings. 2) The extended theory: This describes the extended discursive practices in which language-users engage – i.e., those involving logical vocabulary. More specifically, it does so by showing how basic discursive practices can be extended by the introduction of logical vocabulary. 2) The extended theory: This describes the extended discursive practices in which language-users engage – i.e., those involving logical vocabulary. More specifically, it does so by showing how basic discursive practices can be extended by the introduction of logical vocabulary. Now as we have seen, the overall theory – comprised of both the basic and extended theories – needs to be expressively complete. Failing that, one risks self-defeat prob- lems of sort that stymied verificationism. Yet clearly, participants in the basic practice lack the resources to state the basic theory because they posses no logical vocabulary at all. In which case, the basic theory is self-defeating. That is where the extended theory enters the picture. This theory explains how to introduce logical vocabulary – including modal and normative vocabulary – into the basic practice. So, participants in the extended practice – i.e., the practice described by the extended theory – possess the vocabulary required to state the basic theory. But now the problem recurs. In order to ensure expressive completeness of the extended theory, such practitioners must not merely have access to the basic theory, but to the extended theory as well. Otherwise, we (once more) risk regress. Brandom’s proposed solution is that logical vocabulary is in expressive equilibrium —that it makes explicit not only those norms that confer content on non-logical vocabulary, but those norms that confer content on logical vocabulary as well. And in the parlance of Between Saying and Doing, this is just the claim that logical vocabulary, broadly construed to include normative and modal vocabulary, makes explicit the very practices that one needs to master in order to possess logical vocab- ulary itself. 7.3 An Objection In other words, an adequate semantic theory should explain how linguistic expressions acquire their content in such a way that practitioners are, in principle, able to use the theory in order to explain the practices in which they participate. A corollary of this – one that is central to appreciating the significance of this constraint – is that such a theory will apply to itself. That is, an expressively complete semantic theory will correctly explain how the very linguistic expressions that comprise the theory acquire their meanings. Why endorse such a commitment? Though we will not explore the issue in detail here, the central motivation is to avoid the sorts of self-refutation worries that notori- ously confront various historically influential theories of meaning, most notably early versions of verificationism, but also Wittgenstein’s Tractarian account of meaning.32 Clearly, an expressively complete theory will not confront this problem since such theories are self-applicable – that is, they provide a meaning theory for the expressions in the theory itself. Be that as it may, the assumption of expressive completeness alone fails to explain Brandom’s commitment to the VP-sufficiency of Vmodal for Pobj-modal. This is because the mere commitment to expressive completeness very obviously does not demand that modal expressions make explicit the practices on which they depend. On the face of it, all that is required is that the theory not be self-defeating – that the theory possess the resources to explain how the expressions of the theory themselves acquire their meanings. What does require VP-sufficiency, however, is the strategy that Brandom adopts in order to ensure the expressive completeness of his preferred approach to semantic theorizing – what he calls expressive equilibrium. Expressive Equilibrium To a first approximation, the requirement for expressive equilibrium is the requirement that the broad category of logical vocabulary – which, for Brandom, includes traditional logical connectives and quantifiers, but also norma- tive and modal notions – makes explicit not only those norms that confer content on 1826 Philosophia (2017) 45:1803–1827 non-logical vocabulary, but those norms that confer content on logical vocabulary as well. To see the significance of this notion for Brandom’s semantic project we need to rehearse briefly the overall structure of the approach to semantic properties that Brandom adopts. Roughly put, he advocates a two-part approach: non-logical vocabulary, but those norms that confer content on logical vocabulary as well. 7.3 An Objection In short: the notion of universal LX vocabulary is a way of making more precise the demand for expressive equilibrium: a demand that Brandom thinks is central to an account of meaning that avoids self-defeat. We are now in a position to summarize our major concerns about Brandom’s view of intentionality. First, as noted earlier, our objections to the modal Kant-Sellars thesis in Section 5 show that the view as stated is false. Second, there is no reason internal to the theory of intentionality to assume that modal vocabulary is universal LX. At most the theory of intentionality per se requires arrows 1 and 2 – not arrow 3 – because it is the first two connections that are involved in specifying the discursive practices relevant to explicating the Bworld pole^ of the intentionality relation. Third, arrow 3 is required if one seeks to endorse both the account of intentionality and the strategy for ensuring expressive completeness. But this is just because it is required by the theory of expressive completeness. In other words, arrow 3 comes not from the theory of intentionality but from Brandom’s strategy for achieving expressive completeness for his pragmatist theory of meaning. 1827 Philosophia (2017) 45:1803–1827 Finally, once one sees the problem, it should be clear that arrow 3 is demanded by expressive equilibrium, irrespective of what account of intentionality Brandom en- dorses. That is, once one adopts expressive equilibrium as a way of attaining expressive completeness, it needs to be the case that modal vocabulary is VP-sufficient for Pobj- modal; and more generally, that logical vocabulary, broadly construed, is VP-sufficient for those practices needed to master exactly that logical vocabulary. What our argu- ments in the earlier sections of this paper show is that this strategy cannot succeed. 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 repro- duction 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. Rosen, G. (2001). Brandom on modality, normativity, and intentionality. Philosophy and Phenomenologica Research, 63, 611–623. Brandom, R. (1994). Making it explicit. Cambridge: Harvard University Press. References Brandom, R. (1994). Making it explicit. Cambridge: Harvard University Press. Brandom, R. (2008). Between saying and Doing: Towards an analytic pragmatism. Oxford: Oxfo University Press. Dretske, F. (1981). Knowledge and the flow of information. Cambridge: Cambridge University Press. Fodor, J. (1992). A theory of content and other essays. Cambridge: MIT Press. Lewis, D. (1983). General semantics. In Philosophical papers I. Oxford: Oxford University Press. Rosen, G. (2001). Brandom on modality, normativity, and intentionality. Philosophy and Phenomenological Research, 63, 611–623. Russell, B. (1945). The history of western philosophy. London: George Allen & Unwin. Wittgenstein, L. (1922). Tractatus Logico-Philosophicus. London: Harcourt, Brace & co..
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The Novel Long Noncoding RNA linc00467 Promotes Cell Survival but Is Down-Regulated by N-Myc
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Abstract Competing Interests: The authors have declared that no competing interests exist. Funding: The authors were supported by National Health and Medical Research Council Australia and Cancer Council New South Wa recipient of an ARC Future Fellowship. The funders had no role in study design, data collection and analysis, decision to publish, or pre Competing Interests: The authors have declared that no competing interests exist. * E-mail: tliu@unsw.edu.au Bernard Atmadibrata1, Pei Y. Liu1, Nicolas Sokolowski1, Lihong Zhang1,2, Matthew Wong1, Andrew E. Tee1, Glenn M. Marshall1,3, Tao Liu1,4* 1 Children’s Cancer Institute Australia for Medical Research, Randwick, Sydney, Australia, 2 Department of Anatomy, Histology and Embryology, School of Basic Medical Sciences, Fudan University, Shanghai, China, 3 Kids Cancer Centre, Sydney Children’s Hospital, Randwick, Australia, 4 School of Women’s & Children’s Health, UNSW Medicine, University of New South Wales, Randwick, Sydney, Australia Abstract The worst subtype of neuroblastoma is caused by MYCN oncogene amplification and N-Myc oncoprotein over-expression. Long noncoding RNAs (lncRNAs) are emerging as critical regulators of gene expression and tumourigenesis. While Myc oncoproteins are well-known to exert tumourigenic effects by regulating the expression of protein-coding genes and microRNAs, little is known about which lncRNAs are Myc targets and whether the Myc target lncRNAs play a role in Myc- induced oncogenesis. Here we performed differential gene expression studies using lncRNA microarray in neuroblastoma cells after transfection with control or N-Myc-specific small interfering RNA (siRNA), and identified N-Myc target lncRNAs including the novel lncRNA linc00467, the expression and function of which were completely unknown. RT-PCR, chromatin immunoprecipitation and luciferase assays showed that N-Myc suppressed linc00467 gene expression through direct binding to the linc00467 gene promoter and reducing linc00467 promoter activity. While N-Myc suppressed the expression of RD3, the protein-coding gene immediately down-stream of linc00467 gene, through direct binding to the RD3 gene promoter and reducing RD3 promoter activity, linc00467 reduced RD3 mRNA expression. Moreover, Affymetrix microarray analysis revealed that one of genes significantly up-regulated by linc00467 siRNA was the tumour suppressor gene DKK1. Importantly, knocking-down linc00467 expression with siRNA in neuroblastoma cells reduced the number of viable cells and increased the percentage of apoptotic cells, and co-transfection with DKK1 siRNA blocked the effects. These findings therefore demonstrate that N-Myc-mediated suppression of linc00467 gene transcription counterintuitively blocks N-Myc- mediated reduction in RD3 mRNA expression, and reduces neuroblastoma cell survival by inducing DKK1 expression. Citation: Atmadibrata B, Liu PY, Sokolowski N, Zhang L, Wong M, et al. (2014) The Novel Long Noncoding RNA linc00467 Promotes Cell Survival but Is Down- Regulated by N-Myc. PLoS ONE 9(2): e88112. doi:10.1371/journal.pone.0088112 Editor: Yunli Zhou, Harvard Medical School, United States of America Received June 11, 2013; Accepted January 6, 2014; Published February 19, 2014 Copyright:  2014 Atmadibrata et al. This is an open-access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited. Funding: The authors were supported by National Health and Medical Research Council Australia and Cancer Council New South Wales project grants. TL was a recipient of an ARC Future Fellowship. The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript. Citation: Atmadibrata B, Liu PY, Sokolowski N, Zhang L, Wong M, et al. (2014) The Novel Long Noncoding RNA linc00467 Promotes Cell Survival but Is Down- Regulated by N-Myc. PLoS ONE 9(2): e88112. doi:10.1371/journal.pone.0088112 Introduction intronic and intergenic (lincRNA) [9,10]. lncRNAs are emerging as important regulators of gene transcription, tumour initiation and progression [9,10]. For example, lincRNA-p21 is directly activated by p53 and functions as an inhibitor of the genes that interfere with apoptosis [11], the lincRNA CTBP1-AS promotes both hormone-dependent and castration-resistant prostate cancer growth [12], and the lincRNA MALAT1 and HOTAIR play critical roles in lung and breast cancer invasion and metastasis [13,14]. Neuroblastoma is a solid extracranial paediatric cancer that arises from neural crest cells, and accounts for 15% of cancer- related death in children [1]. Amplification of MYCN oncogene and consequent N-Myc oncoprotein over-expression occur in approximately 40% of high risk neuroblastoma, and is clinically associated with cancer metastasis, resistance to therapies and poor patient outcome [1,2]. Myc oncoproteins, including N-Myc and c-Myc, exert biolog- ical effects through modulating gene transcription. After Myc oncoproteins dimerize with Max, the Myc-MAX complex binds to Myc-responsive element E-boxes at target gene promoters, leading to transcriptional activation [3,4]. On the other hand, Myc oncoproteins repress gene transcription by forming transcriptional repressor complexes with histone deacetylases at Sp1-binding sites of target gene promoters [5,6,7,8]. Identifying N-Myc target genes and understanding the function of the N-Myc target genes are important in developing better anticancer therapies. Myc oncoproteins have been extensively shown to modulate the expression of microRNAs, and targeting the microRNAs is a promising approach for treating Myc-induced cancers (reviewed in [15]). However, little is known about which lincRNAs are Myc targets and whether the Myc target lincRNAs play a role in Myc- induced cancer. Here, we screened for lincRNA targets of N-Myc in neuroblastoma cells by noncoding RNA microarray, and identified linc00467 as an N-Myc target. While linc00467 had not been studied at all in the literature, we discovered that linc00467 suppressed the expression of its downstream protein-coding gene RD3, and induced neuroblastoma cell survival by reducing the expression of the tumour suppressor gene DKK1. Long noncoding RNAs (lncRNAs) are transcripts longer than 200 nucleotides without a functional open reading frame, and can be divided into five different types: sense, antisense, bidirectional, February 2014 | Volume 9 | Issue 2 | e88112 1 February 2014 | Volume 9 | Issue 2 | e88112 PLOS ONE | www.plosone.org linc00467 Promotes Cell Survival genome project). Linc00467 reduces mRNA expression of the linc00467 neighbouring protein-coding gene RD3 g g g g lincRNAs exert biological functions partly through in cis regulation of mRNA expression of their neighbouring protein coding genes through various mechanisms [12,21,22,23]. We therefore examined whether linc00467 regulated the expression of RD3, the gene immediately down-stream of linc00467. BE(2)-C and Kelly cells were transfected with control siRNA, linc00467 siRNA-1 or linc00467 siRNA-2 for 48 hours, followed by RT- PCR analysis of RD3 mRNA expression. As shown in Figure 2A, transfection with linc00467 siRNA-1 or linc00467 siRNA-2 reduced linc00467 RNA expression in the neuroblastoma cells. Importantly, knocking-down linc00467 expression up-regulated RD3 mRNA expression in both BE(2)-C and Kelly cells (Figure 2B). The data indicate that linc00467 reduces mRNA expression of its neighbouring protein-coding RD3. We have shown previously that N-Myc represses gene transcription by recruiting histone deacetylases to Sp1-binding site-enriched regions of target gene promoters [5,6,7,8]. To understand whether N-Myc could directly repress linc00467 gene transcription, we firstly analysed transcription factor binding sites at the linc00467 gene promoter with Gene-Regulation software (http://www.gene-regulation.com/pub/programs/alibaba2/index. html). Results showed that Sp1-binding sites were enriched at 2176 bp to 214 bp upstream of linc00467 gene transcription start site as well as +7 bp to +426 bp in intron 1 (Figure 1D). We then examined a c-Myc chromatin immunoprecipitation-sequencing (ChIP-Seq) dataset, which was generated by Dr. Michael Snyder’s group at Yale University for the ENCODE/SYDH project (The Encyclopedia of DNA Elements/Stanford/Yale/USC/Harvard N-Myc suppresses the expression of the long noncoding RNA linc00467 by direct binding to its gene promoter N-Myc suppresses the expression of the long noncoding RNA linc00467 by direct binding to its gene promoter Myc oncoproteins exert biological effects by modulating gene transcription. However, it is unknown whether N-Myc modulates the transcription of lncRNAs. We therefore performed differential gene expression analysis using NCodeTM Human Non-coding RNA Microarray in BE(2)-C neuroblastoma cells 30 hours after transfection with control siRNA or N-Myc siRNA No. 1 (N-Myc siRNA-1). As shown in Table 1, the microarray gene expression study showed that 5 lncRNAs were down-regulated, and 1 lncRNA was up-regulated, by N-Myc siRNA-1 within 30 hours by more than 2 fold. One of the lncRNAs most significantly up- regulated by N-Myc siRNA-1 was linc00467, which was identified by Human Genome Organisation Gene Nomenclature Commit- tee (HGNC) according to published DNA and cDNA sequencing data [16,17,18,19,20]. To validate the microarray data, we performed siRNA transfections with control siRNA, N-Myc siRNA-1 or N-Myc siRNA-2 for 48 hours in two MYCN oncogene amplified human neuroblastoma cell lines, BE(2)-C and Kelly, followed by real-time RT-PCR study of linc00467. As shown in Figure 1A, transfection with N-Myc siRNA-1 or N-Myc siRNA-2 reduced the expression of both N-Myc mRNA and protein in the two neuroblastoma cell lines. Consistent with the microarray data, down-regulation of N- Myc expression resulted in increased linc00467 expression (Figure 1B). We next performed RT-PCR study of N-Myc and linc00467 in SHEP-21N neuroblastoma cells, which were stably transfected with a tetracycline withdrawal-inducible N-Myc expression construct [6,7], 48 hours after incubation with or without tetracycline. As shown in Figure 1C, withdrawal of tetracycline induced N-Myc expression, and reduced linc00467 RNA expression. Introduction As shown in Figure 1E, the ChIP-seq data showed that c-Myc oncoprotein bound to the linc00467 gene core promoter region matching the Sp1-binding site-enriched region in K562 leukemia cells. Consistently, our own ChIP assays showed that an anti-N-Myc antibody efficiently immunoprecipitated the region of the linc00467 gene core promoter enriched in the Sp1- binding sites in BE(2)-C neuroblastoma cells (Figure 1F), in addition to the gene core promoter of ODC1 (Figure S1), a well- known Myc target gene. To further understand whether the binding of N-Myc to the linc00467 gene promoter region repressed linc00467 gene transcription, the Sp1-binding site-enriched region of the linc00467 gene promoter was cloned into a pLightSwitch_- Prom construct. Luciferase assays were performed in BE(2)-C cells after transfection with control siRNA or N-Myc siRNA-1, followed by transfection with a pLightSwitch_Prom construct expressing empty vector or the linc00467 promoter region. Results showed that knocking-down N-Myc expression significantly up-regulated luciferase activity of the pLightSwitch_Prom construct expressing the linc00467 promoter region (Figure 1G). Taken together, the data suggest that N-Myc represses linc00467 gene transcription by direct binding to the Sp1-binding site-enriched region of the linc00467 gene promoter and reducing linc00467 promoter activity. N-Myc represses RD3 gene expression by direct binding to the RD3 gene promoter (G) BE(2)-C cells were transfected with control siRNA or N-Myc siRNA-1, followed by co-transfection with Cypridina TK control construct plus empty vector or linc00467 gene promoter pLightSwitch_Prom construct. Luciferase activities were measured with a LightSwitch Dual Assay System kit, and expressed as a percentage change relative to control siRNA transfected samples. Error bars represented standard error. *, ** and *** indicated P,0.05, 0.01 and 0.001 respectively. doi:10.1371/journal.pone.0088112.g001 linc00467 Promotes Cell Survival Figure 1. N-Myc represses linc00467 gene expression by direct binding to the linc00467 gene promoter. (A–B). BE(2)-C and Kelly cells were transfected with scrambled control (Cont) siRNA, N-Myc siRNA-1 or N-Myc siRNA-2 for 48 hours, followed by RNA and protein extraction, real- time RT-PCR and immunoblot analyses of N-Myc mRNA, protein expression (A) or linc00467 RNA expression (B). (C) SHEP-21N cells were incubated with or without tetracycline for 48 hours, followed by RNA extraction and RT-PCR analysis of N-Myc and linc00467 RNA expression. (D) Schematic representation of the linc00467 gene promoter. TSS represented transcription start site, and | represented Sp1-binding sites. (E) ChIP-Seq data from Dr. Michael Snyder’s group at Yale University for the ENCODE/SYDH project generated from K562 cells. (F) ChIP assays were performed with a control or anti-N-Myc antibody (Ab) and primers targeting a negative control region or the linc00467 gene core promoter region enriched in Sp1-binding sites in BE(2)-C cells. Fold enrichment was calculated by dividing PCR products from DNA samples immunoprecipitated with the anti-N-Myc Ab by PCR products from DNA samples immunoprecipitated with the control Ab, relative to input. Fold enrichment at the negative control region was artificially set as 1.0. (G) BE(2)-C cells were transfected with control siRNA or N-Myc siRNA-1, followed by co-transfection with Cypridina TK control construct plus empty vector or linc00467 gene promoter pLightSwitch_Prom construct. Luciferase activities were measured with a LightSwitch Dual Assay System kit, and expressed as a percentage change relative to control siRNA transfected samples. Error bars represented standard error. *, ** and *** indicated P,0.05, 0.01 and 0.001 respectively. doi:10 1371/journal pone 0088112 g001 PCR analysis of RD3 mRNA expression was performed in BE(2)- C and Kelly cells 48 hours after transfection with control siRNA, N-Myc siRNA-1 or N-Myc siRNA-2. Surprisingly, opposite to our expectation, knocking-down N-Myc expression with N-Myc siRNA-1 or N-Myc siRNA-2 up-regulated RD3 mRNA expres- sion (Figure 3A). N-Myc represses RD3 gene expression by direct binding to the RD3 gene promoter p53 has been shown to indirectly repress the expression of a subset of genes by inducing the expression of lincRNA-p21 [11]. We therefore examined whether N-Myc increased RD3 mRNA expression indirectly by suppressing linc00467 expression. RT- Table 1. Modulation of lncRNA expression by N-Myc siRNA-1 by more than 2 fold thirty hours after siRNA transfection, as identified by lncRNA microarray. Probe name Target ID Probe Sequence Fold change h13721 AL122062 TACTTCTAAAAAAAGTATTTTGTATCTACTTTTGTAACTTCGTCAGAATAAAATATATTG; TTCGTCAGAATAAAATATATTG 0.33 h03377 AK002005 GTTATCCAGGAAACAATATATATACACTTGTGAACTGTTGTTTGTGATTTAAGCATATAT 0.36 h31926 uc002oww TTGTAGATTGGTTGTGTTTACACAGTTGTATATATTGACACCCTTGAGTGTTATGACTTC 0.37 h33198 uc002vpg AATATTCATTTCTGAAATACTTTAGTATGATAGATAAATTTGGTTAAGTTCTTGTTCATT 0.46 h17938 BC039246 ATGTACTAATAATTTTATCTGACTTCTGTTTATATCATTTGTACAATTACATGGTTGTAA 0.47 h26159 linc00467 GAAACAACCACATATGTCACCTTTCCAAGAGGGACTGAAACTGGGCTGACCCTTTTGATT 2.17 doi:10.1371/journal.pone.0088112.t001 PLOS ONE | www.plosone.org 2 February 2014 | Volume 9 | Issue 2 | e88112 Table 1. Modulation of lncRNA expression by N-Myc siRNA-1 by more than 2 fold thirty hours after siRNA transfection, as identified by lncRNA microarray. Probe name Target ID Probe Sequence Fold change h13721 AL122062 TACTTCTAAAAAAAGTATTTTGTATCTACTTTTGTAACTTCGTCAGAATAAAATATATTG; TTCGTCAGAATAAAATATATTG 0.33 h03377 AK002005 GTTATCCAGGAAACAATATATATACACTTGTGAACTGTTGTTTGTGATTTAAGCATATAT 0.36 h31926 uc002oww TTGTAGATTGGTTGTGTTTACACAGTTGTATATATTGACACCCTTGAGTGTTATGACTTC 0.37 h33198 uc002vpg AATATTCATTTCTGAAATACTTTAGTATGATAGATAAATTTGGTTAAGTTCTTGTTCATT 0.46 h17938 BC039246 ATGTACTAATAATTTTATCTGACTTCTGTTTATATCATTTGTACAATTACATGGTTGTAA 0.47 h26159 linc00467 GAAACAACCACATATGTCACCTTTCCAAGAGGGACTGAAACTGGGCTGACCCTTTTGATT 2.17 doi:10.1371/journal.pone.0088112.t001 February 2014 | Volume 9 | Issue 2 | e88112 2 linc00467 Promotes Cell Survival PCR analysis of RD3 mRNA expression was performed in BE(2)- siRNA-1 or N-Myc siRNA-2 up-regulated RD3 mRNA expres- Figure 1. N-Myc represses linc00467 gene expression by direct binding to the linc00467 gene promoter. (A–B). BE(2)-C and Kelly cells were transfected with scrambled control (Cont) siRNA, N-Myc siRNA-1 or N-Myc siRNA-2 for 48 hours, followed by RNA and protein extraction, real- time RT-PCR and immunoblot analyses of N-Myc mRNA, protein expression (A) or linc00467 RNA expression (B). (C) SHEP-21N cells were incubated with or without tetracycline for 48 hours, followed by RNA extraction and RT-PCR analysis of N-Myc and linc00467 RNA expression. (D) Schematic representation of the linc00467 gene promoter. TSS represented transcription start site, and | represented Sp1-binding sites. (E) ChIP-Seq data from Dr. Michael Snyder’s group at Yale University for the ENCODE/SYDH project generated from K562 cells. (F) ChIP assays were performed with a control or anti-N-Myc antibody (Ab) and primers targeting a negative control region or the linc00467 gene core promoter region enriched in Sp1-binding sites in BE(2)-C cells. Fold enrichment was calculated by dividing PCR products from DNA samples immunoprecipitated with the anti-N-Myc Ab by PCR products from DNA samples immunoprecipitated with the control Ab, relative to input. Fold enrichment at the negative control region was artificially set as 1.0. Reduction in DKK1 expression contributes to linc00467- mediated neuroblastoma cell survival doi:10.1371/journal.pone.0088112.g002 doi:10.1371/journal.pone.0088112.g002 To understand the mechanism through which linc00467 promotes neuroblastoma cell survival, we performed differential gene expression study of linc00467 target genes in BE(2)-C cells 48 hours after transfection with control siRNA or linc00467 siRNA-1. As shown in Table S1, one of the genes significantly up- regulated by linc00467 siRNA-1 was DKK1, a Wnt antagonist tumour suppressor gene known to induce cancer cell apoptosis [24,25]. RT-PCR analysis confirmed that transfection with linc00467 siRNA-1 or linc00467 siRNA-2 considerably up- regulated the expression of DKK1 in BE(2)-C and Kelly cells (Figure 5A). To understand whether N-Myc directly repressed RD3 gene transcription, we firstly analysed transcription factor binding sites at the RD3 gene promoter with Gene-Regulation software (http:// www.gene-regulation.com/pub/programs/alibaba2/index.html.) Results showed that Sp1-binding sites were enriched at +475 bp to +731 bp of RD3 gene intron 1, relative to intron 1 start site (Figure 3C). We then examined a c-Myc ChIP-Seq dataset, which was generated by Dr. Michael Snyder’s group at Yale University for the ENCODE/SYDH project. As shown in Figure 3D, the ChIP-seq data showed that the c-Myc oncoprotein bound to the RD3 gene intron 1 region matching the Sp1-binding site-enriched fragment in K562 leukemia cells. Consistently, our own ChIP assays with primers targeting RD3 intron 1 showed that an anti-N- Myc antibody efficiently immunoprecipitated the RD3 gene intron 1 region enriched in Sp1-binding sites in BE(2)-C neuroblastoma cells (Figure 3E). To further understand whether the binding of N- Myc to the RD3 intron 1 region repressed RD3 gene transcription, the Sp1-binding site-enriched RD3 intron 1 region was cloned into a pLightSwitch_Prom construct. Luciferase assays were performed in BE(2)-C cells after transfection with control siRNA or N-Myc siRNA-1, followed by transfection with a pLightSwitch_Prom construct expressing empty vector or the RD3 intron 1 region. Results showed that knocking-down N-Myc expression signifi- cantly up-regulated luciferase activity of the pLightSwitch_Prom construct expressing the RD3 intron 1 region (Figure 3F). To understand whether N-Myc and linc00467 co-operatively reduce RD3 expression, we transfected BE(2)-C cells with control siRNA, To examine whether up-regulation of the tumour suppressor gene DKK1 contributed to linc00467 siRNA-mediated apoptosis, we transfected BE(2)-C cells with control siRNA, linc00467 siRNA-1, DKK1 siRNA, or combination of linc00467 siRNA-1 and DKK1 siRNA. RT-PCR analysis showed that DKK1 siRNA reduced DKK1 gene expression, and blocked linc00467 siRNA- mediated DKK1 gene up-regulation (Figure 5B). N-Myc represses RD3 gene expression by direct binding to the RD3 gene promoter Consistent with this finding, withdrawal of tetracycline from SHEP-21N neuroblastoma cells, which induced N-Myc expression, reduced linc00467 RNA expression (Figure 3B). February 2014 | Volume 9 | Issue 2 | e88112 PLOS ONE | www.plosone.org PLOS ONE | www.plosone.org 3 linc00467 Promotes Cell Survival Figure 2. linc00467 reduces mRNA expression of its neigh- bouring protein-coding RD3. BE(2)-C and Kelly cells were transfect- ed with scrambled control siRNA, linc00467 siRNA-1 or linc00467 siRNA- 2 for 48 hours, followed by RNA extraction and and real-time RT-PCR analysis of the expression of linc00467 (A) or RD3 (B). Error bars represented standard error. * indicated P,0. 05, and *** indicated P,0.001. doi:10.1371/journal.pone.0088112.g002 N-Myc siRNA, linc00467 siRNA, or combination of N-Myc siRNA and linc00467 siRNA. RT-PCR analysis showed that N- Myc siRNA and linc00467 siRNA did not have co-operative effect in modulating RD3 expression (Figure S2). Taken together, the data suggest that N-Myc represses RD3 gene transcription by direct binding to the Sp1-binding site-enriched region of the RD3 gene promoter and reducing RD3 promoter activity. N-Myc siRNA, linc00467 siRNA, or combination of N-Myc siRNA and linc00467 siRNA. RT-PCR analysis showed that N- Myc siRNA and linc00467 siRNA did not have co-operative effect in modulating RD3 expression (Figure S2). Taken together, the data suggest that N-Myc represses RD3 gene transcription by direct binding to the Sp1-binding site-enriched region of the RD3 gene promoter and reducing RD3 promoter activity. Knocking-down linc00467 expression reduces neuroblastoma cell survival To understand whether repression of linc00467 expression by N-Myc contributed to an N-Myc-induced cancer phenotype, we transfected BE(2)-C and Kelly cells with control siRNA or linc00467 siRNA for 48 hours, followed by Alamar blue assays. As shown in Figure 4A, knocking-down linc00467 expression with siRNA reduced the number of viable BE(2)-C and Kelly cells. Alamar blue assays in BE(2)-C and Kelly cells 0, 24, 72 and 96 hours after transfection with control siRNA or linc00467 siRNA showed that linc00467 siRNA considerably reduced the number of viable cells 72 and 96 hours after siRNA transfection (Figure 4B). To examine whether the effect was due to cell death, we transfected BE(2)-C and Kelly cells with control siRNA or linc00467 siRNA, followed by staining with propidium iodide (PI) and cell cycle study with flow cytometry. We also transfected BE(2)-C and Kelly cells with control siRNA or linc00467 siRNA, followed by staining with the apoptosis marker fluorescein isothiocyanate (FITC)-conjugated Annexin V and analyses with flow cytometry. Data analyses showed that knocking-down linc00467 expression with siRNA increased the proportion of cells at sub-G1 phase of the cell cycle (Figure 4C) and the proportion of apoptotic cells (Figure 4D). Taken together, the data suggest that linc00467 promotes neuroblastoma cell survival. Figure 2. linc00467 reduces mRNA expression of its neigh- bouring protein-coding RD3. BE(2)-C and Kelly cells were transfect- ed with scrambled control siRNA, linc00467 siRNA-1 or linc00467 siRNA- 2 for 48 hours, followed by RNA extraction and and real-time RT-PCR analysis of the expression of linc00467 (A) or RD3 (B). Error bars represented standard error. * indicated P,0. 05, and *** indicated P,0.001. doi:10 1371/journal pone 0088112 g002 Reduction in DKK1 expression contributes to linc00467- mediated neuroblastoma cell survival Luciferase activities were measured with a LightSwitch Dual Assay System kit, and expressed as a percentage change relative to control siRNA transfected samples. Error bars represented standard error. * indicated P,0.05. Figure 4. linc00467 enhances neuroblastoma cell survival. (A) BE(2)-C and Kelly cells were transfected with scrambled control siRNA or linc00467 siRNA-1 for 48 hours, followed by Alamar blue assays. The effect of linc00467 siRNA-1 was expressed as a percentage change in the number of viable cells after transfection with linc00467 siRNA-1, compared with control siRNA-transfected samples. (B) BE(2)-C and Kelly cells were transfected with scrambled control siRNA or linc00467 siRNA- 1 for 0, 72 or 96 hours, followed by Alamar blue assays. The effects of time and siRNAs were expressed as percentages of the number of viable cells after transfection with control siRNA for 0 hour. (C) After transfection with control siRNA or linc00467 siRNA-1 for 72 hours, BE(2)- C and Kelly cells were stained with propodium iodide, and subjected to flow cytometry analyses of the cell cycle. The percentage of cells at sub- G1 phase was calculated. (D) After transfection with control siRNA or linc00467 siRNA-1 for 72 hours, BE(2)-C and Kelly cells were stained with FITC-conjugated Annexin V, and subjected to flow cytometry analyses. The percentage of cells positively stained by Annexin V was calculated. Error bars represented standard error. * indicated p,0.05, and ** p,0.01. Figure 3. N-Myc represses RD3 gene transcription by direct binding to the RD3 gene promoter. (A) BE(2)-C and Kelly cells were transfected with scrambled control (Cont) siRNA, N-Myc siRNA-1 or N- Myc siRNA-2 for 48 hours, followed by RNA extraction and real-time RT- PCR analyses of RD3 mRNA expression. (B) SHEP-21N cells were incubated with or without tetracycline for 48 hours, followed by RNA extraction and RT-PCR analysis of RD3 RNA expression. (C) Schematic representation of the RD3 gene promoter. TSS represented transcription start site, and | represented Sp1-binding sites. (D) ChIP-Seq data from Dr. Michael Snyder’s group at Yale University for the ENCODE/SYDH project generated from K562 cells. (E) ChIP assays were performed with a control or anti-N-Myc antibody (Ab) and primers targeting a negative control region or the RD3 gene core promoter region enriched in Sp1- binding sites in BE(2)-C cells. Reduction in DKK1 expression contributes to linc00467- mediated neuroblastoma cell survival The effect of linc00467 siRNA-1 was expressed as a percentage change in the number of viable cells after transfection with linc00467 siRNA-1, compared with control siRNA-transfected samples. (B) BE(2)-C and Kelly cells were transfected with scrambled control siRNA or linc00467 siRNA- 1 for 0, 72 or 96 hours, followed by Alamar blue assays. The effects of time and siRNAs were expressed as percentages of the number of viable cells after transfection with control siRNA for 0 hour. (C) After transfection with control siRNA or linc00467 siRNA-1 for 72 hours, BE(2)- C and Kelly cells were stained with propodium iodide, and subjected to flow cytometry analyses of the cell cycle. The percentage of cells at sub- G1 phase was calculated. (D) After transfection with control siRNA or linc00467 siRNA-1 for 72 hours, BE(2)-C and Kelly cells were stained with FITC-conjugated Annexin V, and subjected to flow cytometry analyses. The percentage of cells positively stained by Annexin V was calculated. Error bars represented standard error. * indicated p,0.05, and ** p,0.01. doi:10.1371/journal.pone.0088112.g004 Figure 3. N-Myc represses RD3 gene transcription by direct binding to the RD3 gene promoter. (A) BE(2)-C and Kelly cells were transfected with scrambled control (Cont) siRNA, N-Myc siRNA-1 or N- Myc siRNA-2 for 48 hours, followed by RNA extraction and real-time RT- PCR analyses of RD3 mRNA expression. (B) SHEP-21N cells were incubated with or without tetracycline for 48 hours, followed by RNA extraction and RT-PCR analysis of RD3 RNA expression. (C) Schematic representation of the RD3 gene promoter. TSS represented transcription start site, and | represented Sp1-binding sites. (D) ChIP-Seq data from Dr. Michael Snyder’s group at Yale University for the ENCODE/SYDH project generated from K562 cells. (E) ChIP assays were performed with a control or anti-N-Myc antibody (Ab) and primers targeting a negative control region or the RD3 gene core promoter region enriched in Sp1- binding sites in BE(2)-C cells. Fold enrichment was calculated by dividing PCR products from DNA samples immunoprecipitated with the anti-N-Myc Ab by PCR products from DNA samples immunoprecipitated with the control Ab, relative to input. Fold enrichment at the negative control region was artificially set as 1.0. (F) BE(2)-C cells were transfected with control siRNA or N-Myc siRNA-1, followed by co- transfection with Cypridina TK control construct plus empty vector or RD3 gene promoter pLightSwitch_Prom construct. Reduction in DKK1 expression contributes to linc00467- mediated neuroblastoma cell survival Alamar blue assays (Figure 5C) and flow cytometry analyses of Annexin V positively stained cells (Figure 5D) showed that linc00467 siRNA-1 reduced the number of viable cells and increased the proportion of cells positively stained by Annexin V, and that DKK1 siRNA blocked linc00467 siRNA-1-mediated reduction in the number of viable neuroblastoma cells and induction of Annexin V positively stained cells. Taken together, the data suggest that reduction in DKK1 expression contributes to linc00467-mediated neuroblas- toma cell survival. February 2014 | Volume 9 | Issue 2 | e88112 PLOS ONE | www.plosone.org 4 linc00467 Promotes Cell Survival Figure 3. N-Myc represses RD3 gene transcription by direct binding to the RD3 gene promoter. (A) BE(2)-C and Kelly cells were transfected with scrambled control (Cont) siRNA, N-Myc siRNA-1 or N- Myc siRNA-2 for 48 hours, followed by RNA extraction and real-time RT- PCR analyses of RD3 mRNA expression. (B) SHEP-21N cells were incubated with or without tetracycline for 48 hours, followed by RNA extraction and RT-PCR analysis of RD3 RNA expression. (C) Schematic representation of the RD3 gene promoter. TSS represented transcription start site, and | represented Sp1-binding sites. (D) ChIP-Seq data from Dr. Michael Snyder’s group at Yale University for the ENCODE/SYDH project generated from K562 cells. (E) ChIP assays were performed with a control or anti-N-Myc antibody (Ab) and primers targeting a negative control region or the RD3 gene core promoter region enriched in Sp1- binding sites in BE(2)-C cells. Fold enrichment was calculated by dividing PCR products from DNA samples immunoprecipitated with the anti-N-Myc Ab by PCR products from DNA samples immunoprecipitated with the control Ab, relative to input. Fold enrichment at the negative control region was artificially set as 1.0. (F) BE(2)-C cells were transfected with control siRNA or N-Myc siRNA-1, followed by co- transfection with Cypridina TK control construct plus empty vector or RD3 gene promoter pLightSwitch_Prom construct. Luciferase activities were measured with a LightSwitch Dual Assay System kit, and expressed as a percentage change relative to control siRNA transfected samples. Error bars represented standard error. * indicated P,0.05. doi:10 1371/journal pone 0088112 g003 Figure 4. linc00467 enhances neuroblastoma cell survival. (A) BE(2)-C and Kelly cells were transfected with scrambled control siRNA or linc00467 siRNA-1 for 48 hours, followed by Alamar blue assays. Discussion lncRNAs are emerging as critical regulators of gene transcrip- tion, tumour initiation, progression and metastasis [9,10,26]. Myc oncoproteins, including N-Myc and c-Myc, are well-known to exert biological effects by modulating the expression of protein- coding genes and microRNAs [3,4,15]. However, little is known about whether Myc modulates the expression of lncRNAs, and whether regulation of lncRNA expression by Myc plays a role in Myc oncogenesis. In this study, we have performed genome-wide differential expression study with lncRNA microarray in neuro- blastoma cells 30 hours after knocking-down N-Myc gene expression. Data analysis reveals that knocking-down N-Myc gene expression for 30 hours alters the expression of 6 lncRNAs by more than 2 fold. One of the lncRNAs most significantly up- regulated by N-Myc siRNA is linc00467. lncRNAs exert biological effects through in cis and in trans regulation of RNA expression at both transcriptional and post- transcriptional levels. For examples, a number of lncRNAs have been shown to up- or down-regulate the expression of their neighboring protein-coding genes through modulating chromatin structure and gene transcription [14,22,27,28]. The lncRNAs DLEU1 and DLEU2 at 13q14.3 are often deleted in multiple types of cancers, and DLEU1 and DLEU2 modulate nuclear factor B function by down-regulating the transcription of their neighboring protein-coding KPNA3 and the microRNAs miR-15 and miR-16 [23]. Additionally, the lncRNA MALAT1 controls cell cycle progression by regulating the expression of the oncogenic transcription factor B-MYB through altering the binding of splicing factors on B-MYB pre-mRNA and causing aberrant alternative splicing [29], and the PTEN pseudogene expressed noncoding RNA antisense RNA (PTENpg1 asRNA) regulates both PTEN gene transcription and PTEN mRNA stability [30]. In this study, we have confirmed that knocking-down linc00467 up- regulates the expression of its neighbouring protein-coding gene RD3, which encodes a retinal protein responsible for the retinal degeneration disorder Leber congenital amaurosis type 12 [31,32]. linc00467 was identified by Human Genome Organisation Gene Nomenclature Committee (HGNC) based on published DNA and cDNA sequencing data [16,17,18,19,20]. Till today, the biological function of linc00467 is completely unknown. We have found that the linc00467 gene core promoter is enriched in Sp1- binding sites, and that c-Myc binds to the Sp1-binding site- enriched region of the lin00467 gene core promoter in K562 leukemia cells according to a publically available ChIP-Seq dataset. Reduction in DKK1 expression contributes to linc00467- mediated neuroblastoma cell survival Fold enrichment was calculated by dividing PCR products from DNA samples immunoprecipitated with the anti-N-Myc Ab by PCR products from DNA samples immunoprecipitated with the control Ab, relative to input. Fold enrichment at the negative control region was artificially set as 1.0. (F) BE(2)-C cells were transfected with control siRNA or N-Myc siRNA-1, followed by co- transfection with Cypridina TK control construct plus empty vector or RD3 gene promoter pLightSwitch_Prom construct. Luciferase activities were measured with a LightSwitch Dual Assay System kit, and expressed as a percentage change relative to control siRNA transfected samples. Error bars represented standard error. * indicated P,0.05. doi:10.1371/journal.pone.0088112.g003 Figure 3. N-Myc represses RD3 gene transcription by direct binding to the RD3 gene promoter. (A) BE(2)-C and Kelly cells were Figure 4. linc00467 enhances neuroblastoma cell survival. (A) BE(2)-C and Kelly cells were transfected with scrambled control siRNA or linc00467 siRNA-1 for 48 hours, followed by Alamar blue assays. The effect of linc00467 siRNA-1 was expressed as a percentage change in the number of viable cells after transfection with linc00467 siRNA-1, compared with control siRNA-transfected samples. (B) BE(2)-C and Kelly cells were transfected with scrambled control siRNA or linc00467 siRNA- 1 for 0, 72 or 96 hours, followed by Alamar blue assays. The effects of time and siRNAs were expressed as percentages of the number of viable cells after transfection with control siRNA for 0 hour. (C) After transfection with control siRNA or linc00467 siRNA-1 for 72 hours, BE(2)- C and Kelly cells were stained with propodium iodide, and subjected to flow cytometry analyses of the cell cycle. The percentage of cells at sub- G1 phase was calculated. (D) After transfection with control siRNA or linc00467 siRNA-1 for 72 hours, BE(2)-C and Kelly cells were stained with FITC-conjugated Annexin V, and subjected to flow cytometry analyses. The percentage of cells positively stained by Annexin V was calculated. Error bars represented standard error. * indicated p,0.05, and ** p,0.01. doi:10.1371/journal.pone.0088112.g004 February 2014 | Volume 9 | Issue 2 | e88112 PLOS ONE | www.plosone.org 5 linc00467 Promotes Cell Survival Figure 5. Reduction in DKK1 expression contributes to linc00467-mediated neuroblastoma cell survival. (A) BE(2)-C and Kelly cells were transfected with scrambled control siRNA, linc00467 siRNA-1 or linc00467 siRNA-2 for 48 hours, followed by RNA extraction and RT-PCR analysis of DKK1 gene expression. Reduction in DKK1 expression contributes to linc00467- mediated neuroblastoma cell survival In this study, we have confirmed that knocking-down linc00467 up- regulates the expression of its neighbouring protein-coding gene RD3, which encodes a retinal protein responsible for the retinal degeneration disorder Leber congenital amaurosis type 12 [31,32]. data demonstrate that linc00467 gene expression is reduced by N- Myc and up-regulated by N-Myc siRNAs. As N-Myc is well- known to repress gene transcription by direct binding to target gene promoter regions enriched in Sp1-binding sites [5,6,7,8], our data suggest that N-Myc represses linc00467 gene expression by direct binding to the linc00467 gene promoter region enriched in Sp1-binidng sites and suppresses linc00467 gene promoter activity. Reduction in DKK1 expression contributes to linc00467- mediated neuroblastoma cell survival (B) BE(2)-C cells were transfected with scrambled control siRNA, linc00467 siRNA-1, DKK1 siRNA, or combination of linc00467 siRNA-1 and DKK1 siRNA for 48 hours, followed by RNA extraction and RT-PCR analysis of DKK1 gene expression. (C) BE(2)-C cells were transfected with scrambled control siRNA, linc00467 siRNA-1, DKK1 siRNA, or combination of linc00467 siRNA-1 and DKK1 siRNA for 72 hours, followed by Alamar blue assays. The effect of linc00467 siRNA-1 alone, DKK1 siRNA alone, or combination of linc00467 siRNA-1 and DKK1 siRNA was expressed as a percentage change, compared with control siRNA-transfected samples. (D) BE(2)-C cells were transfected with scrambled control siRNA, linc00467 siRNA-1, DKK1 siRNA, or combination of linc00467 siRNA-1 and DKK1 siRNA for 72 hours, followed by staining with FITC-conjugated Annexin V, and subjected to flow cytometry analyses. The percentage of cells positively stained by Annexin V was calculated. Error bars represented standard error. *, ** and *** indicated p,0.05, 0.01 and 0.001 respectively. doi:10.1371/journal.pone.0088112.g005 Figure 5. Reduction in DKK1 expression contributes to linc00467-mediated neuroblastoma cell survival. (A) BE(2)-C and Kelly cells were transfected with scrambled control siRNA, linc00467 siRNA-1 or linc00467 siRNA-2 for 48 hours, followed by RNA extraction and RT-PCR analysis of DKK1 gene expression. (B) BE(2)-C cells were transfected with scrambled control siRNA, linc00467 siRNA-1, DKK1 siRNA, or combination of linc00467 siRNA-1 and DKK1 siRNA for 48 hours, followed by RNA extraction and RT-PCR analysis of DKK1 gene expression. (C) BE(2)-C cells were transfected with scrambled control siRNA, linc00467 siRNA-1, DKK1 siRNA, or combination of linc00467 siRNA-1 and DKK1 siRNA for 72 hours, followed by Alamar blue assays. The effect of linc00467 siRNA-1 alone, DKK1 siRNA alone, or combination of linc00467 siRNA-1 and DKK1 siRNA was expressed as a percentage change, compared with control siRNA-transfected samples. (D) BE(2)-C cells were transfected with scrambled control siRNA, linc00467 siRNA-1, DKK1 siRNA, or combination of linc00467 siRNA-1 and DKK1 siRNA for 72 hours, followed by staining with FITC-conjugated Annexin V, and subjected to flow cytometry analyses. The percentage of cells positively stained by Annexin V was calculated. Error bars represented standard error. *, ** and *** indicated p,0.05, 0.01 and 0.001 respectively. doi:10.1371/journal.pone.0088112.g005 Figure 5. Reduction in DKK1 expression contributes to linc00467-mediated neuroblastoma cell surv Figure 5. Reduction in DKK1 expression contributes to linc00467-mediated neuroblastoma cell survival. Reduction in DKK1 expression contributes to linc00467- mediated neuroblastoma cell survival (A) BE(2)-C and Kelly cells were transfected with scrambled control siRNA, linc00467 siRNA-1 or linc00467 siRNA-2 for 48 hours, followed by RNA extraction and RT-PCR analysis of DKK1 gene expression. (B) BE(2)-C cells were transfected with scrambled control siRNA, linc00467 siRNA-1, DKK1 siRNA, or combination of linc00467 siRNA-1 and DKK1 siRNA for 48 hours, followed by RNA extraction and RT-PCR analysis of DKK1 gene expression. (C) BE(2)-C cells were transfected with scrambled control siRNA, linc00467 siRNA-1, DKK1 siRNA, or combination of linc00467 siRNA-1 and DKK1 siRNA for 72 hours, followed by Alamar blue assays. The effect of linc00467 siRNA-1 alone, DKK1 siRNA alone, or combination of linc00467 siRNA-1 and DKK1 siRNA was expressed as a percentage change, compared with control siRNA-transfected samples. (D) BE(2)-C cells were transfected with scrambled control siRNA, linc00467 siRNA-1, DKK1 siRNA, or combination of linc00467 siRNA-1 and DKK1 siRNA for 72 hours, followed by staining with FITC-conjugated Annexin V, and subjected to flow cytometry analyses. The percentage of cells positively stained by Annexin V was calculated. Error bars represented standard error. *, ** and *** indicated p,0.05, 0.01 and 0.001 respectively. doi:10.1371/journal.pone.0088112.g005 data demonstrate that linc00467 gene expression is reduced by N- Myc and up-regulated by N-Myc siRNAs. As N-Myc is well- known to repress gene transcription by direct binding to target gene promoter regions enriched in Sp1-binding sites [5,6,7,8], our data suggest that N-Myc represses linc00467 gene expression by direct binding to the linc00467 gene promoter region enriched in Sp1-binidng sites and suppresses linc00467 gene promoter activity. lncRNAs exert biological effects through in cis and in trans regulation of RNA expression at both transcriptional and post- transcriptional levels. For examples, a number of lncRNAs have been shown to up- or down-regulate the expression of their neighboring protein-coding genes through modulating chromatin structure and gene transcription [14,22,27,28]. The lncRNAs DLEU1 and DLEU2 at 13q14.3 are often deleted in multiple types of cancers, and DLEU1 and DLEU2 modulate nuclear factor B function by down-regulating the transcription of their neighboring protein-coding KPNA3 and the microRNAs miR-15 and miR-16 [23]. Additionally, the lncRNA MALAT1 controls cell cycle progression by regulating the expression of the oncogenic transcription factor B-MYB through altering the binding of splicing factors on B-MYB pre-mRNA and causing aberrant alternative splicing [29], and the PTEN pseudogene expressed noncoding RNA antisense RNA (PTENpg1 asRNA) regulates both PTEN gene transcription and PTEN mRNA stability [30]. Real-time RT-PCR Following siRNA transfections, RNA was extracted from cells using PureLink RNA Mini kit (Life Technologies) according to the manufacturer’s instructions. RNA samples were then quantified using Nanodrop spectrophotometer and treated with DNAse 1 (Life Technologies) to remove remaining genomic DNA. Synthesis of cDNA from RNA samples was carried out using M-MLV Reverse Transcriptase (Invitrogen). Real-time RT PCR was performed in Applied Biosystems 7900 using SYBR green PCR Master Mix (Life Technologies) as previously described [5,6,7,8]. Discussion Moreover, our own ChIP assays have confirmed that N- Myc indeed binds to the Sp1-binding site-enriched region of the lin00467 gene core promoter, luciferase assays show that N-Myc siRNA enhances linc00467 gene promoter activity, and RT-PCR February 2014 | Volume 9 | Issue 2 | e88112 PLOS ONE | www.plosone.org 6 linc00467 Promotes Cell Survival Surprisingly, we have also confirmed that N-Myc suppresses RD3 gene expression through direct binding to the RD3 gene promoter region enriched in Sp1-binding sites and reducing RD3 gene promoter activity. These data indicate that linc00467 reduces RD3 mRNA expression most likely through an in cis mechanism, and that N-Myc-mediated suppression of linc00467 gene tran- scription counterintuitively blocks N-Myc-mediated reduction in RD3 mRNA expression. In addition, our differential gene expression study with Affymetrix microarray has identified DKK1 as one of the genes significantly up-regulated by linc00467 siRNA, suggesting that linc00467 is also likely to modulate gene expression through in trans mechanisms. lncRNA microarray Neuroblastoma BE(2)-C cells were transfected with scrambled control siRNA or N-Myc siRNA-1. Thirty hours after siRNA transfection, RNA was extracted from the cells with RNeasy mini kit and treated with DNAse 1. Differential gene expression was examined with NCodeTM Human Non-coding RNA Microarray (Invitrogene), according to the manufacturer’s instructions. Results from the microarray hybridization were analysed with GeneSpring software (GeneSpring), and deposited at Gene Expression Omnibus website (accession number: GSE52984) While the biological function of linc00467 is completely unknown in the literature, the Wnt antagonist DKK1 is well- known to induce cancer cell apoptosis and function as a tumour suppressor gene [24,25]. This study reveals that knocking-down linc00467 gene expression reduces the number of viable neuro- blastoma cells, increases the percentage of cells at sub-G1 phase of the cell cycle and induces apoptosis in neuroblastoma cells. Importantly, simultaneous knocking-down DKK1 expression blocks linc00467 siRNA-regulated neuroblastoma cell death. The data suggest that linc00467 may play a role in tumourigenesis through reducing DKK1 expression, leading to enhanced tumour cell viability, and that N-Myc-mediated suppression of linc00467 gene transcription counterintuitively blocks N-Myc-mediated cell survival. Chromatin imunoprecipitation (ChIP) assays ChIP assays were performed with an anti-N-Myc antibody (Merck Millipore) or a control antibody and PCR with primers targeting negative control region or Sp1-binding site-enriched region of the linc00467 or RD3 gene core promoter. Fold enrichment of the linc00467 and RD3 gene core promoter region by the anti-N-Myc antibody was calculated by dividing the PCR product from the linc00467 or RD3 gene core promoter region by the PCR product from the negative control region, relative to input. siRNA transfection Cells were transfected with AllStars negative control siRNA, N- Myc siRNA, linc00467 siRNA or DKK1 siRNA (Qiagen) using Lipofectamine 2000 (Life Technologies) as previously described [5,6,7,8]. Cell culture Neuroblastoma BE(2)-C cells were cultured in Dulbecco’s modified Eagle’s medium supplemented with 10% fetal calf serum [5,6,7,8]. Kelly cells were grown in RPMI 1640 supplemented with 10% fetal calf serum and 1% L-glutamine. Immunoblot For the analysis of protein expression by immunoblot, cells were lysed, protein extracted and separated by gel electrophoresis. After western transfer, membranes were probed with mouse anti-N-Myc antibody (1:1000) (Santa Cruz Biotech), followed by horseradish peroxidase-conjugated anti-mouse (1:10000) antiserum (Santa Cruz Biotech). Protein bands were visualized with SuperSignal (Pierce). The membranes were lastly re-probed with an anti-actin antibody (Sigma) as loading controls. Luciferase assays Sp1-binding site enriched linc00467 gene promoter region (2248 bp upstream of linc00467 gene transcription start site to +567 bp of intron 1) was custom-cloned into the pLightSwitch_ Prom construct by SwitchGear Genomics. Sp1-binding site enriched intron 1 region of RD3 (0 bp to +1043 bp) was also custom-cloned into the pLightSwitch_Prom construct by Switch- Gear Genomics. BE(2)-C neuroblastoma cells were transfected with control siRNA or N-Myc siRNA-1, followed by co- transfection with Cypridina TK control construct plus empty vector, linc00467 or RD3 gene promoter pLightSwitch_Prom construct. Luciferase activities were measured with a LightSwitch Dual Assay System kit (SwitchGear Genomics) according to the manufacturer’s instructions, and expressed as percentage changes relative to control siRNA transfected samples. mRNA microarray Neuroblastoma BE(2)-C cells were transfected with scrambled control siRNA or linc00467 siRNA-1. Fourty-eight hours after siRNA transfection, RNA was extracted from the cells with RNeasy mini kit. Differential gene expression was examined with Affymetrix HuGene-2_0-st Arrays (Affymetrix), according to the manufacturer’s instructions. Results from the microarray hybrid- ization were analysed in R (http://www.r-project.org/) with bioconductor package (http://www.bioconductor.org/), and de- posited at Gene Expression Omnibus website (accession number: GSE52985). In summary, this study identifies lncRNAs as targets of N-Myc in neuroblastoma cells through genome-wide differential gene expression study, and demonstrates that N-Myc suppresses linc00467 gene transcription through direct binding to the linc00467 gene promoter. linc00467 reduces the expression of its neighbouring protein-coding gene RD3, while N-Myc suppresses RD3 gene transcription through direct binding to the RD3 gene promoter. Importantly, linc00467 enhances neuroblastoma cell survival through reducing DKK1 expression. These findings therefore demonstrate that N-Myc-mediated suppression of linc00467 gene transcription counterintuitively blocks N-Myc- mediated reduction in RD3 mRNA expression, and reduces neuroblastoma cell survival by inducing DKK1 expression. References 1. Maris JM, Hogarty MD, Bagatell R, Cohn SL (2007) Neuroblastoma. Lancet 369: 2106–2120. 1. Maris JM, Hogarty MD, Bagatell R, Cohn SL (2007) Neuroblastoma. Lancet 369: 2106–2120. 16. Hosgood HD 3rd, Zhang L, Shen M, Berndt SI, Vermeulen R, et al. (2009) Association between genetic variants in VEGF, ERCC3 and occupational benzene haematotoxicity. Occup Environ Med 66: 848–853. 2. Brodeur GM (2003) Neuroblastoma: biological insights into a clinical enigma. Nat Rev Cancer 3: 203–216. 2. Brodeur GM (2003) Neuroblastoma: biological insights into a clinical enigma. Nat Rev Cancer 3: 203–216. 17. Gregory SG, Barlow KF, McLay KE, Kaul R, Swarbreck D, et al. (2006) The DNA sequence and biological annotation of human chromosome 1. 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(2011) SIRT1 promotes N-Myc oncogenesis through a positive feedback loop involving the effects of MKP3 and ERK on N-Myc protein stability. PLoS Genet 7: e1002135. 20. Strausberg RL, Feingold EA, Grouse LH, Derge JG, Klausner RD, et al. (2002) Generation and initial analysis of more than 15,000 full-length human and mouse cDNA sequences. Proc Natl Acad Sci U S A 99: 16899–16903. y p y 7. Liu T, Tee AE, Porro A, Smith SA, Dwarte T, et al. (2007) Activation of tissue transglutaminase transcription by histone deacetylase inhibition as a therapeutic approach for Myc oncogenesis. Proc Natl Acad Sci U S A 104: 18682–18687. 21. Wang KC, Yang YW, Liu B, Sanyal A, Corces-Zimmerman R, et al. Statistical analysis Experiments were conducted 3 times in duplicates. Data were analysed on Prism 6 software (GraphPad) and presented as mean 6 standard error. Differences were analyzed for significance using ANOVA among groups or two-sided unpaired t-test for two groups. A p value of less than 0.05 was considered statistically significant. Acknowledgments Children’s Cancer Institute Australia is affiliated with University of New South Wales and Sydney Children’s Hospital. Flow cytometry study Figure S2 N-Myc and linc00467 do not have a co-operative effect on RD3 expression in neuroblastoma cells. BE(2)-C cells were transfected with scrambled control siRNA, N-Myc siRNA-1, N-Myc siRNA-2, linc00467 siRNA-1, linc00467 siRNA-2, combination of N-Myc siRNA-1 and linc00467 siRNA-1, or combination of N-Myc siRNA-2 and linc00467 siRNA-2 for 48 hours, followed by RT-PCR analysis of RD3 expression. Error bars represented standard error. * indicated P,0. 01, compared with control siRNA-transfected samples. For the analysis of cells at sub-G1 phase, seventy-two hours after siRNA transfection, cells were harvested, fixed in 80% ethanol, washed and then stained with 50 mg/ml propidium iodide (Sigma) in solution containing 2 mg/ml RNase (Roche). Flow cytometric analysis of the cells was performed using FACS Calibur machine and FACS Diva software (BD Biosciences). The percentage of cells at sub-G1 phase of the cell cycle was analyzed. For the analysis of apoptosis, seventy-two hours after siRNA transfection, cells were incubated with FITC-conjugated Annexin V (BD Biosciences), and then subjected to flow cytometric analysis of FITC-positive cells using FACS Calibur machine and FACS Diva software. Table S1 Modulation of target gene expression by linc00467 siRNA-1 by more than 1.8 fold, as identified by Affymetrix microarray, in BE(2)-C cells 48 hours after transfection with control siRNA or linc00467 siRNA-1. The cut-off was set at 1.80 fold, as linc00467 siRNA-1 reduced the expression of linc00467 by 1.841 fold. Alamar blue assays Alamar blue assays were performed as previously described [33]. Briefly, cells were transfected with various siRNAs in 96 well February 2014 | Volume 9 | Issue 2 | e88112 PLOS ONE | www.plosone.org 7 linc00467 Promotes Cell Survival immunoprecipitated with the anti-N-Myc Ab by PCR products from DNA samples immunoprecipitated with the control Ab, relative to input. Fold enrichment at the negative control region was artificially set as 1.0. Error bars represented standard error. ** indicated P,0. 01. (PDF) plates. After siRNA transfection, cells were incubated with Alamar blue (Invitrogen), and the plates were read on a microplate reader at 570/595 nm. Results were calculated according to the optical density absorbance units and expressed as percentage change in viable cell number. Author Contributions Figure S1 N-Myc directly binds to the ODC1 gene promoter. ChIP assays were performed with a control or anti-N-Myc antibody (Ab) and primers targeting a negative control region or the ODC1 gene core promoter region. 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(2006) Premature truncation of a novel protein, RD3, exhibiting subnuclear localization is associated with retinal degeneration. Am J Hum Genet 79: 1059–1070. 28. Lai F, Orom UA, Cesaroni M, Beringer M, Taatjes DJ, et al. (2013) Activating RNAs associate with Mediator to enhance chromatin architecture and transcription. Nature 494: 497–501. linc00467 Promotes Cell Survival References February 2014 | Volume 9 | Issue 2 | e88112 PLOS ONE | www.plosone.org February 2014 | Volume 9 | Issue 2 | e88112 8 linc00467 Promotes Cell Survival linc00467 Promotes Cell Survival 27. Tsai MC, Manor O, Wan Y, Mosammaparast N, Wang JK, et al. (2010) Long noncoding RNA as modular scaffold of histone modification complexes. Science 329: 689–693. 31. Friedman JS, Chang B, Kannabiran C, Chakarova C, Singh HP, et al. (2006) Premature truncation of a novel protein, RD3, exhibiting subnuclear localization is associated with retinal degeneration. Am J Hum Genet 79: 1059–1070. 28. Lai F, Orom UA, Cesaroni M, Beringer M, Taatjes DJ, et al. (2013) Activating RNAs associate with Mediator to enhance chromatin architecture and transcription. Nature 494: 497–501. 32. Peshenko IV, Olshevskaya EV, Azadi S, Molday LL, Molday RS, et al. (2011) Retinal degeneration 3 (RD3) protein inhibits catalytic activity of retinal membrane guanylyl cyclase (RetGC) and its stimulation by activating proteins. Biochemistry 50: 9511–9519. 29. Tripathi V, Shen Z, Chakraborty A, Giri S, Freier SM, et al. (2013) Long noncoding RNA MALAT1 controls cell cycle progression by regulating the expression of oncogenic transcription factor B-MYB. PLoS Genet 9: e1003368. 33. Liu T, Liu PY, Tee AEL, Haber M, Norris MD, et al. (2009) Over-expression of clusterin is a resistance factor to the anti-cancer effect of histone deacetylase inhibitors. European Journal of Cancer 45: 1846–1854. p g p 30. Johnsson P, Ackley A, Vidarsdottir L, Lui WO, Corcoran M, et al. (2013) A pseudogene long-noncoding-RNA network regulates PTEN transcription and translation in human cells. Nat Struct Mol Biol 20: 440–446. February 2014 | Volume 9 | Issue 2 | e88112 PLOS ONE | www.plosone.org PLOS ONE | www.plosone.org 9
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The Effect of Emotional Intelligence Group Training on Human and Social Capital in Isfahan University of Technology
International letters of social and humanistic sciences
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www.ssoar.info www.ssoar.info The effect of emotional intelligence group training on human and social capital in Isfahan University of technology Abedi, Mohammad Reza; Nilforoushan, Parisa; Charsoughi, Batoul Tadayon Veröffentlichungsversion / Published Version Zeitschriftenartikel / journal article Empfohlene Zitierung / Suggested Citation: Abedi, M. R., Nilforoushan, P., & Charsoughi, B. T. (2015). The effect of emotional intelligence group training on human and social capital in Isfahan University of technology. International Letters of Social and Humanistic Sciences, 50, 91-99. https://doi.org/10.18052/www.scipress.com/ILSHS.50.91 Empfohlene Zitierung / Suggested Citation: Abedi, M. R., Nilforoushan, P., & Charsoughi, B. T. (2015). The effect of emotional intelligence group training on human and social capital in Isfahan University of technology. International Letters of Social and Humanistic Sciences, 50, 91-99. https://doi.org/10.18052/www.scipress.com/ILSHS.50.91 Empfohlene Zitierung / Suggested Citation: Abedi, M. R., Nilforoushan, P., & Charsoughi, B. T. (2015). The effect of emotional intelligence group training on human and social capital in Isfahan University of technology. International Letters of Social and Humanistic Sciences, 50, 91-99. https://doi.org/10.18052/www.scipress.com/ILSHS.50.91 Nutzungsbedingungen: Dieser Text wird unter einer CC BY Lizenz (Namensnennung) zur Verfügung gestellt. Nähere Auskünfte zu den CC-Lizenzen finden Sie hier: https://creativecommons.org/licenses/by/4.0/deed.de Online: 2015-03-23 International Letters of Social and Humanistic Sciences ISSN: 2300-2697, Vol. 50, pp 91-99 doi:10.18052/www.scipress.com/ILSHS.50.91 © 2015 SciPress Ltd., Switzerland ABSTRACT The aim of this study was to investigate the effect of emotional intelligence group training on human and social capital in the students of Isfahan University of Technology. This research was a quasi-experimental study with pretest-posttest design and control group. The research population consists of all the students of Isfahan University of Technology who have been studying in 2012- 2013. For sampling, voluntary sampling method was used that 36 subjects were selected as the sample and were randomly divided into two groups of experiment and control. The subjects of experimental group participated in 8 sessions of 90-minute classes while the control group received no intervention. The study's tool was Mousavi Employability Questionnaire (2013). Analysis of covariance was used to analyze the data. According to the results, emotional intelligence training have had impact upon human capital (P<0.004). However, emotional intelligence training had no effect on social capital. SciPress applies the CC-BY 4.0 license to works we publish: https://creativecommons.org/licenses/by/4.0/ The Effect of Emotional Intelligence Group Training on Human and Social Capital in Isfahan University of Technology Batoul Tadayon Charsoughi1, Mohammad Reza Abedi2, Parisa Nilforoushan3 1 Islamic Azad University, Khomeini Shar Branch, Isfahan, Iran 2 Isfahan University and Islamic Azad University, Khomeini Shar Branch, Isfahan, Iran 3 Psychology Department, Isfahan University, Isfahan, Iran E-mail address: 1bt.td20@yahoo.com 2dr.mr.abedi@gmail.com 3panilfo@yahoo.com Keywords: emotional intelligence; human capital; social capital 1. INTRODUCTION In each community, employment and having a job is important for the people at working age. Given the outcomes of having a job, a large part of the population seeks employment. In this regard, the employed people are more healthy and satisfied with their lives. The importance of employment and its consequent effects have prompted the researchers to make effort for enhancing people employability. Many definitions have been offered for the concept of employability. Employability is defined as one's ability to gain initial employment, job retention, job changes within the same organization, and achieve a new job when needed. This definition includes both unemployed people who are looking for a job and the employed people who are looking for a new job or want to promote their position (Hillage& Pollard, 1998). Fugate et al (2004) describe employability as a psychosocial structure that embodies those personal characteristics that enhances adaptive cognition and behavior, and reinforces one's working relations. Since it involves those characteristics that cover the gaps between the individual and the environment, employability is considered a psychosocial structure. They define employability as one's ability in identifying and specifying career opportunities within and between organizations. According to them, employability includes the four aspects of career adaptability, personal adaptability, human capital, and social capital. Individuals and organizations invest in the field of human and social capitals and predict its future returns in the workplace (Dess& Shaw, 2001). One's ability to identify and realize career opportunities depends on the extent to which one is influenced by such capitals. These factors are often included in a broader concept of knowledge, skills and abilities. However, the discussion here is limited to human and social capitals since these concepts have a specific relationship with iPress applies the CC-BY 4.0 license to works we publish: https://creativecommons.org/licenses/by/4.0/ Volume 50 92 employability. Moreover, human and social capitals are tied to the people's career identities and are embedded in sociability structure (Fugate et al, 2004). employability. Moreover, human and social capitals are tied to the people's career identities and are embedded in sociability structure (Fugate et al, 2004). Social capital emanates from the social networks. It provides an interpersonal and social element for employability, transfers information, and through social networks, provides people with influence, power and information. 1. INTRODUCTION This influence, power and information not only provide the individuals with the possibility to have access to career opportunities but also, they are important components in achieving career aspirations (Fugate, 2006). In this regard, the network's size and power are two important features of the network that determine the potentiality of the created influence and information. The breadth and diversity of an individual network is probably proportional to the amount of information and influence in the network, as well as to the usefulness and influence of the information (Seibret et al, 2001). The benefits of social capital and its impact on the employability are demonstrated in employment-related behaviors of individuals. Individuals with a well-developed social capital, not only benefit from more formal job searching networks (like a company that supports tender services) but also from informal job searching networks as well (e.g a friend of friend). In this regard, Boxman, Graaf and Flap (1991) found out that top managers often search for jobs through informal networks. They also found that social capital has a positive and independent impact on wages that is higher and beyond human capital. Social networks can increase one's ability in identifying and understanding career opportunities between organizations and industries and during the career (Fugate, 2008). An employee's ability to understand job opportunities in the labor market is heavily influenced by his human capital. Human capital refers to a set of factors that affect the variables associated with one's career advancements. These factors include age and education, experience and job training, job performance and organizational tenure, emotional intelligence, cognitive abilities and knowledge, and skills and abilities that are created by such factors. Among human capital factors, education and experience are the strongest predictors of career advancement (Fugate et al, 2004). In many types of employments, educational levels, even if they are not necessary, are preferred. These educational levels include compulsory education, associate degree, bachelor degree, certificate for a specific job and professional degrees. In fact, some researchers believe that the value and rate of return for education is measurable (Goleman, 1988). Experience is also very important. Experience often leads to different levels of skills and tacit knowledge which make the experienced person seem more attractive and prospective in the eyes of employers. In modern workplaces, experience is shown through portable skills and not through industry or exposure to a specific occupation. 1. INTRODUCTION eness of one's emotions (self-evaluation and self-esteem). • Management of the emotions (self-control, trustworthiness, conscientiousness, innovation and adaptability). • Self-stimulation (progress motivation, optimism and commitment). • Self-stimulation (progress motivation, optimism and commitment). • Identification of others' emotions (understanding others, developing others, variety in the influence styles, and political awareness). • Identification of others' emotions (understanding others, developing others, variety in the influence styles, and political awareness). • Regulating the relations with others (influence, communication, conflict management, leadership, bonding and participation, collective capabilities). • Regulating the relations with others (influence, communication, conflict management, leadership, bonding and participation, collective capabilities). There have been many studies about the effects of emotional intelligence training upon the constitutive dimensions of emotional intelligence some of which are mentioned here: William et al (2008), Soleimani and Ali-beigi (2009), Pourfaraj (2008), Karademas (2006), RaghibiabdRabbani (2012), Seifi and Pour-mousavi (2010), and Yaryari, Moradi and Yahyazadeh (2007). However, no research was found emphasizing the impact of emotional intelligence training on social capital or the relationship between these two. In addition, the content of emotional intelligence deals with the issues which are important factors in human and social capital. It seems that through training emotional intelligence, human and social capitals can be influenced. In this regard, doing a research on the effects of emotional intelligence training upon the social and human capitals seems necessary. In other words, the aim of this study is to evaluate how emotional intelligence training influences human and social capitals. Based on the intended variables this study aims to examine two following hypotheses: • Emotional intelligence training has impact upon human capital. otional intelligence training has impact upon • Emotional intelligence training has impact upon social capita 1. INTRODUCTION Portable skills include knowledge, abilities and skills which are obtained from very different areas and are transferable to other areas. Employees often choose individuals according to their experiences and experience is associated with productivity and rewards. In short, human capital refers to one's ability in meeting performance expectations in a particular profession (Protess, 1998). Therefore, one's level of education and experience are powerful predictors for career advancement. Human capital will help both individual and organizational adaptability. Furthermore, investment in human capital throughout a person's career is an adaptive orientation and a commitment to continuous learning that will lead to further strengthening of employability (Fugate, 2004). Researchers have shown that according to some factors, people with high IQ are not necessarily successful in their lives, while those with an average IQ may be placed on the road to success. These factors include another dimension of intelligence called as emotional intelligence by Goleman. The concept of emotional intelligence was first proposed by Salovay and Mayer as a form of social intelligence. Salovay used this concept for describing and understanding of people's feeling, sympathy with the feelings of others, and the ability in optimal management of temperament. In their definition, they introduced emotional intelligence as the ability to process emotional information including the proper assessment of one's and others' emotions, appropriate International Letters of Social and Humanistic Sciences Vol. 50 93 expression of emotions, and adaptive regulation of emotions so that it leads to the improvement of flow of life (Nouri-Emamzadeh, 2009). expression of emotions, and adaptive regulation of emotions so that it leads to the improvement of flow of life (Nouri-Emamzadeh, 2009). In 1990, Goleman argued that emotional intelligence can be used to identify employees who might have the best performance and since then, a practical movement was started to increase research on emotional intelligence. According to Goleman, emotional intelligence refers to individuals' ability in controlling and governing themselves and their relationships. He also believes that 80 to 90 percent of individuals' successes can be attributed to emotional intelligence. In defining emotional intelligence for public, Goleman considers it as a form of social behavior (Whitte, 2004). ( ) According to Goleman's definition (1995, trans. By Parsa, 2004), emotional intelligence is classified into five areas of: ( ) According to Goleman's definition (1995, trans. By Parsa, 2004), emotional intelligence is classified into five areas of: • Awareness of one's emotions (self-evaluation and self-esteem). Volume 50 Volume 50 94 descriptive statistics including the mean, standard deviation, and inferential statistics including ANCOVA. The format of the sessions is summarized as follows: ession: explaining the objectives of the sessions, familiarity with the concept, the positio cessity of employability. The second session: familiarizing the members with the concept of emotional intellig components of emotional intelligence. The third session: informing the members of self-awareness and the factors that p awareness of his emotions. The fourth session: familiarizing the members with 5 barriers preventing one's aware emotions and using some alternatives for these barriers. The fifth session: familiarizing the members with the concept of emotional permeab how we can reduce permeability. The fifth session: familiarizing the members with the concept of emotional permeability and that how we can reduce permeability. p y ession: members' familiarity with the concept of perception and perception barriers. The sixth session: members' familiarity with the concept of perception and perception barriers. The seventh session: members' familiarity with the concept of accepting and expressing emotions, and the barriers of it. The sixth session: members familiarity with the concept of perception and perception barriers. The seventh session: members' familiarity with the concept of accepting and expressing emotions, and the barriers of it. The eighth session: getting feedback from the members on the use and quality of the di The eighth session: getting feedback from the members on the use and quality of the discussions. 1.3. RESEARCH TOOLS To assess the concept of employability, Mousavi Employability Questionnaire (2013) was used in this study. This questionnaire contains 96 questions that the questions 1 to 6 are related to age, sex, education, job and training experiences, and have been coded by alphabets. Social-capital- related questions have been designed in one question that is question 58. The range of scores in this questionnaire is between 57 and 441. Using Likert scale, scoring process was done as follows: 1 C l t l 2 A 3 M di 4 Di 5 C l t l di p y g ; g ; ; g ; p y g It should be noted that for the questions 3, 8, 32, 16, 11, 33, 34, 49, 50, 51, and 53 scoring was done reversely: 1- Completely disagree; 2- Disagree; 3- Medium; 4- Agree; 5- Completely agree. It should be noted that for the questions 3, 8, 32, 16, 11, 33, 34, 49, 50, 51, and 53 scoring was done reversely: 1- Completely disagree; 2- Disagree; 3- Medium; 4- Agree; 5- Completely agree. Mousavi has evaluated the content validity of the questionnaire using the opinions of specialists, and to assess the convergent construct validity, he has reported the correlation of this questionnaire with employment self-efficacy questionnaire (Salehi, 2005). Using a sample of 20 students of Isfahan University of Technology who have been studying in the academic year of 2012-2013, and using Cronbach alpha, Mousavi, has calculated the reliability of the questionnaire as 0.94. Cronback alpha reliability of the questionnaire in this study was estimated to be 0.90 through using a sample of 36 students who were studying in Isfahan University of Technology. 1.2. METHODOLOGY This research is a quasi-experimental study with pretest-posttest design and control group. The study's variables include the independent variable of emotional intelligence training and dependent variables of human and social capitals. The research population consists of all the students of Isfahan University of Technology who have been studying in 2012-2013. For sampling, in this study voluntary sampling method with random assignment was used. In this regard, some declarations were designed declaring: emotional intelligence workshops along with the instructor's name, and the time and place of registration. Then, the interested students were invited to participate in the workshop. The sample size was 36 students. They were randomly divided into two groups of the experimental group (n=18) and the control group (n=18). Criteria for being included in the experimental and control group: a) being a student in Isfahan University of Technology; b) being a student in the academic year of 2012-2013. After sampling, the subjects of both groups were assessed using employability questionnaire. Then, the experimental group received 8 sessions emotional intelligence group training while the control group remained in the waiting list. After the end of the sessions, both groups responded again to the questionnaire. Then, the data obtained from both experimental and control groups were analyzed. For data analyzing, SPSS16 software was used. The statistical method used in this study has been 2. DISCUSSION Given the purpose and method of the research that is based on comparing the two groups of experiment and control in their scores of employability and its dimensions and considering that it was necessary to control pretest, analysis of covariance (ANCOVA) was used. However, to use this test, its pre-assumptions/defaults have to be examined. In continue, at first, these pre-assumptions will be discussed and then the results of analysis of covariance for the scores of both human and social capitals are provided separately. Table 1 summarizes the results of Kolmogorov-Smirnov and Shapiro-Willkie tests to verify the default normal distribution of scores on the dependent variable (posttest score) and the scores of auxiliary random variable (pretest score). Table 1. The summary results of the default normal distribution of the dependent variable (posttest score) and the auxiliary random variables (pretest score) The variable Pretest Posttest Kolmogorov-Smirnov Shapiro-Willkie Kolmogorov-Smirnov Shapiro-Willkie statistic df Significance statistic df Significance statistic df Significance statistic df Significance Human capital 0.12 36 0.18 0.98 36 0.58 0.09 36 0.20 0.98 36 0.80 Social capital 0.10 36 0.20 0.98 36 0.59 0.09 36 0.20 0.97 36 0.52 The summary results of the default normal distribution of the dependent variable (posttest score) and the auxiliary random variables (pretest score) International Letters of Social and Humanistic Sciences Vol. 50 95 As it is shown in table 1, the default normal distribution in each of the dependent variables (posttest scores) and the auxiliary random variables (pretest scores) cannot be rejected. Table 2 summarizes the results of the Levine test to examine the default equality of variances of the dependent variable scores (posttest score) and the scores of auxiliary random variable (pretest score). Table 2. The summary result of Levine test in examining the default equality of variances of the dependent variables (posttest scores) and the auxiliary random variables (pretest score) summary result of Levine test in examining the default equality of variances of the dependent variables (posttest scores) and the auxiliary random variables (pretest score) Table 2. 2. DISCUSSION The summary result of Levine test in examining the default equality of variances of the dependent variables (posttest scores) and the auxiliary random variables (pretest score) The variables Pretest Posttest F df1 df2 Significance F df1 df2 Significance Human capital 3.63 3 32 0.02 1.36 3 32 0.27 Social capital 5.82 3 32 0.000 2.77 3 32 0.06 The variables Pretest Posttest F df1 df2 Significance F df1 df2 Significance Human capital 3.63 3 32 0.02 1.36 3 32 0.27 Social capital 5.82 3 32 0.000 2.77 3 32 0.06 According to the Levine test (Table 2), the default equality of variances cannot be rejected. Now, given the observance of the default, analysis of covariance can be used. 2.1. THE RESULTS OF ANALYSIS OF COVARIANCE FOR HUMAN CAPITAL SCORES 2.1. THE RESULTS OF ANALYSIS OF COVARIANCE FOR HUMAN CAPITAL SCORES Descriptive statistic results for the pretest and posttest scores of human capital are 2.1. THE RESULTS OF ANALYSIS OF COVARIANCE FOR HUMAN CAPITAL SCORES Descriptive statistic results for the pretest and posttest scores of human capital are summarized in table 3. In the posttest phase, the averages are calculated after controlling the effects 2.1. THE RESULTS OF ANALYSIS OF COVARIANCE FOR HUMAN CAPITAL SCORES Descriptive statistic results for the pretest and posttest scores of human capital are summarized in table 3. In the posttest phase, the averages are calculated after controlling the effects of auxiliary random variable. Descriptive statistic results for the pretest and posttest scores of human capital are summarized in table 3. In the posttest phase, the averages are calculated after controlling the effects of auxiliary random variable. Table 3. Descriptive statistics of human capital both in pretest and posttest stages Group belonging of therapy Group belongings of sex Sample size Pretest Posttest Mean Standard deviation Modified mean Standard deviation Experimental Male 13 22.23 3.83 24.25 4.98 Female 5 20.40 1.52 23.76 3.91 Control Male 13 22.46 4.94 21.88 4.35 Female 5 19.00 5.29 21.11 5.03 Total Experimental 18 20.31 3.41 24.01 4.71 Control 18 21.73 5.14 21.49 4.78 Table 3. Descriptive statistics of human capital both in pretest and posttest stages As it can be observed in table 3, both in the experimental group and the control group, there have been differences between the pretest and posttest means, but the differences have been more in the experimental group. 2. DISCUSSION Good criteria for the means and determining the significance and non- significance of the differences are given in figure Volume 50 96 1: Figure 1. Comparison of human capital means in both experimental and control groups in pretest posttest stages and at the confidence level of 95%. The between-line points represent the averages and the lines' extents show the confidence intervals of the means. 12Posttest Control 11.Posttest Exp 10.Pretest Control 9.Pretest Exp 8.Posttest F Control 7.Posttest M Control 6.Posttese F Exp 5.Posttest M Exp 4.Pretest F Control 3.Pretest M Control 2.Pretest female, Exp 1.Pretest male Exp 96 Volume 50 Figure 1. Comparison of human capital means in both experimental and control groups in pretest posttest stages and at the confidence level of 95%. The between-line points represent the averages and the lines' extents show the confidence intervals of the means. In terms of intergroup comparisons, significance of the differences is shown in figure 1. Combined with the results of the table 4, these differences are discussed in continue: Table 4. The summary results of covariance analysis to determine intergroup differences in posttest scores of human capital Effect source Degree of freedom (df) Mean squares F Statistical significance Effect size Statistical power Pretest 1 544.42 118.49 0.0005 0.79 1.00 Intervention 1 45.52 9.91 0.004 0.24 0.86 According to the results of table 4 human capital posttest significant difference between the two Table 4. The summary results of covariance analysis to determine intergroup differences in po human capital ummary results of covariance analysis to determine intergroup differences in posttest scores of human capital According to the results of table 4, human capital posttest significant difference between the two groups of experiment and control (P=0.004; F=9.91; R2=0.24) indicates the effectiveness of emotional intelligence on the human capital (the comparison of lines 11 and 12 of figure 1 also suggest the same significant difference). Therefore, the first research hypothesis is confirmed. Emotional intelligence training has been able to predict 24 percent increase in human capital, suggesting the effectiveness of the intervention (R2>0.10). 2.2.ANALYSIS OF COVARIANCE FOR SOCIAL CAPITAL SCORES Descriptive statistic results for the pretest and posttest scores of social capital are summarized in table 5. In the posttest phase, the averages are calculated after controlling the effects of auxiliary random variable. Table 5. Descriptive statistics of social capital both in pretest and posttest stages Group belonging of therapy Group belongings of sex Sample size Pretest Posttest Mean Standard deviation Modified mean Standard deviation Experimental Male 13 63.77 24.87 66.36 26.09 Female 5 79.00 9.41 61.13 11.59 International Letters of Social and Humanistic Sciences Vol. 50 International Letters of Social and Humanistic Sciences Vol. 50 Control Male 13 72.54 29.28 67.87 29.93 Female 5 57.60 25.88 65.47 27.62 Total Experimental 18 71.39 22.51 63.75 23.03 Control 18 65.07 28.46 66.67 29.50 International Letters of Social and Humanistic Sciences Vol. 50 97 97 Control Male 13 72.54 29.28 67.87 29.93 Female 5 57.60 25.88 65.47 27.62 Total Experimental 18 71.39 22.51 63.75 23.03 Control 18 65.07 28.46 66.67 29.50 As it can be observed in table 5, there have been differences between the pretest and posttest means both in the experimental group and the control group, but the differences have been more in the experimental group. Good criteria for the means and determining the significance and non- significance of the differences are given in figure 2: Figure 2. Comparison of social capital means in both experimental and control groups in pretest posttest stages and at the confidence level of 95%. The between-line points represent the averages and the lines' extents show the confidence intervals of the means. 12Posttest Control 11.Posttest Exp 10.Pretest Control 9.Pretest Exp 8.Posttest F Control 7.Posttest M Control 6.Posttese F Exp 5.Posttest M Exp 4.Pretest F Control 3.Pretest M Control 2.Pretest female, Exp 1.Pretest male Exp Figure 2. Comparison of social capital means in both experimental and control groups in pretest posttest stages and at the confidence level of 95%. The between-line points represent the averages and the lines' extents show the confidence intervals of the means. T bl 6 Th lt f i l i t d t i i t diff i tt t f 12Posttest Control 11.Posttest Exp 10.Pretest Control 9.Pretest Exp 8.Posttest F Control 7.Posttest M Control 6.Posttese F Exp 5.Posttest M Exp 4.Pretest F Control 3.Pretest M Control 2.Pretest female, Exp 1.Pretest male Exp Figure 2. 2.2.ANALYSIS OF COVARIANCE FOR SOCIAL CAPITAL SCORES Comparison of social capital means in both experimental and control groups in pretest posttest stages and at the confidence level of 95%. The between-line points represent the averages and the lines' extents show the confidence intervals of the means. Table 6. The summary results of covariance analysis to determine intergroup differences in posttest scores of human capital Effect source Degree of freedom (df) Mean squares F Statistical significance Effect size Statistical power Pretest 1 18733.17 153.76 0.0005 0.83 1.00 Intervention 1 60.91 0.50 0.48 0.02 0.11 Table 6. The summary results of covariance analysis to determine intergroup differences in posttest scores of human capital Effect source Degree of freedom (df) Mean squares F Statistical significance Effect size Statistical power Pretest 1 18733.17 153.76 0.0005 0.83 1.00 Intervention 1 60.91 0.50 0.48 0.02 0.11 ummary results of covariance analysis to determine intergroup differences in posttest scores of human capital According to the results of table 6, human capital posttest non-significant difference between the two groups of experiment and control (P=0.48; F=0.50; R2=0.02) suggests the non-effectiveness of emotional intelligence on the social capital (the comparison of lines 11 and 12 of figure 2 also suggests this significant difference). Therefore, this hypothesis is rejected. Emotional intelligence training has been not able to predict the increase in social capital, suggesting the practical non- effectiveness of the intervention (R2<0.10). 3. CONCLUSION The results obtained from the analysis of covariance showed that there is a significant difference between the posttest of the experimental group and the control group in terms of human capital. Therefore, it can be concluded that emotional intelligence training has had impact on the human capital of students in the Isfahan University of Technology. Moreover, the results obtained from the analysis of covariance showed that there is no significant difference between the posttests of the experimental and control groups in the social capital. Hence, it can be concluded that emotional intelligence training has had no impact on the social capital of students in the Isfahan University of Technology. Volume 50 98 To explain the first hypothesis, it can be said that regardless of the conducted researches, the effect of emotional intelligence training is evident on the human capital. As mentioned in the methodology part, training pattern of increasing emotional intelligence in this intervention has been based on increasing the dimensions of emotional intelligence. Thus, it is clear that this intervention has had effect on the various aspects of human capital (such as self-awareness, self-regulation, motivation, empathy and social skills), and finally has led to the increase of human capital in this study. It is also noteworthy that the effects of an intervention are measured by its measuring tools. Regarding this study's tools, to measure human capital some questions have been used about emotions control, emotions expression and emotions management. Hence, it is obvious that this intervention has been more effective on human capital. In the course of this study, as a result of interaction with each other, the members gained new learning during these training sessions. At these sessions, the members defined their experiences with regard to the controlling and expressing their emotions. In this process, members, through observing each other's experiences and integration of assigned duties, gain the ability to manage, know and express their emotions. To explain the second hypothesis, it can be said that although no research was found on the effect of emotional intelligence training on social capital and the relationship between these two, this non-effectiveness can be explained in this way that by social capital in the Fugate et al model (2004), it means the networks that give influence and information to the individuals and help them to identify and realize career opportunities. 3. CONCLUSION A group of these capitals such as parents, siblings, relatives and so on are with a person at his/her birth, and another group such as school, university, friends and so forth are created over the time. The second group of capitals can be created under the influence of the first group. For example, educated parents can make their children familiar with books or enroll them in high level schools. In this regard, the first group of capitals is to a large extent out of one's control and is controlled by the deterministic powers. The second category of capitals, as it is formed by the first group, is also, to some extent, out of one's control but it cannot be claimed definitely that they are completely out of one's control. Since these capitals have been established in the individual from years ago and out of the individual's control, they cannot be increased in the process of the intervention. For example, some parts of these capitals are created by media and their contents cannot be changed, or they are established by the family, relatives and friends networks which cannot be influenced directly by the intervention. Hence, this intervention cannot in a short time change or increase the capitals whose processes of creation have been from many years ago. However, perhaps if the intervention followed up, we could see the effect of the intervention on the increase of such capitals; since, on the one hand, this intervention was itself a social capital and, on the other hand, according to the dimension of social skills in emotional intelligence, the individual sought information, trainings and measures to increase such capitals in oneself, but, due to the limited scope of the study it was impossible for us to do it. In conclusion, it can be said that the efficacy of an intervention can be measured by its measuring tools. In the employability questionnaire, social capital is measured by such questions as "how much have your parents contributed to the creation of employment opportunities in you?" Since for increasing the scores of these questions, such factors as trainings, family counseling and social capitals should be considered and due to the fact that such trainings were impossible in our intervention, the intervention could not increase the members' social capital. Acknowledgement At the end we express our endless gratitude and indebtedness to all people who help us in this project. In addition, we would like to thank Mr. Roohollah Datli Beigi, M.A. in English Language and Literature, University of Isfahan, whose linguistic and grammatical comments were helpful and constructive in preparation of this paper. International Letters of Social and Humanistic Sciences Vol. 50 International Letters of Social and Humanistic Sciences Vol. 50 99 References [1] Dess, G.G., & Shaw, S.D.(2001). “Voluntary turnover, Social capital, and organizational performance”, Academy of Management Review, 26,446-456.Fugate, M. (2006).Employability in the new millennium.In Greenhaus, J. H., &Callanan, G. A. (Eds.).Encyclopedia of Career Development. Sage: London. [2] Fugate, M., Kinicki, A. J., &Ashforth, B. E. (2004). “Employability: A psycho-social construct, its dimensions, and applications”, Journal of Vocational Behavior, 65(1), 14-38. [3] Fugate, M., &Kinicki, A.J. (2008).“A dispositional approach to employability: Development of a measure and test of implications for employee reactions to organizational change”, Journal of Occupational and Organizational Psychology, 81(3), 503-527. [4] Goleman, D. (1998). Working with Emotional Intelligeace. New York: Ban tam books. [5] Goleman, D (2004). Emotional Intelligence, Self-awareness, Self-control, Empathy, and Helping Others.Trans. NasrinParsa, Tehran. [6] Hillage, J. & Pollard, E. (1998).”Employability: Developing a Framework for Policy Analysis.London: FfEE. [7] Karademas, E.C. (2006). Self –efficacy, social support and well- being. The mediating role of optimism.Personality and Individual Difterences, 40, 1281-1290. ] Nouri-Emamzadeh, A (2009). Emotional Intelligence in the Workplace.Isfahan. Printe [9] Pourfaraj-Omran, M (2008). "An Investigation of the Relationship between Innovation, Emotional Intelligence, and self-efficacy".The First National Conference of Creativity, Engineering and Innovation Management.Iran. [10] Protess, A.(1998). Social capital: Its origins and applications in modern sociology. In J.Hagan& K.S. cook (Eds), Annual Review of Sociology, 24, 1-24. [11] Raghibi, M and Rabbani, M (2012)."The Effect of Optimism Skills Training on the Emotional Intelligence of the Boys in Keman Juvenile Institution".The Journal of Educational- Psychological Studies.9 (15): 107-124. [12] Seibert, S. E., Kraimer, M. L., &Crant, J. M. (2001). “What do proactive people do? A longitudinal model linking proactive personality and career success”, Personnel Psychology, 54(4), 845-874. [13] Seifi, M. Y. and Pour-mousavi, M (2009). Investigation of the Relationship between Emotional Intelligence and Locus of Control (Internal-External).BA Thesis, Psychology,Payam Noor University of Boroujen. [14] Soleimani, N and Ali-beigi, F (2009)."Relationship betweenEmotional Intelligence and Self- efficacy of the Managers in the Branches of Islamic Azad University".Journal of Educational Leadership and Management.(10): 137-154. [15] Whitte, R. (2004). Emotional intelligence and career success among the highly intelligent. Journal of career Assessment. [16] Yaryari, F. Moradi, A. R. and Yahyazadeh, S (2007)."Relationship of Emotional Intelligence and Control Locus with Mental Health of the Students in the University of Mazandaran".Journal of Psychological Studies.(1): 120-128.
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Zerumbone, a cyclic sesquiterpene, exerts antimitotic activity in HeLa cells through tubulin binding and exhibits synergistic activity with vinblastine and paclitaxel
Cell proliferation
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cc-by
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O R I G I N A L A R T I C L E O R I G I N A L A R T I C L E Funding information g This work is partly supported by the grant from the DST/SERB, Government of India (SR/SO/BB‐0013/2010), to Dr Rathinasamy K and partly by TEQIP‐II, NIT Calicut, for the Student Project to SMA (Ref. NITC/TEQIP‐ II/R&D/2014). Results: Zerumbone selectively inhibited the proliferation of HeLa cells with an IC50 of 14.2 ± 0.5 μmol/L through enhanced cellular uptake compared to the normal cell line L929. It induced a strong mitotic block with cells exhibiting bipolar spindles at the IC50 and monopolar spindles at 30 μmol/L. Docking analysis indicated that tubulin is the principal target of zerumbone. In vitro studies indicated that it bound to goat brain tubulin with a Kd of 4 μmol/L and disrupted the assembly of tubulin into micro‐ tubules. Zerumbone and colchicine had partially overlapping binding site on tubulin. Zerumbone synergistically enhanced the anti‐proliferative activity of vinblastine and paclitaxel through augmented mitotic block. Conclusion: Our data suggest that disruption of microtubule assembly dynamics is one of the mechanisms of the anti‐cancer activity of zerumbone and it can be used in combination therapy targeting cell division. Shabeeba M. Ashraf | Jomon Sebastian | Krishnan Rathinasamy Shabeeba M. Ashraf | Jomon Sebastian | Krishnan Rathinasamy School of Biotechnology, National Institute of Technology Calicut, Calicut, Kerala, India Abstract Objectives: The aim of this study was to elucidate the antimitotic mechanism of zerumbone and to investigate its effect on the HeLa cells in combination with other mitotic blockers. Correspondence Krishnan Rathinasamy, School of Biotechnology, National Institute of Technology Calicut, Calicut, Kerala, India. Email: rathin@nitc.ac.in Funding information This work is partly supported by the grant from the DST/SERB, Government of India (SR/SO/BB‐0013/2010), to Dr Rathinasamy K and partly by TEQIP‐II, NIT Calicut, for the Student Project to SMA (Ref. NITC/TEQIP‐ II/R&D/2014). Materials and methods: HeLa cells and fluorescence microscopy were used to ana‐ lyse the effect of zerumbone on cancer cell lines. Cellular internalization of zerum‐ bone was investigated using FITC‐labelled zerumbone. The interaction of zerumbone with tubulin was characterized using fluorescence spectroscopy. The Chou and Talalay equation was used to calculate the combination index. Received: 28 July 2018  |  Revised: 19 September 2018  |  Accepted: 10 October 2018 DOI: 10.1111/cpr.12558 Received: 28 July 2018  |  Revised: 19 September 2018  |  Accepted: 10 October 2018 DOI: 10.1111/cpr.12558 y 2018  |  Revised: 19 September 2018  |  Accepted: 10 October 2018 Received: 28 July 2018  |  Revised: 19 September 2018  |  Accepted: 10 October 2018 DOI: 10.1111/cpr.12558 Cell Proliferation. 2019;52:e12558. https://doi.org/10.1111/cpr.12558 Cell Proliferation. 2019;52:e12558.  |  1 of 17 wileyonlinelibrary.com/journal/cpr This is an open access article under the terms of the Creative Commons Attribution License, which permits use, distribution and reproduction in any medium, provided the original work is properly cited © 2018 The Authors. Cell Proliferation Published by John Wiley & Sons Ltd 2.1 | Materials Paclitaxel, vinblastine sulphate, podophyllotoxin, colchicine, 5,5′‐ dithiobis‐2‐nitrobenzoic acid (DTNB), sulforhodamine B (SRB), Hoechst 33342, guanosine 5′‐triphosphate (GTP), propidium iodide, EGTA, MgCl2, piperazine‐N,N′‐bis (2‐ethanesulphonic acid) (PIPES), mouse monoclonal anti‐α‐tubulin IgG and FITC‐conjugated anti‐ mouse IgG, fluorescein isothiocyanate isomer (FITC) and dimeth‐ ylformamide were purchased from Sigma‐Aldrich (St. Louis, MO, USA). Foetal bovine serum (FBS) and Alexa Fluor 568‐conjugated anti‐mouse IgG were purchased from Invitrogen (Thermo Scientific, Massachusetts, USA). Acridine orange (AO), hydroxysuccinimide, minimal essential medium (MEM), cell culture tested antibiotic so‐ lution, and phosphate‐buffered saline (PBS) were purchased from HiMedia (Mumbai, India). Dichloromethane, n‐hexane, hydroxy‐ lamine hydrochloride and triethylamine were purchased from Merck, India. 1‐Ethyl‐3‐(3‐dimethylaminopropyl) carbodiimide hydrochlo‐ ride was purchased from SRL chemicals. All other reagents used in the study were of analytical grade. The clinically successful antitubulin agents such as the pacli‐ taxel and the vinblastine are obtained from plants. Natural product research is gaining a huge attention because many of the phyto‐ chemicals exhibit excellent chemopreventive and chemotherapeutic potential in addition to their selectivity against cancer cells and low cost of production.10 Natural products such as genistein, apigenin, quercetin, curcumin, berberine, limonene, coumarin, indirubin, bras‐ sinin, indole‐3‐carbinol, lycopene and resveratrol are in clinical/ preclinical trials either alone or in combination therapy for the treat‐ ment of cancer.11-13 In the present study, we have investigated the anti‐proliferative mechanism of the natural product zerumbone iso‐ lated from the plant Zingiber zerumbet belonging to the ginger family of flowering plants (Zingiberaceae). Zerumbone is a sesquiterpene and is reported to exhibit anti‐cancer potential and other pharma‐ cological activities such as anti‐inflammatory, antibacterial, antima‐ larial and antioxidant properties.14-17 Zerumbone was found to be effective in preventing tumour angiogenesis by inhibiting the VEGF expression and NF‐κB activity.18 It was reported to induce apoptosis in various cancer cell lines by modulating the FAS and TRAIL signal‐ ling pathways, through enhanced expression of TNF and modulating Bax/Bcl‐2 ratio .19-21 Recently, zerumbone was reported to block the cell cycle at mitosis17,22 and induce apoptosis in cancer cells through inhibition of microtubule assembly.23 However, the mechanism be‐ hind the mitotic block was not clearly established, and hence, we have performed this study to elaborate its anti‐cancer mechanism through biophysical, biochemical and cell culture studies. kinases such as the Aurora family of proteins; the Polo‐like kinase; and the mitotic checkpoint proteins.5,7 its inhibitory effects on tubulin and induction of mitotic block, and combination of zerumbone and other anti‐cancer drugs might pro‐ vide a therapeutic advantage in controlling the growth of cancer cells. Microtubules, the key components of the cytoskeleton, are com‐ posed of alpha and beta tubulin heterodimer. They are highly dy‐ namic polymers that undergo polymerization and depolymerization in a short span of time and play essential role in the maintenance of cell shape, intracellular trafficking, cell motility and cell signalling apart from cell division and mitosis.8 Tubulin has two nucleotide (GTP) binding sites and three well‐characterized drug binding sites such as the colchicine binding site, the paclitaxel binding site and the Vinca alkaloid binding site. The GTP binding site is located at the N‐terminal region of the α and the β subunits, and the colchicine binding site is present at the interface of the α‐ β subunit.8,9 The paclitaxel binding site is located at the β‐tubulin, and the Vinca alka‐ loids binding site is located in the N‐terminal region of the β‐tubulin subunit close to the GTP binding site.9 2.2 | Isolation, purification and characterization of zerumbone Fresh rhizomes of Zingiber zerumbet were collected from the farms of the Indian Institute of Spice Research (IISR), Calicut, Kerala (India), and it was authenticated by Dr D Prasath, Principal Scientist, IISR, Calicut. Zerumbone was extracted and isolated from the rhizomes of Zingiber zerumbet as described earlier.24 Briefly, 1 kg of fresh rhi‐ zomes was washed under running tap water and cut into slices. The slices were then shade‐dried at 37°C for 5 days. The dried samples were then soaked in methanol for 3 days, and the methanolic ex‐ tract was concentrated by using the rotary evaporator (Heidolph Instruments, GmbH & CO. KG, Schwabach, Germany). The extract was then fractionated by silica gel (mesh size 200) column chroma‐ tography using organic solution mixture of hexane: ethyl acetate (8:2; v/v). Zerumbone thus obtained was further purified by crystal‐ lization. The purity of zerumbone was analysed and confirmed using the Shimadzu liquid chromatography‐mass spectrometry (LC‐MS) and Bruker Avance III Nuclear Magnetic Resonance spectroscopy (NMR) using the standard procedure. Cell culture studies showed an excellent observation that zerum‐ bone exhibited selective toxicity against HeLa cells through enhanced internalization of the compound and inhibited their migration. The anti‐proliferative effect of zerumbone in HeLa cells correlated well with its ability to inhibit the cell cycle at mitosis through tubulin bind‐ ing. Zerumbone bound to tubulin at the colchicine binding site and inhibited the polymerization of tubulin into microtubules. Zerumbone exhibited excellent synergistic antimitotic and anti‐proliferative ac‐ tivity in HeLa cells when combined with clinically established drugs such as paclitaxel and vinblastine. Together, the results suggest that the anti‐proliferative effects of zerumbone could be partly through 2 of 17 ASHRAF et al. 1 | INTRODUCTION 1 and invasion. Hence, chemotherapeutic agents that target cell di‐ vision are highly valuable in cancer chemotherapy. Mammalian cell division requires the coordinated actions of the cytoskeleton, the membrane proteins, the motor proteins and the cell cycle regulatory proteins which are precisely controlled in space and time.4,5 Among the various players in cell division, tubulin is indispensable for mito‐ sis and chromosome segregation, and antimitotic agents targeting tubulin are the most successful in the treatment of various types of tumours.5,6 The other essential antimitotic targets include the mi‐ totic kinesins such as Eg5, CENP‐E, MCAK, and MKLP1; the mitotic Cancer is one of the leading causes of death all over the world, and cancer treatment remains one of the greatest challenges in front of the healthcare professionals.1,2 Most of the anti‐cancer agents cur‐ rently available in the market are non‐selective and could destroy the normal healthy cells. In addition, they could pose greater risk of developing drug resistance in the tumour cells and are ineffective in inhibiting metastasis.3 Cancer is a complex disease characterized by an overactive cell cycle that promotes increased cell proliferation |  1 of 17 wileyonlinelibrary.com/journal/cpr 1 of 17 wileyonlinelibrary.com/journal/cpr (EDF) to characterize the binding site of zerumbone on tubulin. Zerumbone oxime was synthesized as described earlier.25 In brief, zerumbone (0.3 g) was dissolved in 10 mL of ethanol containing 0.9 g of hydroxylamine hydrochloride and 1.8 g potassium carbonate. The mixture was stirred for 5 hours at room temperature. The reaction mixture was then filtered, and the residue was washed with methanol. The filtrate was concentrated under reduced pressure and was then mixed with dichloromethane (10 mL). The organic layer was collected and washed with water. The resultant mixture was concentrated and dried to get crystalline zerumbone oxime, which was subjected to FTIR analysis. Fluoresceinthiocarbamyl ethylenediamine (EDF) was synthesized as described earlier.26 Zerumbone oxime (20 mg), 1‐ethyl‐3‐(3‐dimethylaminopropyl) carbodiimide hydrochloride (20 mg) and hydroxysuccinimide (6 mg) were dissolved in 1 mL of dimethylformamide. The mixture was stirred continuously for 1 hour; and to this mixture, 5 mL of dimethylformamide containing 25 mg of EDF was added dropwise over a period 30 minutes. The reaction mixture was allowed to resolve on a preparative silica gel using ethyl acetate/methanol/acetic acid (90/8/2 v/v/v) as mobile phase. The spots on the preparative TLC plate were identified in a UV chamber, and the spot corresponding to FITC‐conjugated zerumbone (Rf 0.49) was eluted using methanol.26 estimated as mmol/cell.29 Standard curve of fluorozerumbone was obtained using the standard solution in the range of 1‐100 µmol/L. Spectral scan was analysed using Systronics AU‐2701 UV‐visible double beam spectrophotometer at 200‐800 nm. estimated as mmol/cell.29 Standard curve of fluorozerumbone was obtained using the standard solution in the range of 1‐100 µmol/L. Spectral scan was analysed using Systronics AU‐2701 UV‐visible double beam spectrophotometer at 200‐800 nm. 2.8 | Mitotic index assay and Immunofluorescence microscopy For determination of mitotic index (MI), HeLa cells were grown on poly‐l‐lysine‐coated glass coverslips (12 mm) in 24‐well tissue culture plates and were subsequently treated with different concentrations of zerumbone for 24 hours. The cells were then fixed with 3.7% (v/v) formaldehyde solution in PBS for 30 minutes at 37°C. The cells were permeabilized with cold methanol at −20°C for 30 minutes. Cells were then stained with Hoechst 33342 (1.5 µg/mL). The coverslips were washed twice with PBS and were mounted on clean glass slides with the mounting medium containing 1,4‐diazabicyclo [2.2.2] octane (DABCO) as anti‐quenching agent. The number of mitotic and interphase cells was counted using a Nikon ECLIPSE Ti‐E inverted fluorescent microscope (Tokyo, Japan). The MI was calculated as the percentage of cells blocked at mitosis.31 At least 1000 cells were counted for each concentrations of zerumbone. The HeLa cells that were treated with different concentrations of zerumbone for 24 hours were fixed with formaldehyde and processed to visualize the interphase and mitotic microtubules using mouse monoclonal alpha‐tubulin antibody and goat %Cell migration %Cell migration = [ 1− (width of scratch at specific time point t width of the scratch at zero time )] ×100 %Cell migration = [ 1− (width of scratch at specific time point t width of the scratch at zero time )] ×100 2.7 | Cell migration assay HeLa cells (1 × 106 cells/mL) were grown in minimum essential medium supplemented with 10% FBS in 35 mm cell culture dishes. At 90% confluence, a wound was made using a sterile micropipette tip.30,31 The floating cells were removed immediately after wounding, and the media were changed with fresh one containing different concentra‐ tions of zerumbone (0, 5, 10 and 15 μmol/L). Cells were observed at 24, 48 and 72 hours of intervals, and the bright‐field images of the wound closure were recorded using the Nikon ECLIPSE Ti inverted micro‐ scope. Percentage wound healing was calculated by using the formula: 2.6 | Calculating the percentage of apoptotic cell death using AO staining HeLa cells (0.5 × 105 cells/mL) grown on poly‐l‐lysine‐coated glass coverslips (12 mm) in 24‐well tissue culture plates were treated with either 0.1% DMSO or different concentrations of zerumbone (10, 20 and 30 μmol/L) for 24 hours. The live cells were immediately viewed under an inverted Nikon ECLIPSE Ti (Tokyo, Japan) fluorescent mi‐ croscope after adding AO (2 μg/mL), and the images were captured using the CoolSNAP digital camera. 2.3 | Fluorescent labelling of zerumbone Zerumbone does not have any characteristic fluorescence; hence, we labelled it with fluorescein isothiocyanate (FITC) by conjugating zerumbone oxime with fluoresceinthiocarbamyl ethylenediamine ASHRAF et al. 3 of 17 3 of 17 2.4 | Cell culture and cell proliferation assay Human cervical cancer cell line (HeLa) and mouse fibroblast cell line (L929) were obtained from the National Centre for Cell Science, Pune, India. The cells were grown in 25 cm2 tissue culture flasks in a humidified atmosphere containing 5% CO2 and 95% air at 37°C. HeLa cells were grown and maintained in minimal essential me‐ dium (MEM) supplemented with 10% (v/v) FBS, sodium bicarbonate and antibiotic solution containing 100 units of penicillin, 100 µg of streptomycin and 0.25 µg of amphotericin B per mL. L929 cells were grown and maintained in Dulbecco’s modified Eagle’s medium (DMEM) supplemented with 10% FBS, sodium bicarbonate and anti‐ biotic solution. The cytotoxic effect of zerumbone on HeLa cells and L929 cells was determined in 96‐well tissue culture plates using the standard SRB assay.27,28 2.13 | Determination of Kd For calculating the dissociation constant, tubulin (1 µmol/L) was in‐ cubated with varying concentrations of zerumbone in PEM buffer at 37°C for 30 minutes. The samples were excited at 295 nm to spe‐ cifically excite the tryptophan residues of tubulin, and the emission spectrum was recorded. The fraction of binding sites (X) occupied by zerumbone was evaluated using the equation X = (Fo − Fc)/ΔFmax, where Fo and Fc represent the fluorescence intensity of tubulin in the absence and presence of varying concentrations of zerumbone. The maximum change in the fluorescence intensity, ΔFmax, was calculated from the Y‐intercept of the graph 1/ΔF vs 1/[zerumbone]. Assuming a single binding site of zerumbone per tubulin dimer, the dissociation constant (Kd) was estimated using the relationship, 1/X = 1 + (Kd/Lf), where Lf is the concentration of free zerumbone.30,39 The experi‐ ment was repeated three times. 2.14 | Sedimentation assay The in vitro microtubule sedimentation assay was performed to detect the effect of zerumbone on the polymerization of tubulin. Different concentrations of zerumbone were incubated with tubu‐ lin (12 μmol/L) in PEM buffer containing 0.8 mol/L glutamate and 1 mmol/L GTP at 37°C for 1 hour. The reaction mixture was then subjected to centrifugation at 50 000 × g for 1 hour. The superna‐ tant and pellet were collected separately, and the protein concentra‐ tion in the supernatant was measured using Bradford assay.30 protein was stored in aliquots at −80°C until further use. All the experiments with tubulin were performed in PEM buffer (25 mmol/L PIPES, 1 mmol/L EGTA, 3 mmol/L MgCl2, pH 6.8). anti‐mouse IgG conjugated to Alexa Fluor 568. The DNA was stained with Hoechst 33342 to visualize the DNA. Gamma tubulin staining was performed using rabbit monoclonal anti‐gamma tubulin antibody at 1:1000 dilutions as described earlier.27,32,33 Immunofluorescence images were acquired using the CoolSNAP digital camera and were processed by using ImageJ (NIH, USA). 2.10 | Prime/MM‐GBSA scoring The binding energy (ΔG binding) of zerumbone to each protein was calculated using Prime/MM‐GBSA method (Schrodinger, LLC) using the equation ΔGbind = ΔEMM + ΔGSolv + ΔGSA, where ΔEMM is the difference in the minimized energies between pro‐ tein‐zerumbone complex and the sum of the minimized energies of unbound protein and zerumbone, ΔGSolv is the difference in the GBSA solvation energy of protein‐zerumbone complex and the sum of the solvation energies of unbound protein and zerumbone, and ΔGSA is the difference in the surface area energies of complex and the sum of the surface area energies of unbound protein and zerumbone. The Prime/MM‐GBSA calculations were performed based on the protein‐zerumbone complexes obtained from Glide docking using OPLS‐2005 forcefield and VSGB2.0 solvent model. 2.9 | Molecular docking study The interaction of zerumbone with tubulin dimer and other cell divi‐ sion proteins such as Eg5, Aurora A, Plk1, Kif2 and Nek2 was analysed through molecular docking. The 3D crystal coordinates of tubulin heter‐ odimer (5J2U), Eg5 motor domain (1X88), Aurora A (5LXM), Polo‐box do‐ main of Plk1(4WHK), Kif2 motor domain (2GRY) and Nek2 (2W5A) were obtained from Protein Data Bank (http://www.rcsb.org/pdb/home/ home.do). The protein preparation wizard of Glide, Schrodinger Maestro v11.1 (Schrodinger, LLC, New York, NY, USA) was used to prepare the protein structures. The structures were refined by adding missing side chains and removing water molecules, ions, cofactors and inhibitors. Then, they were energy‐minimized until the average RMSD of the non‐ hydrogen atoms reached 0.3 Å. The 3D structure of zerumbone was obtained from PubChem (https://pubchem.ncbi.nlm.nih.gov, PubChem CID: 5470187). The low energy conformations of zerumbone were pre‐ pared using LigPrep module of Schrodinger. The molecular docking of zerumbone with different proteins was performed using Grid‐Based Ligand Docking with Energetics (Glide) module of Schrodinger. A grid box covering the entire protein was generated to perform blind docking of zerumbone using extra precision (XP) mode of Glide script. The best docking pose was selected based on Glide scoring function, Glide en‐ ergy, Emodel energy, EvdW and Ecoul using Glide XP Visualizer. 2.12 | Spectral measurements All the absorbance measurements were carried out in Systronics AU‐2701 UV‐visible double beam spectrophotometer using a cuvette of 1 cm path length. FTIR spectrometer (PerkinElmer, PerkinElmer Inc., Waltham, MA, USA) was used to record the FTIR spectra. The fluorescence measurements are performed in JASCO FP‐8300 spectrofluorometer (Tokyo, Japan) equipped with a ther‐ mostatted cell holder directly connected to a circulating water bath for maintaining constant temperature. For all the fluorescence measurements, the inner filter correction was done according to the equation F = Fobs × antilog [(Aex + Aem)/2], where Aex is the absorb‐ ance of ligand at the excitation wavelength, and Aem is the absorb‐ ance of ligand at the emission wavelength.37,38 The background fluorescence exhibited by buffer and free ligands was routinely sub‐ tracted from all the samples. 2.11 | Purification of tubulin Goat brain tubulin was isolated by two cycles of polymerization and depolymerization in the presence of glutamate as described earlier.34,35 Bradford assay was used to estimate the tubulin concentration using bovine serum albumin as the standard.36 The 4 of 17 4 of 17 ASHRAF et al. 2.5 | Cellular uptake of fluorescein zerumbone (fluorozerumbone) HeLa cells and L929 cells (∼1 × 106 cells/mL) were incubated with 0.1% DMSO or 40 µmol/L zerumbone or 40 µmol/L fluorozerumbone for 4 hours. After the incubation period, the cells were collected by trypsinization and counted. Cells were then centrifuged at 800 x g for 10 minutes and washed three times with cold PBS. The cell pellet was then dried and suspended in 800 µL of methanol and sonicated till fluorozerumbone is completely extracted into the methanol fraction. The cell lysate was centrifuged at 2000 x g for 5 minutes. The absorbance and fluorescence spectra (excitation at 494; emission at 500‐600) of the supernatant containing flourozerumbone were recorded. The total cellular uptake was 2.17 | Determination of combination index HeLa cells were incubated with zerumbone or vinblastine or pacli‐ taxel alone or zerumbone (5, 10 and 12 µmol/L) and paclitaxel (5 and 10 nmol/L) in combination or zerumbone (5, 10 and 12 µmol/L) and vinblastine (6 and 1.2 nmol/L) in combination for 24 hours. The CI was calculated to understand the effect of zerumbone on the cy‐ totoxic activity of paclitaxel and vinblastine. The CI was calculated using the Chou and Talalay41 equation: 3.1 | Isolation and characterization Colchicine has a very weak fluorescence in aqueous buffers but ex‐ hibits a strong fluorescence after binding to tubulin.40 This fluores‐ cence property of colchicine is exploited in binding site competition assays to predict the binding site of unknown compounds. Tubulin (1 μmol/L) was incubated with colchicine (10 μmol/L) for 1 hour at 37°C to form a stable tubulin‐colchicine (T‐C) complex which has sev‐ eral fold higher fluorescence than unbound colchicine.40 Different concentrations of zerumbone were then added to the T‐C complex and incubated for further 30 minutes at 37°C. The samples were excited at 360 nm, and the emission spectra were recorded.30,39 Alternatively, competition assay was also done using the fluores‐ cence of the tubulin‐fluorozerumbone complex. Tubulin (2 µmol/L) was incubated with 10 µmol/L fluorozerumbone and incubated for 20 minutes at 37°C. After the incubation period, different con‐ centrations of zerumbone (10 µmol/L or 20 µmol/L), or 20 µmol/L colchicine, or 5 µmol/L vinblastine were added to the tubulin‐ fluorozerumbone complex and incubated under dark at 37°C for fur‐ ther 30 minutes. The samples were excited at 494 nm, and emission spectra were recorded. The competition assay was repeated using EDF in place of fluorozerumbone with different concentrations of zerumbone (10 and 20 µmol/L) and 20 µmol/L colchicine. The methanolic extract of 1 kg of fresh Zingiber zerumbet extract yielded 1.8 g of crystalline zerumbone. The isolated product showed M+ ion at 219 when analysed by LC‐MS (Figure 1A). The compound was further analysed and confirmed by 1H‐NMR (500 MHz, CDCl3) δ = 6‐6.03 (1H,d), 5.95 (1H,s), 5.88‐5.84(1H,s), 5.23‐5.27(1H,m), 2.47‐2.28 (3H,m), 2.19‐2.25(2H,d), 1.88‐1.91(3H,d), 1.54 (3H, s), 1.79(3H, s), 1.2(3H,s), 1.07(3H,s) (Figure 1B). 3.2 | Selective toxicity of zerumbone on the proliferation of cancer cells Zerumbone inhibited the proliferation of human cervical cancer cell line (HeLa) in a concentration‐dependent manner. After 24 hours of incubation, the half‐maximal inhibitory concentration (IC50) of zerumbone on HeLa cells was found to be 14.2 ± 0.5 μmol/L (Figure 2A). Cells treated with 20, 40, 60 and 80 μmol/L zerum‐ bone inhibited the cell proliferation by 63%, 86%, 98% and 100%, respectively. We analysed the effect of zerumbone on L929 cells since it is generally used as an ideal in vitro model to test the chemi‐ cal toxicity, drug cytotoxicity and material biocompatibility.43,44 Interestingly, zerumbone exhibited less cytotoxic effect on nor‐ mal mouse fibroblast cells (L929) with an IC50 of 30.5 ± 1.5 μmol/L (Figure 2B). Cells treated with 10, 20, 40 and 80 μmol/L zerumbone inhibited the growth of L929 cells by 20%, 33%, 70% and 83%, respectively. Different concentrations of zerumbone were added to 12 μmol/L tubulin in the polymerization buffer containing 25 mmol/L PIPES, 1 mmol/L EGTA, 3 mmol/L MgCl2 and 0.8 mol/L glutamate. The assembly reaction was initiated by adding 1 mmol/L GTP and incubated at 37°C.38 The polymerization of tubulin was monitored by light scattering at 550 nm for 15 minutes using JASCO FP‐8300 spectrofluorometer (Tokyo, Japan) connected with circulating water bath maintained at 37°C. where Dm is the median dose, fa is the fraction affected, and fu is the fraction unaffected (fu = 1 − fa). The median dose (Dm) was cal‐ culated as described earlier.42 A CI < 1 indicates synergism, CI = 1 shows additivity, and CI > 1 specifies antagonism. HeLa cells grown on coverslips in 24‐well tissue culture plate were treated with zerum‐ bone in combination with paclitaxel or vinblastine and processed to visualize microtubules and DNA. 2.15 The effect of zerumbone on the assembly of microtubule was also analysed by monitoring the kinetics of tubulin polymerization. 5 of 17 ASHRAF et al. 3.3 | Internalization of fluorozerumbone by HeLa and L929 cells The fluorozerumbone internalized by HeLa and L929 cells treated with 40 µmol/L fluorozerumbone for 4 hours was extracted using methanol and quantified based on the absorption spectra of the standard fluorozerumbone. We found that the uptake of fluorozerumbone by HeLa cells was 26 nmol/cell and that by L929 cells was 14.8 nmol/cell. The methanolic extracts were further subjected to fluorometric analysis by exciting them at 494 nm as explained in Materials and methods. As shown in Figure 2C, the fluorescence intensity of the extract obtained from HeLa cells was 16% higher than that of the extract obtained from L929 cells. These results indicate that the cellular uptake of fluorozerumbone is much higher in tumour cells. CI=[(D) 1∕(Dx) 1]+[(D) 2∕(Dx) 2] CI=[(D) 1∕(Dx) 1]+[(D) 2∕(Dx) 2] Where, (D)1 and (D)2 are the concentrations of drug 1 (zerum‐ bone) and drug 2 (vinblastine or taxol) in combination that produces a given effect, (Dx)1 and (Dx)2 are the concentrations of drug 1 and drug 2 that also produces the same effect when used alone. (Dx), the concentration of the drug which produces any particular effect, was calculated from the median effect equation of the Chou and Talalay41: (Dx)=Dm [fa∕fu ]1∕m (Dx)=Dm [fa∕fu ]1∕m 6 of 17  |     ASHRAF et al 3.4 | Zerumbone‐induced apoptosis in HeLa cells Acridine orange staining is a common method used to detect apoptotic cell death. After 24 hours, the control cells remained which indicated the characteristic of apoptosis (Figure 3A) Approximately 20% and 36% apoptotic cells were detected in the cells treated with 10 and 20 μmol/L zerumbone for 24 hours (Figure 3B). At higher concentration of zerumbone (30 μmol/L) FI G U R E 1 Characterization of zerumbone isolated from Zingiber zerumbet. A, LC‐MS analysis of zerumbone. Zerumbone exhibited an M+ ion at 219.17. The inset shows the chemical structure of zerumbone [(2E,6E,10E)‐2,6,9,9‐tetramethylcycloundeca‐2,6,10‐trien‐1‐one]. B, 1H‐ NMR spectrum of zerumbone (500 MHz, CDCl3) 6 of 17  |     ASHRAF et al. ASHRAF et al. 6 of 17 FI G U R E 1 Characterization of zerumbone isolated from Zingiber zerumbet. A, LC‐MS analysis of zerumbone. Zerumbone exhibited an M ion at 219.17. The inset shows the chemical structure of zerumbone [(2E,6E,10E)‐2,6,9,9‐tetramethylcycloundeca‐2,6,10‐trien‐1‐one]. B, 1H FI G U R E 1 Characterization of zerumbone isolated from Zingiber zerumbet. A, LC‐MS analysis of zerumbone. Zerumbone exhibited an M+ ion at 219.17. The inset shows the chemical structure of zerumbone [(2E,6E,10E)‐2,6,9,9‐tetramethylcycloundeca‐2,6,10‐trien‐1‐one]. 3.3 | Internalization of fluorozerumbone by HeLa and L929 cells B, 1H‐ NMR spectrum of zerumbone (500 MHz, CDCl3) which indicated the characteristic of apoptosis (Figure 3A). Approximately 20% and 36% apoptotic cells were detected in the cells treated with 10 and 20 μmol/L zerumbone for 24 hours (Figure 3B). At higher concentration of zerumbone (30 μmol/L), ~50% cells were found to be in the later stages of apoptotic cell death with the characteristic features such as membrane blebbing migration of cancer cells even at concentrations lower than the IC50. After 24 hours, the control cells have shown 30% migration; during the same time, 5, 10 and 15 μmol/L zerumbone exhibited 20%, 11% and 9% migration, respectively. After 48 hours, the control cells displayed ~56% wound healing, and in the cells treated with zerumbone 5, 10 and 15 μmol/L, the wound healing was found to be 41%, 36% and 15%, respectively. Similar concentration‐dependent inhibition of wound healing was observed after 72 hours of treatment, and the complete wound healing was observed in control cells after 96 hours, while 63%, 56% and 35% wound healing were observed in the cells treated with 5, 10 and 15 μmol/L zerumbone (Figure 4B). The results suggest that zerumbone can effectively prevent the migration of cancer cells in a concentration‐ and time‐dependent manner even at concentrations lower than the IC50. 3.6 | Zerumbone caused depolymerization of interphase and mitotic microtubules in HeLa cells and blocked the cells at mitosis Zerumbone induced significant depolymerization of interphase and mitotic microtubules in HeLa cells. The control cells treated with vehicle (0.1% DMSO) exhibited typical microtubule or‐ ganization with the microtubules spread over the entire cell. Zerumbone at the IC50 (15 μmol/L) and lower concentrations such as 10 μmol/L did not alter the interphase microtubule net‐ work (Figure 5A). However, higher concentrations such as 30 and 100 μmol/L caused significant disruption of the interphase microtubules with shrunken cells. Similarly, zerumbone did not cause significant disruption of the mitotic spindles at the IC50 (15 μmol/L) and lower concentrations but perturbed the organi‐ zation of the chromosomes at the metaphase plate (Figure 5B). Surprisingly, we found that significant number of the mitotic cells had monopolar spindles. Hence, the mitotic cells were further analysed by observing the centrosome using gamma tubulin stain‐ ing. As shown in Figure 5C, zerumbone inhibited the centrosome separation and caused the formation of monopolar spindles with rosette‐like chromosomes around it. The estimation of HeLa cells blocked at mitosis 24 hours of post‐treatment with zerumbone revealed a strong mitotic block with both bipolar and monopolar spindles. In the cells treated with 10, 15, and 30 μmol/L zerum‐ bone, the number of cells with monopolar spindles was calculated to be 3%, 7% and 27%, respectively, and the MI, which is the ratio of the total number of the mitotic cells to the total cells, was found to be 15%, 20% and 34%, respectively. Under similar conditions, the MI of the control cells was 3.5% (Figure 5D). FI G U R E 2 Zerumbone differentially inhibited the proliferation of HeLa (A) and L929 (B) cells. The inhibition of proliferation was determined by the standard SRB assay after treating the cells with different concentration of zerumbone. C, Fluorescence spectra of fluorozerumbone extracted from (▲) L929 and (∆) HeLa cells treated with 40 µmol/L fluorozerumbone. Control L929 (●) and HeLa (○) cells were treated with 0.1% DMSO and cell shrinkage. Under similar conditions, the number of apoptotic cells in the control was 8%. 3.4 | Zerumbone‐induced apoptosis in HeLa cells Acridine orange staining is a common method used to detect apoptotic cell death. After 24 hours, the control cells remained viable and healthy and the zerumbone‐treated cells displayed brightly stained hypercondensed nucleus and membrane blebbing, ASHRAF et al. FI G U R E 2 Zerumbone differentially inhibited the proliferatio of HeLa (A) and L929 (B) cells. The inhibition of proliferation was determined by the standard SRB assay after treating the cells wi different concentration of zerumbone. C, Fluorescence spectra o fluorozerumbone extracted from (▲) L929 and (∆) HeLa cells trea with 40 µmol/L fluorozerumbone. Control L929 (●) and HeLa (○) cells were treated with 0.1% DMSO 7 of 17 ASHRAF et al. 3.5 | Zerumbone inhibited the migration of HeLa cells in a concentration‐dependent manner Since zerumbone treatment produced cells with mitotic abnormalities, we investigated the interaction of the zerumbone with cell division proteins such as tubulin, Eg5, Aurora A, Plk1, Wound healing assay was used to check the migration of HeLa cells upon treatment with different concentrations of zerumbone. As shown in Figure 4A, zerumbone effectively inhibited the 8 of 17  |     ASHRAF et al FI G U R E 3 Zerumbone‐induced apoptosis in HeLa cells in a dose‐dependent manner. A, HeLa cells (0.5 × 105 cells/mL) were incubated with different concentrations of the zerumbone for 24 h. After the incubation period, AO was added and the live cells were viewed under the fluorescent microscope using FITC filter. Apoptotic cells appeared brightly stained with hypercondensed nucleus. Scale bar represents 20 μm. B, Graph represents percentage of apoptotic cells observed after 24 h of treatment with different concentrations of zerumbone. At least, 600 cells were counted for each concentration. The experiment was repeated thrice, and data represent mean ± SD (A) (B) ASHRAF et al. 8 of 17 (B) (B) FI G U R E 3 Zerumbone‐induced apoptosis in HeLa cells in a dose‐dependent manner. A, HeLa cells (0.5 × 105 cells/mL) were incubated with different concentrations of the zerumbone for 24 h. After the incubation period, AO was added and the live cells were viewed under the fluorescent microscope using FITC filter. Apoptotic cells appeared brightly stained with hypercondensed nucleus. Scale bar represents 20 μm. B, Graph represents percentage of apoptotic cells observed after 24 h of treatment with different concentrations of zerumbone. At least, 600 cells were counted for each concentration. The experiment was repeated thrice, and data represent mean ± SD spectrofluorometer by measuring the intrinsic tryptophan fluorescence of tubulin. Zerumbone quenched the intrinsic fluorescence of tubulin in a concentration manner (Figure 7A). Figure 7B shows the change in the fluorescence intensity of tubulin incubated with different concentrations of zerumbone and the analysis of the reduction in the fluorescence of tubulin as a function of zerumbone concentration yielded a dissociation constant (Kd) of 4 μmol/L (Figure 7B inset). Kif2 and Nek2 that play role in the organization of mitotic spindle and organization of chromosomes at the metaphase plate through computational docking. Results of the docking analysis with tubulin implied that zerumbone bound at the interface of the α/β‐tubulin dimer with a Glide docking score of −3.608 kcal/ mol (Figure 6). 3.5 | Zerumbone inhibited the migration of HeLa cells in a concentration‐dependent manner The MM‐GBSA scoring has shown that zerumbone has bound to tubulin dimer at this position with a strong affinity (ΔG = −50.638 kcal/mol). The number of hydrogen bonds and the list of protein residues interacting with zerumbone are given in the Table 1. The interaction between tubulin dimer and zerumbone was stabilized by one hydrogen bond with Valβ355 and many hydrophobic interactions with residues Valα177, Proα222, Tyrα224, Leuβ248 and Metβ325. Comparative analysis of the binding sites of the ligands such as paclitaxel,45 colchicine46 and vinblastine47 on the tubulin heterodimer gave an inference that zerumbone binding site partially overlaps with the DAMA colchicine binding site. The residues that both zerumbone and DAMA colchicine interact in the tubulin heterodimer are Serα178, Thrα179, Leuβ248 and Alaβ354 (Table 1). The docking results of other cell division proteins such as Eg5, Aurora kinase A, Polo‐ like kinase 1 (Plk1), Kif2A and NIMA‐related kinase 2 (Nek2) are shown in Figure 6B‐F. Based on the Glide docking score (Table 1) and MM‐GBSA scoring, it is possible to speculate that Eg5 and Aurora kinase A could also be the potential target for zerumbone in addition to tubulin. 3.9 | Zerumbone inhibited the polymerization of tubulin in vitro The effect of zerumbone on tubulin assembly was analysed by using the sedimentation assay and the light scattering assay. Tubulin (12 µmol/L) was allowed to polymerize in the presence or absence of different concentrations of zerumbone as explained in Materials and methods. Zerumbone inhibited the polymerization of tubulin in a concentration‐dependent manner (Figure 8A). The polymer mass of tubulin treated with 20, 40 and 80 µmol/L zerumbone was found to be 82.6%, 78.5% and 74.3%, respectively, compared to the control, which is considered as 100%. The kinetics of tubulin polymerization upon treatment with zerumbone was analysed using light scattering assay. Similar to the results obtained in the sedimentation assay, light scattering assay also indicated inhibition of tubulin polymerization by zerumbone (Figure 8B), in a concentration‐dependent manner. At 20 μmol/L zerumbone, the polymer mass was inhibited by 12%, and at 40 μmol/L, the polymer mass was decreased by 20% compared to control. The steady‐state reading at the saturation point of polymerization was taken for the calculation of inhibition of polymerization. FI G U R E 4 Effect of zerumbone on the migration of HeLa cells. A, Migration of HeLa cells in the absence and presence of the indicated concentrations of zerumbone at different time intervals (24, 48 72 and 96 h) was monitored as mentioned in Section 2. The scale bar represents 200 µm. B, Percentage of cell migration at specific time intervals in the absence and presence of 5, 10 and 15 µmol/L zerumbone was calculated as described in Section 2. The data shown are mean ± SD of three independent experiments (*P < 0.001; ‡ P < 0.01; †P < 0.05) (A) (B) (A) Addition of 20 µmol/L colchicine to the tubulin‐fluorozerumbone reduced the fluorescence by 44%, confirming that colchicine and zerumbone might share their binding site. Since zerumbone induced depolymerization of microtubules, we also analysed its competition with vinblastine and found that vinblastine (5 µmol/L) enhanced the fluorescence of fluorozerumbone by 44%. The results suggest that binding of vinblastine to tubulin might stabilize the tubulin‐ fluorozerumbone complex and both the ligands could bind simulta‐ neously to tubulin. To confirm that the observed effects are not due to the fluorescence tag (EDF), we repeated the same competition assay with EDF in place of fluorozerumbone and found that both zerumbone and colchicine could not reduce the EDF fluorescence (Figure 9C). (B) 3.11 | Zerumbone inhibited the proliferation of HeLa cells synergistically in combination with vinblastine and paclitaxel The scale bar represents 200 µm. B, Percentage of cell migration at specific time intervals in the absence and presence of 5, 10 and 15 µmol/L zerumbone was calculated as described in Section 2. The data shown are mean ± SD of three independent experiments (*P < 0.001; ‡ P < 0.01; †P < 0.05) 9 of 17 9 of 17 ASHRAF et al. 3.11 | Zerumbone inhibited the proliferation of HeLa cells synergistically in combination with vinblastine and paclitaxel Vinblastine and paclitaxel are FDA‐approved clinically used drugs for the treatment of various types of tumours.48 The effect of zerumbone was similar to other classical antimitotic drugs; hence, to further explore its potential cancer therapeutics we performed the combination studies with vinblastine and paclitaxel. Vinblastine and paclitaxel inhibited the HeLa cell proliferation with an IC50 of 1.2 and 10 nmol/L and a median dose of 1.10  and 9.21 nmol/L, respectively (Figure 10A‐D). The logarithmic plot of the cytotoxic data yielded a median dose of 13.68 µmol/L for zerumbone (Figure 10E). Zerumbone synergistically inhibited the proliferation of HeLa cells when combined with vinblastine and paclitaxel. When 0.6 nmol/L vinblastine was combined with zerumbone of 5, 10 and 12 µmol/L, the inhibition of proliferation of HeLa cell was determined to be 65%, 82% and 86%, respectively. When 5, 10 and 12 µmol/L zerumbone was combined with 1.2 nmol/L vinblastine, the inhibition of proliferation was found to be 84%, 98% and 100%, respectively (Figure 10F). The combination index was calculated based on the Chou and Talalay equation as explained in Materials and methods to demonstrate quantitatively the relationship between the combination of zerumbone with vinblastine and paclitaxel. The CI for the combination of 0.6 nmol/L vinblastine and 5, 10 and 12 µmol/L zerumbone was calculated to be 0.55, 0.48 and 0.45, respectively (Figure 10G) and CI for the combination of 1.2 nmol/L vinblastine with 5 and 10 µmol/L zerumbone was found to be 0.39 and 0.10, respectively (Figure 10G). Combination of 5 nmol/L paclitaxel with 5, 10 and 12 µmol/L of zerumbone inhibited the proliferation of HeLa cells by 82%, 85% and 92%, respectively (Figure 10H). When paclitaxel 10 nmol/L was combined with 5 µmol/L zerumbone, the inhibition of proliferation of HeLa cell was found to be 98%. The CI of 5, 10, and 12 µmol/L zerumbone with 5 nmol/L paclitaxel was calculated to be 0.18, 0.25 and 0.15, respectively. When 5 µmol/L zerumbone was combined with 10 nmol/L paclitaxel, the CI was calculated to be 0.04 (Figure 10I). All the calculated combination indices were found to be lesser than 1, signifying that the combination FI G U R E 4 Effect of zerumbone on the migration of HeLa cells. A, Migration of HeLa cells in the absence and presence of the indicated concentrations of zerumbone at different time intervals (24, 48 72 and 96 h) was monitored as mentioned in Section 2. 3.8 | Binding of zerumbone to tubulin Results from the cell culture studies and docking analysis indicated that tubulin could be one of the primary targets for Zerumbone. Hence, binding of zerumbone on tubulin was analysed using 3.10 | Zerumbone competes with colchicine for the binding site on tubulin The data represent mean ± SD FI G U R E 5 Effect of different concentrations of zerumbone on interphase and mitotic microtubules in HeLa cells. A, HeLa cells were incubated with the indicated concentrations of the zerumbone for 24 h. Microtubules (red) and DNA (blue) were visualized as mentioned in Section 2. Scale bar represents 20 μm. B, Effect of zerumbone on the spindle microtubules and chromosome organization in HeLa cells. Zerumbone induced abnormal spindles and misalignment of chromosomes at the metaphase plate. Scale bar represents 10 μm. C, Zerumbone induced the formation of monopolar spindles in HeLa cells. HeLa cells were incubated with different concentrations of zerumbone for 24 h, and the cells were then fixed and processed to visualize centrosomes, DNA and microtubules. Scale bar represents 5 μm. D, Zerumbone treatment increased the number of mitotic cells with monopolar spindles in HeLa cells. Percentage of cells with bipola (black) and monopolar (grey) spindles post‐treatment with zerumbone for 24 h are shown in the graph. Inset shows the percentage of total mitotic cells. All the experiments were performed three times. The data represent mean ± SD with 10 and 12 µmol/L zerumbone, the MI was found to be 32% and 37%, respectively (Figure 11B), and when 10 nmol/L paclitaxel was combined with 10 and 12 µmol/L zerumbone, the MI was in‐ creased to 54% and 60%, respectively, while paclitaxel alone at 5 and 10 nmol/L induced 15% and 31% mitotic block (Figure 11B). In addition to the enhanced mitotic arrest, the combined addition of two drugs induced drastic mitotic abnormalities in the organiza‐ tion of the mitotic spindle and alignment of chromosomes (Figures 11C,D). of zerumbone‐vinblastine and zerumbone‐paclitaxel is strongly synergistic in inhibiting the proliferation of HeLa cells. The effect of the combination of zerumbone with vinblastine and paclitaxel on mitotic cells was analysed by calculating the MI. Zerumbone synergistically increased the MI in combination with vinblastine and paclitaxel. As shown in Figure 11A, vinblastine when used alone induced a mitotic block of 9.4%; however, when combined with zerumbone 5, 10 and 12 µmol/L, the mitotic block was increased to 20%, 25% and 31%, respectively. Similarly, vin‐ blastine 1.2 nmol/L when used alone induced a mitotic block of 19%, and when combined with zerumbone 5, 10 and 12 µmol/L, the mitotic block was found to be increased 28%, 38% and 46%, respectively. 3.10 | Zerumbone competes with colchicine for the binding site on tubulin The effect of zerumbone on the binding of colchicine to tubulin was analysed by monitoring the T‐C fluorescence in the presence of increasing concentrations of zerumbone. As shown in Figure 9A, zerumbone quenched the T‐C fluorescence in a concentration‐de‐ pendent manner. Zerumbone at 5 µmol/L quenched the T‐C fluores‐ cence by 29%, and it is clear from the figure that at concentrations more than 5 µmol/L zerumbone, the fluorescence quenching was nearly equal to 30%. This suggests that 5 µmol/L zerumbone is the saturating concentration. Under similar conditions, 40 µmol/L po‐ dophyllotoxin, which is reported to bind on the colchicine site of tu‐ bulin, quenched the fluorescence of T‐C complex by 32%. To further confirm the binding site of zerumbone on tubulin, we have carried out the competition assay using fluorozerumbone, zerumbone and colchicine by analysing the fluorescence exhibited by fluorozerum‐ bone. As shown in Figure 9B, zerumbone 10 µmol/L quenched the fluorescence of fluorozerumbone by 33%. Approximately 60% quenching of fluorescence was observed with 20 µmol/L zerum‐ bone, indicating that fluorozerumbone binds to the zerumbone binding site and labelling with FITC has not altered its binding site. ASHRAF et al. 10 of 17 10 of 17  |     ASHRAF et a FI G U R E 5 Effect of different concentrations of zerumbone on interphase and mitotic microtubules in HeLa cells. A, HeLa cells were incubated with the indicated concentrations of the zerumbone for 24 h. Microtubules (red) and DNA (blue) were visualized as mentioned in Section 2. Scale bar represents 20 μm. B, Effect of zerumbone on the spindle microtubules and chromosome organization in HeLa cells. Zerumbone induced abnormal spindles and misalignment of chromosomes at the metaphase plate. Scale bar represents 10 μm. C, Zerumbone induced the formation of monopolar spindles in HeLa cells. HeLa cells were incubated with different concentrations of zerumbone for 24 h, and the cells were then fixed and processed to visualize centrosomes, DNA and microtubules. Scale bar represents 5 μm. D, Zerumbone treatment increased the number of mitotic cells with monopolar spindles in HeLa cells. Percentage of cells with bipolar (black) and monopolar (grey) spindles post‐treatment with zerumbone for 24 h are shown in the graph. Inset shows the percentage of total mitotic cells. All the experiments were performed three times. 3.10 | Zerumbone competes with colchicine for the binding site on tubulin Zerumbone induced a significant hike in the mitotic cells when combined with paclitaxel, similar to its synergistic ac‐ tivity with vinblastine. When 5 nmol/L paclitaxel was combined 4 | DISCUSSION Several secondary metabolites from plants have successful application as chemotherapeutic agents either in their unmodified |  11 of 17 ASHRAF et al. FI G U R E 6 Computational docking analysis of zerumbone with different cell division proteins. Interaction of zerumbone with A, tubulin heterodimer (5J2U), B, Eg5 motor domain (1X88), C, Aurora A (5LXM), D, Polo‐box domain of Plk1(4WHK), E, Kif2 motor domain (2GRY) and F, Nek2 (2W5A) TA B LE 1 Computational docking analysis of zerumbone with tubulin, Eg5 motor domain, Aurora A, Polo‐box domain of Plk1, Kif2 motor domain and Nek2 ASHRAF et al. |  11 of 17 TA B LE 1 Computational docking analysis of zerumbone with tubulin, Eg5 motor domain, Aurora A, Polo‐box domain of Plk1, Kif2 motor domain and Nek2 TA B LE 1 Computational docking analysis of zerumbone with tubulin, Eg5 motor domain, Aurora A, Polo‐box domain of Plk1, Kif2 motor d i d N k2 TA B LE 1 Computational docking analysis of zerumbone with tubulin, Eg5 motor domain, Aurora A, Polo‐box domain of Plk1, Kif2 motor domain and Nek2 Protein Docking score (kcal/mol) ΔG binding (kcal/mol) No. of H‐ bonds Interacting residues Tubulin heterodimer −3.608 −50.638 1 (Valβ355) Glnα176, Valα177, Serα178, Thrα179, Argα221, Proα222, Thrα223, Tyrα224, Glnβ247, Leuβ248, Metβ325, Thrβ353, Alaβ354, Valβ355 Eg5 −4.672 −42.675 1 (Val194) Ser159, Leu161, Asp187, Arg189, Asn190, Gly193, Val194, Ile195, Ile196, Leu199, Glu201, Val238, Ser240, Thr242, Lys260, Asn262, Ile319 Aurora A −3.657 −50.326 ‐ Leu139, Gly140, Lys141, Lys143, Val147, Lys162, Leu210, Gly216, Thr217, Arg220, Glu260, Asn261, Leu263, Ala273, Asp274 Plk1 −2.636 −30.562 1 (Trp514) Ser418, Leu435, Phe436, Asn437, Ser439, Thr513, Trp514 Kif2a −2.590 −30.574 1 (Asn207) Arg204, Pro205, Asn207, Gly289, Ser290, Gly291, Hie294 Nek2 −2.445 −38.151 ‐ Val97, Lys100, Gly101, Glu104, Gln106, Tyr107, Leu108, Asp109, Phe112 Glide docking score, ΔG binding, number of hydrogen bonds and interacting residues are shown. Glide docking score, ΔG binding, number of hydrogen bonds and interacting residues are shown. form such as paclitaxel, vinblastine, vincristine, camptothecin and podophyllotoxin or in the synthetically modified form such as docetaxel, vinorelbine, vinflunine, topotecan and etoposide.49,50 cell culture studies indicated that zerumbone exerted selective toxicity against HeLa cells compared to L929 cells. The half‐maxi‐ mal inhibitory concentration for L929 (30 μmol/L) cells was 2‐fold higher than that for the HeLa cells (15 μmol/L). 4 | DISCUSSION To further under‐ stand the preferential killing of HeLa cells, we made an attempt to measure the quantitative uptake of zerumbone using the flu‐ orescently labelled compound fluorozerumbone. Although there are several reports on the anti‐cancer activity of zerumbone,14,15 the internalization of zerumbone was not studied as it does not have a characteristic absorbance or fluorescence. Results from the internalization studies indicated that the uptake of fluo‐ rozerumbone was higher in the HeLa cells compared to the L929 cells. Thus, this property is highly favourable for zerumbone to Results from LC‐MS and NMR were in conformity with the mo‐ lecular weight (218.34) and the structure of the compound. The data were in agreement with the earlier published reports.25 Our 12 of 17  |     FI G U R E 7 Zerumbone bound to tubulin and quenched the intrinsic tryptophan fluorescence in a concentration‐dependent manner. A, Tubulin (1 µmol/L) was incubated with zerumbone 0 (●), 5 (○), 10 (▼), 20 (∆), 40 (■) and 80 (□) µmol/L for 30 min at 37°C. The samples were then excited at 295 nm, and the emission spectrum was recorded. B, The change in the intrinsic tryptophan fluorescence intensity was plotted against different concentrations of zerumbone. Inset shows the double reciprocal plot, which yielded a Kd of 4 μmol/L 12 of 17  | ASHRAF et al. 12 of 17 Microtubules play a very important role in cell migration,54 and most of the potent tubulin‐targeted drugs inhibit the migration of the cell at concentrations lower than their IC50.55,56 Since zerumbone showed excellent mitotic block and inhibited the migration of cancer cells, we analysed the effect of zerumbone on interphase and mitotic microtubules using immunofluorescence microscopy. In our study, we observed that zerumbone at IC50 in‐ duced moderate depolymerization of interphase microtubules in HeLa cells, while at 30 μmol/L (2 × IC50) and higher concentrations, it strongly depolymerized the interphase microtubules. Its effect on mitotic cells was more visible and dramatic as both bipolar and mo‐ nopolar cells were observed depending on the concentration used. At the IC50, most of the mitotic cells had bipolar spindles, while at 30 μmol/L, most of the mitotic cells exhibited monopolar spindles, indicating that at the IC50, microtubules are the preferential target; however, at higher concentrations, it is quite possible that it might have additional targets. 4 | DISCUSSION At the IC50, most of the mitotic cells had bipolar spindles, while at 30 μmol/L, most of the mitotic cells exhibited monopolar spindles, indicating that at the IC50, microtubules are the preferential target; however, at higher concentrations, it is quite possible that it might have additional targets. Our results are in agreement with previous report in which zerumbone inhibited the assembly of microtubule and induced apoptosis in PC‐3 and DU‐145 cells.23 But the capabil‐ ity of zerumbone to induce monopolar spindles was not explored earlier. The mitotic cells with monopolar spindles had condensed chromosomes in a rosette‐like configuration similar to that of the monastrol‐treated cells. Monopolar spindles are generally induced by drugs, which target the mitotic kinesins and mitotic kinases that are involved in the organization of mitotic spindle.57 Hence, we performed the docking analysis with tubulin, Eg5, Aurora kinase, Plk1, Nek2 and Kif2A. Our molecular docking results indicated that zerumbone has strong affinity towards Eg5 and Aurora A in addi‐ tion to tubulin. It has been well documented that Eg5 and Aurora A play important role in the separation of centrosomes and inhibition of them could result in the formation monopolar mitotic cells.58-60 FI G U R E 7 Zerumbone bound to tubulin and quenched the intrinsic tryptophan fluorescence in a concentration‐dependent manner. A, Tubulin (1 µmol/L) was incubated with zerumbone 0 (●), 5 (○), 10 (▼), 20 (∆), 40 (■) and 80 (□) µmol/L for 30 min at 37°C. The samples were then excited at 295 nm, and the emission spectrum was recorded. B, The change in the intrinsic tryptophan fluorescence intensity was plotted against different concentrations of zerumbone. Inset shows the double reciprocal plot, which yielded a Kd of 4 μmol/L be used as a relatively less toxic, safe and effective chemothera‐ peutic agent. Curcumin, the dihydroxy polyphenol from Curcuma longa, was also reported to induce selective toxicity in cancer cells due to preferential uptake by the cancer cells compared to nor‐ mal cells.29 AO staining was used to analyse whether the cytotoxic effect of zerumbone was due to necrotic cell death or apoptosis. 4 | DISCUSSION Our results are in agreement with previous report in which zerumbone inhibited the assembly of microtubule and induced apoptosis in PC‐3 and DU‐145 cells.23 But the capabil‐ ity of zerumbone to induce monopolar spindles was not explored earlier. The mitotic cells with monopolar spindles had condensed chromosomes in a rosette‐like configuration similar to that of the monastrol‐treated cells. Monopolar spindles are generally induced by drugs, which target the mitotic kinesins and mitotic kinases that are involved in the organization of mitotic spindle.57 Hence, we performed the docking analysis with tubulin, Eg5, Aurora kinase, Plk1, Nek2 and Kif2A. Our molecular docking results indicated that zerumbone has strong affinity towards Eg5 and Aurora A in addi‐ tion to tubulin. It has been well documented that Eg5 and Aurora A play important role in the separation of centrosomes and inhibition of them could result in the formation monopolar mitotic cells.58-60 The nearly equal affinity of zerumbone towards Eg5 and Aurora A obtained by docking analysis indicated that it might target both the proteins or any one which can be confirmed only through in vitro analysis. It is important to note that zerumbone at its IC50 had more bipolar mitotic cells and at higher concentration such as 30 μmol/L had more of monopolar mitotic cells. The results suggest that tubulin could be the primary target of tubulin at the IC50, and it might target Eg5 or Aurora A or both at 30 μmol/L. At concentrations higher than 30 μmol/L, the number of mitotic cells decreased with simultaneous increase in the number of apoptotic cells. This behaviour is similar to other antimitotic drugs, which at higher concentration will activate the apoptotic pathways much earlier in the cell cycle.32,61,62 Since zerumbone showed excellent mitotic block and inhibited the migration of cancer cells, we analysed the effect of zerumbone on interphase and mitotic microtubules using immunofluorescence microscopy. In our study, we observed that zerumbone at IC50 in‐ duced moderate depolymerization of interphase microtubules in HeLa cells, while at 30 μmol/L (2 × IC50) and higher concentrations, it strongly depolymerized the interphase microtubules. Its effect on mitotic cells was more visible and dramatic as both bipolar and mo‐ nopolar cells were observed depending on the concentration used. 4 | DISCUSSION B, Tubulin (2 μmol/L) was incubated with 10 μmol/L flourozerumbone for 20 min at 37°C. This was followed by the addition of 0 (●), 10 (○) and 20 (▼) µmol/L zerumbone to the tubulin‐fluorozerumbone complex. Colchicine 20 (∆) µmol/L and vinblastine 5 (■) µmol/L were used as positive controls. All the samples were excited at 494 nm, and emission spectra were recorded. (C) The competition assay was repeated using EDF in place of fluorozerumbone with different concentrations of zerumbone 0 (●), 10 (○) 20 (▼) and 20 (∆) µmol/L colchicine FI G U R E 8 Effect zerumbone on the assembly of tubulin. A, A FI G U R E 8 Effect zerumbone on the assembly of tubulin. A, A sedimentation assay was carried out with different concentrations of zerumbone (20, 40 and 80 µmol/L) to determine the percentage of polymer mass of tubulin. The experiment was done three times, and the data represent mean ± SD. B, Zerumbone inhibited the glutamate‐induced polymerization of tubulin. The assembly kinetics of tubulin in the presence and absence of zerumbone 0 (●), 20 (○) and 40 (▼) µmol/L was monitored by recording the light scattering at 550 nm for 15 min as described in Section 2 FI G U R E 8 Effect zerumbone on the assembly of tubulin. A, A sedimentation assay was carried out with different concentrations of zerumbone (20, 40 and 80 µmol/L) to determine the percentage of polymer mass of tubulin. The experiment was done three times, and the data represent mean ± SD. B, Zerumbone inhibited the glutamate‐induced polymerization of tubulin. The assembly kinetics of tubulin in the presence and absence of zerumbone 0 (●), 20 (○) and 40 (▼) µmol/L was monitored by recording the light scattering at 550 nm for 15 min as described in Section 2 of tubulin are also analysed in a cell‐free system using goat/bovine brain tubulin isolated in vitro .37,38 The results from the sedimenta‐ tion assay and the light scattering assay of tubulin polymerization in the presence of glutamate are in excellent agreement with the results obtained from the cell culture studies, where zerumbone caused strong depolymerization of the interphase and mitotic micro‐ tubules in HeLa cells. 4 | DISCUSSION FI G U R E 8 Effect zerumbone on the assembly of tubulin. A, A sedimentation assay was carried out with different concentrations of zerumbone (20, 40 and 80 µmol/L) to determine the percentage of polymer mass of tubulin. The experiment was done three times, and the data represent mean ± SD. B, Zerumbone inhibited the glutamate‐induced polymerization of tubulin. The assembly kinetics of tubulin in the presence and absence of zerumbone 0 (●), 20 (○) and 40 (▼) µmol/L was monitored by recording the light scattering at 550 nm for 15 min as described in Section 2 |  13 o G U R E 9   Competition experiment of zerumbone and orozerumbone with colchicine, podophyllotoxin and blastine for the localization of binding site on tubulin. A, lchicine (10 μmol/L) was allowed to form complex with bulin (2 μmol/L) for 1 h at 37°C. Different concentrations of umbone 0 (●), 5 (○), 10 (▼) and 20 (∆) µmol/L were added the complex. Podophyllotoxin 40 (■) µmol/L was used as a sitive control. The samples were excited at 360 nm, and the ission spectrum was recorded. B, Tubulin (2 μmol/L) was ubated with 10 μmol/L flourozerumbone for 20 min at 37°C ASHRAF et al. 13 of 17 | FI G U R E 8 Effect zerumbone on the assembly of tubulin. A, A sedimentation assay was carried out with different concentrations of zerumbone (20, 40 and 80 µmol/L) to determine the percentage of polymer mass of tubulin. The experiment was done three times, and the data represent mean ± SD. B, Zerumbone inhibited the glutamate‐induced polymerization of tubulin. The assembly kinetics of tubulin in the presence and absence of zerumbone 0 (●), 20 (○) and 40 (▼) µmol/L was monitored by recording the light scattering at 550 nm for 15 min as described in Section 2 F I G U R E 9   Competition experiment of zerumbone and fluorozerumbone with colchicine, podophyllotoxin and vinblastine for the localization of binding site on tubulin. A, Colchicine (10 μmol/L) was allowed to form complex with tubulin (2 μmol/L) for 1 h at 37°C. Different concentrations of zerumbone 0 (●), 5 (○), 10 (▼) and 20 (∆) µmol/L were added to the complex. Podophyllotoxin 40 (■) µmol/L was used as a positive control. The samples were excited at 360 nm, and the emission spectrum was recorded. 4 | DISCUSSION Since zerumbone induced depolymerization of microtubules, and the docking analysis predicted colchicine bind‐ ing domain as the binding site of zerumbone, we decided to confirm the binding site using competition binding assay using two different approaches. In one method, the change in the fluorescence of col‐ chicine‐tubulin complex upon addition of increasing concentrations of zerumbone was monitored. In the other approach, the change in the fluorescence of tubulin‐fluorozerumbone upon addition of dif‐ ferent compounds was monitored. Fluorescently labelled molecules are widely used to characterize the binding interactions and binding site of the ligand on the protein.63 Unlabelled zerumbone effectively quenched the fluorescence of the T‐C complex; similarly, colchicine could quench the fluorescence of fluorozerumbone through com‐ petitive displacement, indicating that fluorozerumbone, zerumbone and colchicine have overlapping binding sites. To further confirm F I G U R E 9   Competition experiment of zerumbone and F I G U R E 9   Competition experiment of zerumbone and fluorozerumbone with colchicine, podophyllotoxin and vinblastine for the localization of binding site on tubulin. A, Colchicine (10 μmol/L) was allowed to form complex with tubulin (2 μmol/L) for 1 h at 37°C. Different concentrations of zerumbone 0 (●), 5 (○), 10 (▼) and 20 (∆) µmol/L were added to the complex. Podophyllotoxin 40 (■) µmol/L was used as a positive control. The samples were excited at 360 nm, and the emission spectrum was recorded. B, Tubulin (2 μmol/L) was incubated with 10 μmol/L flourozerumbone for 20 min at 37°C. This was followed by the addition of 0 (●), 10 (○) and 20 (▼) µmol/L zerumbone to the tubulin‐fluorozerumbone complex. Colchicine 20 (∆) µmol/L and vinblastine 5 (■) µmol/L were used as positive controls. All the samples were excited at 494 nm, and emission spectra were recorded. (C) The competition assay was repeated using EDF in place of fluorozerumbone with different concentrations of zerumbone 0 (●), 10 (○) 20 (▼) and 20 (∆) µmol/L colchicine F I G U R E 9   Competition experiment of zerumbone and fluorozerumbone with colchicine, podophyllotoxin and vinblastine for the localization of binding site on tubulin. A, Colchicine (10 μmol/L) was allowed to form complex with tubulin (2 μmol/L) for 1 h at 37°C. Different concentrations of zerumbone 0 (●), 5 (○), 10 (▼) and 20 (∆) µmol/L were added to the complex. Podophyllotoxin 40 (■) µmol/L was used as a positive control. 4 | DISCUSSION Apoptotic cells will appear brightly stained, hypercondensed and often fragmented chromatin in spherical or irregular shapes under fluorescent microscope.52 It was clearly evident that by the end of one cell cycle, most of the zerumbone‐treated cells underwent apoptosis since their characteristics were similar to those of the apoptotic cells.52 Molecules preventing metastasis are highly valuable in cancer chemotherapy as they can prevent the cancer cells spreading to other tissues. Results from the cell migration assays indicate that zerumbone strongly inhibited the migration of cancer cells at 5 and 10 μmol/L, which are much lower concen‐ trations than its IC50. This result is in agreement with the previous report suggesting the anti‐metastatic property of zerumbone.53 be used as a relatively less toxic, safe and effective chemothera‐ peutic agent. Curcumin, the dihydroxy polyphenol from Curcuma longa, was also reported to induce selective toxicity in cancer cells due to preferential uptake by the cancer cells compared to nor‐ mal cells.29 AO staining was used to analyse whether the cytotoxic effect of zerumbone was due to necrotic cell death or apoptosis. Apoptotic cells will appear brightly stained, hypercondensed and often fragmented chromatin in spherical or irregular shapes under fluorescent microscope.52 It was clearly evident that by the end of one cell cycle, most of the zerumbone‐treated cells underwent apoptosis since their characteristics were similar to those of the apoptotic cells.52 Molecules preventing metastasis are highly valuable in cancer chemotherapy as they can prevent the cancer cells spreading to other tissues. Results from the cell migration assays indicate that zerumbone strongly inhibited the migration of cancer cells at 5 and 10 μmol/L, which are much lower concen‐ trations than its IC50. This result is in agreement with the previous report suggesting the anti‐metastatic property of zerumbone.53 Results from the docking studies and the cell culture studies in which zerumbone induced a strong mitotic block and depolymeriza‐ tion of interphase microtubules motivated us to study its effect on purified tubulin isolated from goat brains using fluorescence spec‐ troscopy. Tubulin heterodimer contains eight tryptophan residues, and interaction of small molecules will disturb the conformation of tubulin, leading to change in the intrinsic fluorescence contrib‐ uted by the tryptophan residues.38 Zerumbone binding to tubulin with a Kd of 4 μmol/L indicates that it has a high affinity for tubulin. The effects of microtubule targeted drugs on the polymerization |  13 of 1 ASHRAF et al. 4 | DISCUSSION All the experiments were performed three independent times, and the error bars represent mean ± SD |  15 of 17 |  15 of 17 ASHRAF et al. FI G U R E 11 Zerumbone potentiated the mitotic block in HeLa cells in combination with vinblastine and paclitaxel. Zerumbone increased the number of mitotic cells in HeLa cells in combination with vinblastine (A) and paclitaxel (B). All the experiments were performed three times. The data represents mean ± SD. Effect of zerumbone on the spindle microtubule and chromosome alignment in combination with vinblastine (C) and paclitaxel (D). HeLa cells treated with indicated concentrations of zerumbone, vinblastine and paclitaxel were fixed and processed for immunofluorescence microscopy as described in Section 2. The scale bar represents 10 μm ASHRAF et al. FI G U R E 11 Zerumbone potentiated the mitotic block in HeLa cells in combination with vinblastine and paclitaxel. Zerumbone increased the number of mitotic cells in HeLa cells in combination with vinblastine (A) and paclitaxel (B). All the experiments were performed three times. The data represents mean ± SD. Effect of zerumbone on the spindle microtubule and chromosome alignment in combination with vinblastine (C) and paclitaxel (D). HeLa cells treated with indicated concentrations of zerumbone, vinblastine and paclitaxel were fixed and processed for immunofluorescence microscopy as described in Section 2. The scale bar represents 10 μm that the observed quenching of fluorozerumbone is not due to arte‐ fact induced by the labelled molecule, we have used EDF in the place of fluorozerumbone and found that colchicine could not quench the FITC fluorescence. microtubule depolymerizing drug colchicine, it exhibited strong mitotic block when combined with a MT‐stabilizing drug such as paclitaxel or a MT‐destabilizing drug such as vinblastine at very low concentrations. It is well documented that microtubule polymerization inducing agents and microtubule depolymerizing agents inhibit the cell proliferation at the IC50 only by suppress‐ ing the microtubule dynamics.34,65 It is reasonable to assume that the synergistic activity of zerumbone with vinblastine and taxol could be due to the strong suppression of microtubule dynamics. Combination therapy which uses two or more drugs is excel‐ lent for cancer treatment because it is more effective against tumour growth and metastasis. 4 | DISCUSSION The samples were excited at 360 nm, and the emission spectrum was recorded. B, Tubulin (2 μmol/L) was incubated with 10 μmol/L flourozerumbone for 20 min at 37°C. This was followed by the addition of 0 (●), 10 (○) and 20 (▼) µmol/L zerumbone to the tubulin‐fluorozerumbone complex. Colchicine 20 (∆) µmol/L and vinblastine 5 (■) µmol/L were used as positive controls. All the samples were excited at 494 nm, and emission spectra were recorded. (C) The competition assay was repeated using EDF in place of fluorozerumbone with different concentrations of zerumbone 0 (●), 10 (○) 20 (▼) and 20 (∆) µmol/L colchicine 14 of 17  | 14 of 17  |     ASHRAF et al. 14 of 17  | 14 of 17  |     ASHRAF et FI G U R E 10 Zerumbone inhibited th proliferation of HeLa cells in synergism with vinblastine and paclitaxel. HeLa cells were treated with different concentrations of vinblastine (A) and paclitaxel (B) for 24 h, and the inhibition of cell proliferation was determined using SRB assay as described in Section 2. Median effect plot for the inhibition of cell proliferation by vinblastine (C), paclitaxel (D) and zerumbone (E). Zerumbone synergistically inhibited the proliferation of HeLa cells in combinatio with vinblastine (F) and paclitaxel (G). The combined effect of zerumbone with vinblastine (H) and paclitaxel (I) on the proliferation of HeLa cells was calculate using the Chou and Talalay equation, an the CI at different drug concentrations was calculated. All the experiments wer performed three independent times, and the error bars represent mean ± SD ASHRAF et al. 14 of 17 FI G U R E 10 Zerumbone inhibited the proliferation of HeLa cells in synergism with vinblastine and paclitaxel. HeLa cells were treated with different concentrations of vinblastine (A) and paclitaxel (B) for 24 h, and the inhibition of cell proliferation was determined using SRB assay as described in Section 2. Median effect plot for the inhibition of cell proliferation by vinblastine (C), paclitaxel (D) and zerumbone (E). Zerumbone synergistically inhibited the proliferation of HeLa cells in combination with vinblastine (F) and paclitaxel (G). 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Wood Waste Turned Into Value Added Products: Thermal Plasticization by Benzylation Process
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ner, Köse, Yürümez, Ümit Yalçın, Akgül: Wood Waste Turned Into Value Added Products ner, Köse, Yürümez, Ümit Yalçın, Akgül: Wood Waste Turned Into Value Added Products Birol Üner1, Gökhan Köse1, Yeşim Yürümez1, Ömer Ümit Yalçın2, Mehmet Akgül3 1 Autori su izvanredni profesor, dodiplomski student i doktorand Sveučilišta Suleyman Demirel, Šumarski fakultet, Zavod za inženjerstvo proizvoda baziranih na šumi, Isparta, Turska. 2Autor je profesor Sveučilišta Suleyman Demirel, Doktorska škola prirodnih i primijenjenih znanosti, Isparta, Turska. 3Autor je profesor Sveučilišta Necmettin Erbakan, Fakultet inženjerstva, Zavod za inženjerstvo materijala, Seydişehir/Konya, Turska. 1 Authors are associate professor, undergraduate student and PhD student at Suleyman Demirel University, Faculty of Forestry Department of Forest Products Engineering, Isparta, Turkey. 2Author is professor at Suleyman Demirel University, Graduate School of Natural and Applied Sciences, Isparta, Turkey. 3Author is professor at Necmettin Erbakan University, Engineering Faculty, Material Engineering Department., Seydişehir/Konya, Turkey. 1 Authors are associate professor, undergraduate student and PhD student at Suleyman Demirel University, Faculty of Forestry Department of Forest Products Engineering, Isparta, Turkey. 2Author is professor at Suleyman Demirel University, Graduate School of Natural and Applied Sciences, Isparta, Turkey. 3Author is professor at Necmettin Erbakan University, Engineering Faculty, Material Engineering Department., Seydişehir/Konya, Turkey. 1 Autori su izvanredni profesor, dodiplomski student i doktorand Sveučilišta Suleyman Demirel, Šumarski fakultet, Zavod za inženjerstvo proizvoda baziranih na šumi, Isparta, Turska. 2Autor je profesor Sveučilišta Suleyman Demirel, Doktorska škola prirodnih i primijenjenih znanosti, Isparta, Turska. 3Autor je profesor Sveučilišta Necmettin Erbakan, Fakultet inženjerstva, Zavod za inženjerstvo materijala, Seydişehir/Konya, Turska. Pretvorba drvnog otpada u proizvod dodane vrijednosti: toplinsko plastificiranje primjenom procesa benzilacije Original scientifi c paper • Izvorni znanstveni rad Received – prispjelo: 16. 9. 2015. Accepted – prihvaćeno: 6. 9. 2016. UDK: 630*839.8; 630*812.14; 630*871.1 doi:10.5552/drind.2016.1534 ABSTRACT • Sawdust is usually considered as waste in wood-using industries. These materials can be converted into value added thermoplastics by means of benzylation. Products can be utilized in different applications where plastics are used. Thermoplasticization process was carried out with benzyl chloride under different alkaline conditions, 15, 25 and 35 %, respectively. Alkali ions and concentration affect the substitution reaction. Tailored material structure was remarkably changed. In order to detect changes, crystallinity index of the materials and thermal properties were analyzed. In FTIR spectra, the peaks appeared at 698 cm-1 and 740 cm-1, which indicates the aromatic C-C angular deformation. Multiple peak appears at 3030 cm-1, which indicates benzylation of the ma- terials. The peak increase can be observed at 1596 cm-1 due to the aromatic deformation. Benzyl groups attached to hydroxyl to form ether groups increase the peak intensity. As a result of that, hydrogen bond energy changes and crystallinity of the materials is reduced. This substitution of functional groups changes the decomposition temperature of modifi ed materials. It reduces the decomposition temperature to between 330 and 350 ºC. Thermo- gravimetric analysis revealed that modifi ed products were characterized by poorer thermal stability compared to raw materials. Keywords: thermoplastic resin, hydrogen bond energy, crystallinity, thermal properties SAŽETAK • Piljevina se u drvoprerađivačkoj industriji često smatra otpadom. Međutim, ona se postupkom benzi- lacije može pretvoriti u termoplastični materijal kao proizvod dodane vrijednosti. Tako dobiveni materijal moguće je upotrijebiti za različite namjene za koje se obično rabe plastične mase. Toplinski postupak plastifi ciranja pro- vodi se benzil kloridom pri različitim lužnatim uvjetima (15, 25 ili 35 %). Koncentracija lužnatih iona utječe na reakciju supstitucije. Struktura materijala značajno se mijenja. Radi otkrivanja promjene, analizirani su indeks kristaliničnosti materijala i toplinska svojstva. U infracrvenom dijelu spektra pikovi su se pojavili na 698 cm-1 DRVNA INDUSTRIJA 67 (4) 315-322 (2016) 315 ner, Köse, Yürümez, Ümit Yalçın, Akgül: Wood Waste Turned Into Value Added Products i 740 cm-1, što upućuje na aromatske C-C kutne deformacije. Višestruki maksimum pojavljuje se pri 3030 cm-1, što označava benzilaciju materijala. Zbog aromatske deformacije porast pika može se primijetiti na 1596 cm-1. 1 INTRODUCTİON 1. UVOD plasticity was achieved by benzylation process, and characterization of the material was accomplished with crystallinity index and thermogravimetric analy- sis. Increased utilization of wood results in more waste due to debarking, cutting, shaving, sanding, etc. How to deal with this waste is an important issue. En- vironmental concern and defi ciency in natural resourc- es impose solutions that involve reducing waste and recovering and recycling this material to conserve nat- ural resources. Progress has been made in efforts to reduce waste, but additional works need to be done to solve this problem. Pretvorba drvnog otpada u proizvod dodane vrijednosti: toplinsko plastificiranje primjenom procesa benzilacije Benzilne skupine vezane na hidroksilne skupine tvore eterske skupine i povećavaju intenzitet pika, zbog čega se mijenja energija vodikove veze i kristaliničnost materijala se smanjuje. Taj postupak supstitucije na funkcionalnim skupinama mijenja temperaturu razgradnje modifi ciranih materijala. Smanjuje se temperatura dekompozicije na 330 i 350 °C. Termogravimetrijska analiza pokazala je da modifi cirani proizvodi imaju lošiju toplinsku stabilnost nego sirovina od koje su proizvedeni. Ključne riječi: termoplastične smole, energija vodikove veze, kristaliničnost, toplinska svojstva 2.2.1 Alkali treatment 2.2.1. Lužnata obrada Sawdust was treated with different concentra- tions of sodium, lithium, potassium hydroxide and guanidine for 24 hrs at room temperature. After treat- ment, materials were rinsed and dried at 102±3 ºC and tested with Fourier transform infrared spectroscopy (FTIR). 2.1 Materials 2 1 Materijali 2.1 Materials 2.1. Materijali Turkish red pine (TRP) sawdust were obtained from the Isparta region in Turkey. Materials were dried under room temperature. Meal samples were prepared using a Wiley mill and ground to pass various mesh screens (60-80 mesh). Sawdust and wood shavings are produced by a number of sectors and this raw material is part of the municipal waste stream. A tremendous range of prod- ucts can be obtained from these lignocellulosic waste materials due to complexity of cell wall structure. Lig- nocellulosic materials refer to woody and nonwoody plants that are composed of cellulose, hemicellulose, and lignin. Chemical properties of these materials make them suitable for a large number of products. The utilization of lignocellulosic materials in the produc- tion of plastic and composites is becoming more and more attractive (Biswas et al., 2006; Bodirlau et al., 2008; Çöpür et al., 2007; Ebringerova and Heinze, 2000; Hassan et al., 2001; Hon and Luis, 1989; Hon and Ou, 1989; Pereira et al.,1997; Rowell, 1990; Su et al., 2015). 2.2.2 Benzylation process 2.2.2. Proces benzilacije Plastics are being favored for many applications in place of other materials due to formability, light weight and strength properties (Hon and Shiraishi, 2001). In order to plasticize, lignocelullosic materials are chemically modifi ed. Chemical modifi cation of cell wall polymers is usually carried out either in acidic or alkali conditions (Chen et al., 2012; Huang et al., 2014; Qu et al., 2014). Chemical alteration of hydroxyl groups can reduce the interaction among the chain seg- ments, creates free volume and changes the thermal properties of the material (Nakano, 1994). New modi- fi ed lignocellulosic materials have thermoplasticity, when large or many side-chains are introduced by chemical modifi cation. Benzylation process was used similarly to Hon and Ou process. Dried and ground samples (3 grams) were pretreated with various alkali concentrations (15, 25, 35 %) for preswelling. The slurry was transferred to a 250 ml round bottom fl ask containing benzyl chlo- ride (BC) (27.6 ml). The reaction was conducted at 110 ºC for 6 hours with continuous stirring. The crude ben- zylated material was collected by fi ltration and exhaus- tively washed with water to remove any residuals and fi nally washed with ethanol to remove any residual benzyl alcohol. The fi nal product was dried overnight at 40 ºC. 2 RESULTS AND DISCUSSION 2. REZULTATI I RASPRAVA The degree of crystallinity is an important pa- rameter for plasticization of lignocellulosic materials. Cell wall polymers contain signifi cant amounts of hy- droxyl groups. Chemical modifi cation of lignocellu- losic materials usually takes place between these hy- droxyl groups of the cell wall polymers and chemical reagent with or without catalyst (Rowell, 1990). Tai- lored hydroxyl groups can help lignocellulosic materi- als to exhibit thermoplastic properties due to change in hydrogen bonding ability. Figure 1 Assignment of hydroxyl bands in FTIR spectra for untreated Turkish red pine Slika 1. Raspored hidroksilnih skupina na infracrvenom dijelu spektra za netretiranu piljevinu od drva turskoga crvenog bora of the bonded OH groups, and K is a constant (1/ K = 2.625 · 102 kJ). In order to increase reactivity and swell lignocel- lulosic materials before modifi cation, the material was treated with alkaline solution. Alkaline treatments con- tracts microfi brils in the cell wall and changes the di- mension (Nakano, 2010). The mechanism of microfi - bril contraction and anisotropic dimensional changes of cells in wood are treated with aqueous NaOH solu- tion. As a result, chemical reagent can reach and react with hydroxyl groups on cell wall polymers. In untreat- ed materials, there are free, intra and inter hydrogen bonded hydroxyl groups available (Figure 1). The in- tramolecular hydrogen bond in lignin can be observed at 3560-3580 cm-1 (Kondo, 1997; Poletto et al., 2012). Intramolecular hydrogen bond in cellulose appears around 3430 cm-1 and 3340 cm-1 (Kondo, 1997; Poletto et al., 2014). After alkaline treatment, these hydroxyl groups affected crystalline structure changes. Table 1 shows the crystallinity and hydrogen bond energy change after alkaline treatment (Table 1). Introducing new chemicals to cell wall polymers causes breaking up of some hydrogen bonds and changes crystallinity. This change indicates the possi- bility of plasticization and can be detected with FTIR spectroscopy. In order to detect the change in crystal- linity, different peak ratios and hydrogen bond energy can be used (Poletto et al., 2013; Akgul et al., 2007). The ratio between the heights of the bands at 1370 cm-1 and 2900 cm-1 was used to determine the total crystal- line index (TCI), and the ratio between the areas of the bands at 1430 cm-1 and 896 cm-1 was used to determine lateral order index (LOI) of the materials (Akerholm, et al., 2004; Ma, 2007). 2.2.4 Thermal properties 2.2.4. Toplinska svojstva 3 2.5 2 1.5 1 0.5 0 3800 3700 3600 3500 3400 3300 3200 3100 3000 2900 2800 Wavenumber / valni broj, cm–1 Absorbance / apsorpcija, a.u Figure 1 Assignment of hydroxyl bands in FTIR spectra for untreated Turkish red pine Slika 1. Raspored hidroksilnih skupina na infracrvenom dijelu spektra za netretiranu piljevinu od drva turskoga crvenog bora Perkin Elmer Diamond model thermogravimetric analyzer (TGA) was used to study the thermal proper- ties of etherifi ed samples. The heating rate was set at 10 ºC/min and the temperature ranged from 25 to 600 ºC. Measurements on 4 mg samples were carried out under nitrogen atmosphere (100 ml/min). 2.2.3 Fourier transform infrared spectroscopy (FTIR) Thermoplastic materials are usually produced from petrochemicals, which is dependent on limited source of fossil fuels whose price is fl uctuating and may be running out. In addition to that, fossil fuels release gases which cause the greenhouse effect. One solution is to use tailored renewable resources. There- fore, the objectives of this paper were to explore the chemical modifi cation of Turkish red pine sawdust via etherifi cation to impart thermoplasticity. Thermo- ( ) 2.2.3. Fourierova transformacija infracrvene spektroskopije FTIR spectra were obtained on untreated and benzylated substrates from KBr pellet using Perkin Elmer spectrum one model FTIR spectrometer. Each sample was scanned 10 times between 4000 and 400 cm-1 and changes in the chemical structure were re- corded. DRVNA INDUSTRIJA 67 (4) 315-322 (2016) 316 ner, Köse, Yürümez, Ümit Yalçın, Akgül: Wood Waste Turned Into Value Added Products DRVNA INDUSTRIJA 67 (4) 315-322 (2016) 317 2 RESULTS AND DISCUSSION 2. REZULTATI I RASPRAVA The energy of the hydrogen bonds EH for the OH stretching band was calculated using equation 1: (1) (1) Where νo is the standard frequency correspond- ing to free OH groups (3650 cm-1), ν is the frequency Where νo is the standard frequency correspond- ing to free OH groups (3650 cm-1), ν is the frequency There is a small difference in crystallinity index and hydrogen bond energy (EH) of the materials. TCI There is a small difference in crystallinity index and hydrogen bond energy (EH) of the materials. TCI Table 1 Crystallinity of alkaline treated materials of Turkish red pine (TRP) Tablica 1. Kristaliničnost materijala piljevine turskog crvenog bora (TPR) tretiranog lužinama Table 1 Crystallinity of alkaline treated materials of Turkish red pine (TRP) Tablica 1. Kristaliničnost materijala piljevine turskog crvenog bora (TPR) tretiranog lužinama Crystallinity of raw materials / Kristaliničnost sirovine TCI LOI EH (kJ) δʋ (cm-1) 2 δʋ (cm-1) 3 δʋ (cm-1) 6 Untreated Turkish red pine 0.43 0.36 15.75 2.80 2.79 2.77 15 % NaOH TRP sawdust 7.99 2.18 13.59 2.80 2.78 2.77 25 % NaOH TRP sawdust 5.56 2.81 12.87 2.80 2.78 2.77 35 % NaOH TRP sawdust 5.04 21.63 14.38 2.80 2.78 2.77 15 % KOH TRP sawdust 1.50 9.43 16.54 2.80 2.78 2.77 25 % KOH TRP sawdust 2.07 2.73 17.69 2.80 2.78 2.77 35 % KOH TRP sawdust 2.24 10.74 17.04 2.80 2.78 2.77 15 % LiOH TRP sawdust 3.61 1.17 16.54 2.80 2.78 2.77 25 % LiOH TRP sawdust 1.96 1.37 17.98 2.80 2.78 2.77 35 % LiOH TRP sawdust 2.14 1.44 20.35 2.80 2.78 2.77 15 % Guanidine TRP sawdust 3.60 20.20 16.54 2.80 2.78 2.77 25 % Guanidine TRP sawdust 4.70 0.25 17.40 2.80 2.78 2.77 35 % Guanidine TRP sawdust 0.89 4.05 16.54 2.80 2.78 2.77 317 öse, Yürümez, Ümit Yalçın, Akgül: Wood Waste Turned Into Value Added Products... .. transition take place under the alkaline solution. As the concentration of alkali, in particular NaOH, increases, TCI decreases. In contrast to that, LOI increased with the NaOH, KOH and LiOH concentration. It is probably due to the transition between cellulose polymorph and the effect of alkali ion (Nakano, 2010; Borysiak and Doczekalska, 2005). The lowest TCI and LOI values were observed on untreated materials, which may indi- cate that the amorphous region is higher in untreated materials than after alkali treatment when mercerization takes place. 2 RESULTS AND DISCUSSION 2. REZULTATI I RASPRAVA Hydrogen bond energy indicates the interac- tion between the adjacent cellulose chains (Poletto et al., 2012). It increases when cellulose chains approach each other, and the packing density also increases. There was a slight difference among bond distances in alkali treat- ed materials. Hydrogen bond energy increased with al- kali concentration. 0 0.5 1 1.5 2 2.5 3 400 1400 2400 3400 Untreated TRP Alkali Treated TRP Benzylated TRP * Wavenumber / valni broj, cm–1 Absorbance / apsorpcija, a.u Figure 2 FTIR spectra of untreated and chemically treated TRP sawdust of Turkish red pine (TRP) Slika 2. FTIR spektar netretirane i kemijski tretirane piljevine od drva turskoga crvenog bora Figure 2 FTIR spectra of untreated and chemically treated TRP sawdust of Turkish red pine (TRP) Slika 2. FTIR spektar netretirane i kemijski tretirane piljevine od drva turskoga crvenog bora After alkali treatment, the slurry was transferred to a round bottom fl ask containing benzyl chloride. The benzylation process proceeded with continuous stirring. Untreated and chemically treated samples show different FTIR spectra (Figure 2). Benzylation of the materials caused a reduction of hydroxyl peaks at 3400 cm-1and an increase of the aromatic C-C axial de- formations around 1595 cm-1 (Figure 3). In addition to that, aromatic multiple peak appeared at 3030-3100 cm-1. This could be due to monosubstituted benzyl rings. Guanidine showed spectra similar to those of un- treated materials. and LOI of the untreated sample were lower than those of alkaline treated materials. In contrast, hydrogen bond energy was high considering the alkaline treated materi- als. Alkaline treated samples change the crystalline structure and hydrogen bond energy and the degree of this change increases as NaOH < KOH ≤ Guanidine < LiOH at the same concentration. Wood powder merceri- zation under alkaline conditions and the polymorphic and LOI of the untreated sample were lower than those of alkaline treated materials. In contrast, hydrogen bond energy was high considering the alkaline treated materi- als. Alkaline treated samples change the crystalline structure and hydrogen bond energy and the degree of this change increases as NaOH < KOH ≤ Guanidine < LiOH at the same concentration. Wood powder merceri- zation under alkaline conditions and the polymorphic 318 DRVNA INDUSTRIJA 67 (4) 315-322 (2016) Figure 3 Aromatic stretch at 3030 - 3050 cm-1 due to monosubstituted benzyl rings Slika 3. 2 RESULTS AND DISCUSSION 2. REZULTATI I RASPRAVA Aromatski dio na 3030 – 3050 cm-1 zbog monosupstitucije benzilskih prstenova 0.22 0.72 1.22 1.72 2.22 2800 3300 3800 Absorbance / apsorpcija, a.u Wavenumber / valni broj, cm–1 NaOH Untreated 15 % 25 % 35 % + x X 0 0.5 1 1.5 2 2800 3300 3800 Absorbance / apsorpcija, a.u Wavenumber / valni broj, cm–1 KOH 0 0.5 1 1.5 2 2800 3300 3800 Absorbance / apsorpcija, a.u Wavenumber / valni broj, cm–1 LiOH -0.1 0,4 0.9 1.4 1.9 2800 3300 3800 Absorbance / apsorpcija, a.u Wavenumber / valni broj, cm–1 GuOH 2.4 0.22 0.72 1.22 1.72 2.22 2800 3300 3800 Absorbance / apsorpcija, a.u Wavenumber / valni broj, cm–1 NaOH Untreated 15 % 25 % 35 % + x X 0 0.5 1 1.5 2 2800 3300 3800 Absorbance / apsorpcija, a.u Wavenumber / valni broj, cm–1 KOH Absorbance / apsorpcija, a.u Wavenumber / valni broj, cm–1 Wavenumber / valni broj, cm–1 0 0.5 1 1.5 2 2800 3300 3800 Absorbance / apsorpcija, a.u Wavenumber / valni broj, cm–1 LiOH j, -0.1 0,4 0.9 1.4 1.9 2800 3300 3800 Absorbance / apsorpcija, a.u Wavenumber / valni broj, cm–1 GuOH 2.4 Wavenumber / valni broj, cm–1 Figure 3 Aromatic stretch at 3030 - 3050 cm-1 due to monosubstituted benzyl rings Slika 3. Aromatski dio na 3030 – 3050 cm-1 zbog monosupstitucije benzilskih prstenova DRVNA INDUSTRIJA 67 (4) 315-322 (2016) DRVNA INDUSTRIJA 67 (4) 315-322 (2016) 318 ner, Köse, Yürümez, Ümit Yalçın, Akgül: Wood Waste Turned Into Value Added Products ..Üner, Köse, Yürümez, Ümit Yalçın, Akgül: Wood Waste Turned Into Value Added Produ 0 0.2 0.4 0.6 0.8 1 1.2 1400 1500 1600 1700 Absorbance / apsorpcija, a.u Wavenumber / valni broj, cm–1 0 0.2 0.4 0.6 0.8 1 1.2 1400 1500 1600 1700 Absorbance / apsorpcija, a.u Wavenumber / valni broj, cm–1 -0.1 0.1 0.3 0.5 0.7 0.9 1.1 1.3 1400 1500 1600 1700 Absorbance / apsorpcija, a.u Wavenumber / valni broj, cm–1 0.22 0.42 0.62 0.82 1.02 1.22 1400 1500 1600 1700 Absorbance / apsorpcija, a.u Wavenumber / valni broj, cm–1 NaOH KOH LiOH GuOH x Untreated Ƒ 15% + 25% o 35% Figure 4 Increase of aromatic C-C axial deformations around 1595 cm-1 due to benyzlation Slika 4. 2 RESULTS AND DISCUSSION 2. REZULTATI I RASPRAVA Porast aromatske C-C aksijalne deformacije oko 1595 cm-1 zbog benzilacije 0.22 0.72 1.22 1.72 2.22 625 725 825 925 Absorbance / apsorpcija, a.u Wavenumber / valni broj, cm–1 0 0.5 1 1.5 2 625 725 825 925 Absorbance / apsorpcija, a.u Wavenumber / valni broj, cm–1 0 0.5 1 1.5 2 625 725 825 925 Absorbance / apsorpcija, a.u Wavenumber / valni broj, cm–1 0 0.5 1 1.5 2 625 725 825 925 Absorbance / apsorpcija, a.u Wavenumber / valni broj, cm–1 x Untreated Ƒ 15% + 25% o 35% NaOH KOH LiOH GuOH Figure 5 Appearance of bands at 698 cm-1, 740 cm-1 Slika 5. Pojava skupina na 698 cm-1, 740 cm-1 0 0.2 0.4 0.6 0.8 1 1.2 1400 1500 1600 1700 Absorbance / apsorpcija, a.u Wavenumber / valni broj, cm–1 0 0.2 0.4 0.6 0.8 1 1.2 1400 1500 1600 1700 Absorbance / apsorpcija, a.u Wavenumber / valni broj, cm–1 -0.1 0.1 0.3 0.5 0.7 0.9 1.1 1.3 1400 1500 1600 1700 Absorbance / apsorpcija, a.u Wavenumber / valni broj, cm–1 0.22 0.42 0.62 0.82 1.02 1.22 1400 1500 1600 1700 Absorbance / apsorpcija, a.u Wavenumber / valni broj, cm–1 NaOH KOH LiOH GuOH x Untreated Ƒ 15% + 25% o 35% Figure 4 Increase of aromatic C-C axial deformations around 1595 cm-1 due to benyzlation Slika 4. Porast aromatske C-C aksijalne deformacije oko 1595 cm-1 zbog benzilacije 0 0.2 0.4 0.6 0.8 1 1.2 1400 1500 1600 1700 Absorbance / apsorpcija, a.u Wavenumber / valni broj, cm–1 0 0.2 0.4 0.6 0.8 1 1.2 1400 1500 1600 1700 Absorbance / apsorpcija, a.u Wavenumber / valni broj, cm–1 -0.1 0.1 0.3 0.5 0.7 0.9 1.1 1.3 1400 1500 1600 1700 Absorbance / apsorpcija, a.u Wavenumber / valni broj, cm–1 0.22 0.42 0.62 0.82 1.02 1.22 1400 1500 1600 1700 Absorbance / apsorpcija, a.u Wavenumber / valni broj, cm–1 NaOH KOH LiOH GuOH x Untreated Ƒ 15% + 25% o 35% Figure 4 Increase of aromatic C-C axial deformations around 1595 cm-1 due to benyzlation Slika 4. 2 RESULTS AND DISCUSSION 2. REZULTATI I RASPRAVA Porast aromatske C-C aksijalne deformacije oko 1595 cm-1 zbog benzilacije 0.22 0.42 0.62 0.82 1.02 1.22 1400 1500 1600 1700 Absorbance / apsorpcija, a.u Wavenumber / valni broj, cm–1 NaOH 0 0.2 0.4 0.6 0.8 1 1.2 1400 1500 1600 1700 Absorbance / apsorpcija, a.u Wavenumber / valni broj, cm–1 KOH -0.1 0.1 0.3 0.5 0.7 0.9 1.1 1.3 1400 1500 1600 1700 Absorbance / apsorpcija, a.u Wavenumber / valni broj, cm–1 GuOH Figure 4 Increase of aromatic C-C axial deformations around 1595 cm-1 due to benyzlation Slika 4. Porast aromatske C-C aksijalne deformacije oko 1595 cm-1 zbog benzilacije 0.22 0.72 1.22 1.72 2.22 625 725 825 925 Absorbance / apsorpcija, a.u Wavenumber / valni broj, cm–1 0 0.5 1 1.5 2 625 725 825 925 Absorbance / apsorpcija, a.u Wavenumber / valni broj, cm–1 0 0.5 1 1.5 2 625 725 825 925 Absorbance / apsorpcija, a.u Wavenumber / valni broj, cm–1 0 0.5 1 1.5 2 625 725 825 925 Absorbance / apsorpcija, a.u Wavenumber / valni broj, cm–1 x Untreated Ƒ 15% + 25% o 35% NaOH KOH LiOH GuOH Figure 5 Appearance of bands at 698 cm-1, 740 cm-1 Slika 5. Pojava skupina na 698 cm-1, 740 cm-1 DRVNA INDUSTRIJA 67 (4) 315-322 (2016) 0.22 0.72 1.22 1.72 2.22 625 725 825 925 Absorbance / apsorpcija, a.u Wavenumber / valni broj, cm–1 0 0.5 1 1.5 2 625 725 825 925 Absorbance / apsorpcija, a.u Wavenumber / valni broj, cm–1 0 0.5 1 1.5 2 625 725 825 925 Absorbance / apsorpcija, a.u Wavenumber / valni broj, cm–1 0 0.5 1 1.5 2 625 725 825 925 Absorbance / apsorpcija, a.u Wavenumber / valni broj, cm–1 x Untreated Ƒ 15% + 25% o 35% NaOH KOH LiOH GuOH Figure 5 Appearance of bands at 698 cm-1, 740 cm-1 Slika 5. 2 RESULTS AND DISCUSSION 2. REZULTATI I RASPRAVA Pojava skupina na 698 cm-1, 740 cm-1 0 0.5 1 1.5 2 625 725 825 925 Absorbance / apsorpcija, a.u Wavenumber / valni broj, cm–1 KOH 0.22 0.72 1.22 1.72 2.22 625 725 825 925 Absorbance / apsorpcija, a.u Wavenumber / valni broj cm–1 NaOH Wavenumber / valni broj, cm 0 0.5 1 1.5 2 625 725 825 925 Absorbance / apsorpcija, a.u Wavenumber / valni broj, cm–1 GuOH Wavenumber / valni broj, cm 0 0.5 1 1.5 2 625 725 825 925 Absorbance / apsorpcija, a.u Wavenumber / valni broj, cm–1 LiOH Absorbance / apsorpcija, a.u x Untreated Ƒ 15% + 25% o 35% Figure 5 Appearance of bands at 698 cm-1, 740 cm-1 Slika 5. Pojava skupina na 698 cm-1, 740 cm-1 Figure 5 Appearance of bands at 698 cm-1, 740 cm-1 Slika 5. Pojava skupina na 698 cm-1, 740 cm-1 DRVNA INDUSTRIJA 67 (4) 315-322 (2016) DRVNA INDUSTRIJA 67 (4) 315-322 (2016) 319 319 öse, Yürümez, Ümit Yalçın, Akgül: Wood Waste Turned Into Value Added Products... .. 0.22 0.72 1.22 1.72 2.22 900 1000 1100 1200 1300 Absorbance / apsorpcija, a.u Wavenumber / valni broj, cm–1 0 0.5 1 1.5 2 2.5 900 1000 1100 1200 1300 Absorbance / apsorpcija, a.u Wavenumber / valni broj, cm–1 0 0.5 1 1.5 2 2.5 900 1000 1100 1200 1300 Absorbance / apsorpcija, a.u Wavenumber / valni broj, cm–1 0 0.5 1 1.5 2 2.5 900 1000 1100 1200 1300 Absorbance / apsorpcija, a.u Wavenumber / valni broj, cm–1 x Untreated Ƒ 15% + 25% o 35% NaOH KOH LiOH GuOH Figure 6 C-O stretch of the substituted benzyl ether Wavenumber / valni broj, cm 0 0.5 1 1.5 2 2.5 900 1000 1100 1200 1300 Absorbance / apsorpcija, a.u Wavenumber / valni broj, cm–1 LiOH j, 0 0.5 1 1.5 2 2.5 900 1000 1100 1200 1300 Absorbance / apsorpcija, a.u Wavenumber / valni broj, cm–1 GuOH x Untreated Ƒ 15% + 25% o 35% Figure 6 C-O stretch of the substituted benzyl ether Slika 6. C-O dio etera koji je supstituiran benzilom The peak at 670 cm–1gives information on C–OH out of plane bending (Ma, 2007). Spectra of the sam- ples showed a reduction of peak intensity at 670 cm-1. In contrast, peak appears at 698 cm-1due to aromatic C-C angular deformation in treated materials (Figure 5). 2 RESULTS AND DISCUSSION 2. REZULTATI I RASPRAVA In addition to that, benzyl group can be detected at 740 cm-1. The guanidine peak is lower compared to others. Even though it is strong alkali, the size of the molecule may restrict the entrance into the cell wall. After the reaction, an increase of aryl ether band at 1265cm-1 and of alkyl ether band at 1155-1060 cm-1 (due to C-O stretch from the substituted benzyl ether) was observed. (Figure 5). This absorbance band im- plied the substitution of hydroxyl groups in materials with benzyl groups. weight loss curve can be used to tell the point at which weight loss is most apparent (Figure 8). According to the results obtained, raw materials begin to decompose at a higher temperature (Ti = 380 °C) when compared to modifi ed materials (Ti = 330-340 °C). It was seen that there was a signifi cant difference between modifi ed and untreated materials in relation to their respective thermal decomposition temperatures, which may be related to the difference in crystallinity between untreated and modifi ed materials. Tailored hy- droxyl groups change the crystalline structure of the materials, when the intra and intermolecular hydrogen bonds between chains are broken. Rearrangement of the bonding ability of the treated materials reduces the decomposition temperature. As a result, the crystalline domains in the untreated materials show a higher ther- mal stability, when compared to modifi ed materials. Cellulose is the main component in wooden ma- terials and it controls the thermal properties of wood (Hon and Shiraishi, 2001). Hydrogen bonds between cellulose chain and in the chain shift to melting tem- perature over decomposition temperature. Modifi ca- tion of wood helped to disrupt any hydrogen bonds between adjacent chains and in the chain. Therefore, it helps wood to become thermoformable. 4 CONCLUSION 4. ZAKLJUČAK Sawdust is a very promising source of raw materi- als for commercial products such as cellulose, plastics, etc. for many countries due to its availability and abun- dance. However, it is considered as waste. Thermoplas- ticization can be achieved by benzylation. Alkaline spe- cies and alkali concentration affect the substitution reaction and change the material properties. These changes are signifi cant with NaOH, and it can be de- tected with crystallinity, hydrogen bond energy change and thermal analysis. Hydroxyl groups were substituted Figure 7 shows differential thermogravimetric analysis (DTGA) curves of alkali treated with different alkali ions, concentration and untreated materials. The fi rst step of weight loss, from room temperature to 120 °C, was related to the evaporation of absorbed water. The second step ends near 410 °C, and can be described by an abrupt mass loss stage that was related to the main thermal decomposition process. A derivative DRVNA INDUSTRIJA 67 (4) 315-322 (2016) 320 r, Köse, Yürümez, Ümit Yalçın, Akgül: Wood Waste Turned Into Value Added Products... ner, Köse, Yürümez, Ümit Yalçın, Akgül: Wood Waste Turned Into Value Added Products -0.04 0.01 0.06 0.11 0.16 0.21 0.26 0.31 0 100 200 300 400 500 600 Deriv. Weight / promjena mase, %/min Temperature / temperatura, °C -0.05 0 0.05 0.1 0.15 0.2 0.25 0.3 0 100 200 300 400 500 600 Deriv. Weight / promjena mase, %/min Temperature / temperatura, °C x Untreated Ƒ 15% + 25% o 35% NaOH KOH Figure 7 DTGA thermogram of untreated and benzylated wood with different alkali Slika 7. DTGA termogrami netretiranog drva i drva benziliranoga različitim lužinama -0.04 0.01 0.06 0.11 0.16 0.21 0.26 0.31 0 100 200 300 400 500 600 Deriv. Weight / promjena mase, %/min Temperature / temperatura, °C NaOH Temperature / temperatura, °C -0.05 0 0.05 0.1 0.15 0.2 0.25 0.3 0 100 200 300 400 500 600 Deriv. Weight / promjena mase, %/min Temperature / temperatura, °C x Untreated Ƒ 15% + 25% o 35% KOH Figure 7 DTGA thermogram of untreated and benzylated wood with different alkali Slika 7. DTGA termogrami netretiranog drva i drva benziliranoga različitim lužinama Figure 7 DTGA thermogram of untreated and benzylated wood with different alkali Slika 7. 5 REFERENCES 5. LITERATURA 1. Akgul, M; Gumuskaya, E.; Korkut, S., 2007: Crystalline structure of heat-treated Scots pine [Pinus sylvestris L.] and Uludag˘ fi r [Abies nordmanniana (Stev.) subsp. born- muelleriana (Mattf.)] wood. Wood Science and Technol- ogy, 41: 281-289. http://dx.doi.org/10.1007/s00226-006-0110-9. 18. Pereira, R.; Campana Filho, S. P.; Curvelo, A. A. S., 1997: Benzylated Pulps From Sugar Cane Bagasse. Cel- lulose, 4: 21-31. http://dx.doi.org/10.1023/A:1018459016966. http://dx.doi.org/10.1023/A:1018459016966. 19. Poletto, M.; Pistor, V.; Zanetta, A. J., 2013: Structural Characteristics and Thermal Properties of Native Cellu- lose, in Cellulose, Fundamental Aspects Edited by Theo Van de Ven and Louis Godbout published by In Tech Croatia, 2013. 2. Akerholm, M.; Hinterstoisser, B.; Salmen, L., 2004: Characterization of the crystalline structure of cellulose using static and dynamic FT-IR spectroscopy. Carbohy- drate Research, 339: 569-578. http://dx.doi.org/10.1016/j.carres.2003.11.012. 20. Poletto, M.; Junior, H. L. O.; Zattera, A. J., 2014: Native Cellulose: Structure Characterization and Thermal Prop- erties. Materials, 7: 6105-6119. http://dx.doi.org/10.3390/ma7096105. 3. Biswas, A.; Saha, B. C.; Lawton, J. W.; Shogren, R. L.; Willet, J. L., 2006: Process for obtaining cellulose acetate from agricultural by-products. Carbohydrate Polymers, 64:134-137. http://dx.doi.org/10.1016/j.carbpol.2005.11.002. 21. Poletto, M.; Zattera, A. J.; Santana, R. M. C., 2012: Stru- cural differences between wood species: Evidence from Chemical Composition, FTIR Spectroscopy and Ther- mogravimetric Analysis. Journal of Applied Polymer Science, 126, E336-E343. http://dx.doi.org/10.1002/app.36991. http://dx.doi.org/10.1016/j.carbpol.2005.11.002. 4. Bodirlau, R.; Teaca, C. A.; Spiridon, I., 2008: Chemical Modifi cation Of Beech Wood: Effect On Thermal Stabil- ity. Bioresources, 3 (3): 789-800. 5. Borysiak, S.; Doczekalska, B., 2005: X-ray Diffraction Study of Pine Wood Treated with NaOH. Fibres & Tex- tiles in Eastern Europe January / December 2005, 13, No: 5 (53), 87-89. 22. Qu, B.; Qin, T.; Chu, F.; 2014: Cyanoethylation of sev- eral fi ber materials and their utilization as adhesive in wood-based panels. Wood Science Technology, 48: 519- 531. http://dx.doi.org/10.1007/s00226-014-0623-6. 6. Çöpür, Y.; Güler, C.; Akgül, M.; Taşçıoğlu, C., 2007: Some chemical properties of hazelnut husk and its suita- bility for particleboard production. Building and Envi- ronment, 42: 2568-2572. http://dx.doi.org/10.1016/j.buildenv.2006.07.011. 23. Rowell, R. M., 1990: Chemical Modifi cation of Ligno- cellulosic Fibers To Produce High-Performance Com- posites. In: Glass, J. Edward; Swift, Graham (eds.). Agri- cultural and syntheticpoly mers-Biode gradabilityan- dutilization. ACSsymposium series 433. American Chemical Society197th national meeting; 1989 April 9-14; Dallas, TX. Washington, DC: American Chemical Society; 1990. Chapter 21. https://doi.org/10.1021/bk-1990-0433.ch021. 7. 4 CONCLUSION 4. ZAKLJUČAK DTGA termogrami netretiranog drva i drva benziliranoga različitim lužinama DRVNA INDUSTRIJA 67 (4) 315-322 (2016) 321 -0.05 0 0.05 0.1 0.15 0.2 0.25 0 10 20 30 40 50 60 70 80 90 100 0 100 200 300 400 500 600 Weight / masa, % Temperature / temperatura, °C -0.05 0 0.05 0.1 0.15 0 10 20 30 40 50 60 70 80 90 100 0 100 200 300 400 500 600 Deriv.Weight, %/min promjena mase, %/min Weight / masa, % Temperature / temperatura, °C -0.2 -0.1 0 0.1 0.2 0.3 0 10 20 30 40 50 60 70 80 90 100 0 200 400 600 Weight / masa, % Temperature / temperatura, °C -0.1 -0.05 0 0.05 0.1 0.15 0.2 0.25 0.3 0 20 40 60 80 100 0 100 200 300 400 500 600 Weight / masa, % Temperature / temperatura, °C 25 % LiOH 25 % GuOH 25 % KOH 25% NaOH Deriv.Weight, %/min promjena mase, %/min Deriv.Weight, %/min promjena mase, %/min Deriv.Weight, %/min promjena mase, %/min Figure 8 DTG thermograms of benzylated materials under different alkali concentrations Slika 8. DTG termogrami benziliranog materijala pri različitim koncentracijama lužine -0.05 0 0.05 0.1 0.15 0.2 0.25 0 10 20 30 40 50 60 70 80 90 100 0 100 200 300 400 500 600 Weight / masa, % Temperature / temperatura, °C 25 % KOH Deriv.Weight, %/min promjena mase, %/min -0.05 0 0.05 0.1 0.15 0 10 20 30 40 50 60 70 80 90 100 0 100 200 300 400 500 600 Deriv.Weight, %/min promjena mase, %/min Weight / masa, % Temperature / temperatura, °C 25% NaOH -0.1 -0.05 0 0.05 0.1 0.15 0.2 0.25 0.3 0 20 40 60 80 100 0 100 200 300 400 500 600 Weight / masa, % Temperature / temperatura, °C 25 % LiOH Deriv.Weight, %/min promjena mase, %/min -0.2 -0.1 0 0.1 0.2 0.3 0 10 20 30 40 50 60 70 80 90 100 0 200 400 600 Weight / masa, % Temperature / temperatura, °C 25 % GuOH Deriv.Weight, %/min promjena mase, %/min Figure 8 DTG thermograms of benzylated materials under different alkali concentrations Slika 8. DTG termogrami benziliranog materijala pri različitim koncentracijama lužine DRVNA INDUSTRIJA 67 (4) 315-322 (2016) 321 DRVNA INDUSTRIJA 67 (4) 315-322 (2016) DRVNA INDUSTRIJA 67 (4) 315-322 (2016) 321 321 öse, Yürümez, Ümit Yalçın, Akgül: Wood Waste Turned Into Value Added Products... .. 5 REFERENCES 5. LITERATURA Chen, C.; Cho, M.; Kim, B.-W.; Namb, J.-D.; Lee, Y., 2012: Thermo plasticization and characterization of ke- naf fi ber by benzylation. Journal of Industrial Engineer- ing and Chemistry, 18: 1107-1111. http://dx.doi.org/10.1016/j.jiec.2011.12.012. http://dx.doi.org/10.1016/j.jiec.2011.12.012. 24. Su, M.; Cheng, J.; Pan, Z.; Li, X.; Xu, A.; Hong, J., 2015: Study on the Preparation and Mechanical Properties of Injection-Moulded Wood-Based Plastics. Journal of Ap- plied Polymer Science, 132 (5): 41376. http://dx.doi. org/10.1002/app.41376. 8. Ebringerova, A.; Heinze, T., 2000: Xylan and xylan de- rivatives – biopolymers with valuable properties. Macro- molecular Rapid Communication, 21: 542-556. 9. Hassan, M. L.; El-Wakil, N. A.; Sefain, M. Z., 2001: Thermoplasticization of Bagasse by Cyanoethylation. Journal of Applied Polymer Science, 79: 1965-1978. 4 CONCLUSION 4. ZAKLJUČAK 12. Hon, D. N.-S.; Shiraishi, N., 2001: Wood and Cellulosic Chemistry, second edition. Marcel Dekker Inc. New York, Basel. with benzyl groups and this caused the change of FTIR spectra. Guanidine shows different trends. This could be due to the molecule size. It cannot penetrate the pore on the cell wall, because the pore size could be smaller than the molecule size. Na Li and K are effective on thermo- plasticization. Multiple peaks appear at around 3030 cm-1 indicating benzylation of the materials. The appear- ance of the peak at 698 cm-1 and 740 cm-1 indicates the aromatic C-C angular deformation. Thermogravimetric analysis revealed that modifi ed products were character- ized by poorer thermal stability compared to raw materi- als. Deformed crystallinity of the materials reduced sig- nifi cantly the decomposition temperature, and the reduction of the decomposition temperature ranged be- tween 40 to 60 oC. 13. Huang, K.; Zhang, M.; Zhang, G.; Jiang, X.; Huang, D., 2014: Acetylation Modifi cation of Rice Straw Fiber And Its Thermal Properties. Cellulose Chemistry and Tech- nology, 48 (3-4): 199-207. 14. Kondo, T., 1997: The assignment of IR absorption bands due to free hydroxyl groups in cellulose. Cellulose, 4: 281-292. http://dx.doi.org/10.1023/A:1018448109214. 15. Ma, L., 2007: Plasticization of Wood By Benzylation. MSc. Thesis University of Idaho. 16. Nakano, T., 1994: Mechanism Of Thermoplasticity for Chemically-Modifi ed Wood. Holzforschung-Internation- al Journal of the Biology, Chemistry, Physics and Tech- nology of Wood, 48 (4): 318-324. http://dx.doi.org/10.1515/hfsg.1994.48.4.318. 17. Nakano, T., 2010: Mechanism of microfi bril contraction and anisotropic dimensional changes for cells in wood treated with aqueous NaOH solution. Cellulose, 17: 711- 719. http://dx.doi.org/10.1007/s10570-010-9414-x. Corresponding address: 10. Hon, D. N-S.; Louis, J. M. S., 1989: Thermoplasticiza- tion of Wood. II. Cyanoethylation. Journal of Polymer Science. Part A: Polymer Chemistry, 27: 4143-4160. http://dx.doi.org/.1002/pola.1989.080271221. Assoc. prof. BIROL ÜNER, Ph.D. Assoc. prof. BIROL ÜNER, Ph.D. Suleyman Demirel University, Faculty of Forestry Department of Forest Products Engineering Isparta 32260, TURKEY e-mail: biroluner09@gmail.com Suleyman Demirel University, Faculty of Forestry 11. Hon, D. N.-S.; Ou, N.-H., 1989: Thermoplasticization of Wood. I. Benyzlation of wood. Journal of Polymer Sci- ence. Part A: Polymer Chemistry, 27: 2457-2482. http://dx.doi.org/10.1002/pola.1989.080270725. 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Gamificação e gestão de pessoas: um estudo de caso sobre treinamento e ambiente de diversidade cultural
Revista de Carreiras e Pessoas
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RESUMO Este artigo tem como objetivo discutir a aplicação de gamificação na gestão de pessoas, mais especificamente no treinamento e desenvolvimento de competências de colaboradores de diferentes níveis culturais dentro de uma grande organização. Para tal estudo, iremos trabalhar, principalmente, com as teorias de carreira sem fronteiras e de gamificação voltada para o aprendizado ligados a um estudo de caso do desenvolvimento de projeto piloto de treinamento corporativo em empresa de grande porte, com mais de 4 mil colaboradores, ligada ao setor de infraestrutura, com atuação na região metropolitana de São Paulo. O estudo de caso utilizou entrevistas não somente dos profissionais do departamento de treinamento da empresa, como também coletou dados da empresa terceirizada responsável pelo processo de gamificação além de entrevistas com os desenvolvedores do game/ treinamento. Os resultados da pesquisa mostraram as motivações da escolha desta dinâmica e como a empresa se preparou para a implementação do projeto. ABSTRACT ABSTRACT This article aims to discuss the application of gamification in people management, more specifically in the training and development of skills of employees of different cultural levels within a large organization. For this study, we will work mainly with theories of career without frontiers and gamification focused on learning linked to a case study of the development of pilot project of corporate training in large company, with more than 4 thousand employees, linked to the infrastructure sector, operating in the metropolitan region of São Paulo. The case study used interviews not only from the professionals of the training department of the company, but also collected data from the outsourced company responsible for the gamification process in addition to interviews with the developers of the game / training. The results of the research showed the motivations of choosing this dynamic and how the company prepared for the implementation of the project. Dados para contato: Matheus Matsuda Marangoni Escola Superior de Propaganda e Marketing. Rua Dr. Álvaro Alvim, 123 - Vila Mariana, 04018-010, São Paulo, SP, Brasil. URL da Homepage: https://www.espm.br/ Recebido em: 06/02/2017 Aprovado em: 14/12/2017 DOI: http://dx.doi.org/10.20503/recape. v8i3.37285 Mauro Miguel Rodrigues Berimbau Professor de Graduação e de Pós-Graduação Lato Sensu da Escola Superior de Propaganda e Marketing. Coordenador do Game Lab ESPM (Brasil) mauroberimbau@gmail.com Palavras-chave: carreira sem fronteiras; gamificação; gestão de pessoas; treinamento. ISSN: 2237-1427 Vol. 8 | Nº 3 | Ano 2018 Páginas 431-444 Matheus Matsuda Marangoni GAMIFICACIÓN Y GESTIÓN DE PERSONAS: UN ESTUDIO DE CASO SOBRE ENTRENAMIENTO Y AMBIENTE DE DIVERSIDAD CULTURAL Professor de Graduação e de Pós-Graduação Lato Sensu da Escola Superior de Propaganda e Marketing (Brasil) matheus.marangoni@espm.br RESUMO RESUMEN Este artículo tiene como objetivo discutir la aplicación de gamificación en la gestión de perso- nas, más específicamente en el entrenamiento y desarrollo de competencias de colaboradores de diferentes niveles culturales dentro de una gran organización. Para este estudio, vamos a trabajar principalmente con las teorías de carrera sin fronteras y de gamificación orientada al aprendizaje vinculado a un estudio de caso del desarrollo de un proyecto piloto de entrenamiento corporativo en una empresa de gran tamaño, con más de 4 mil colaboradores, vinculada al sector de infraestructura, con actuación en la región metropolitana de São Paulo. El estudio de caso utilizó entrevistas no sólo de los profesionales del departamento de entrenamiento de la empresa, como también recolectó datos de la empresa tercerizada responsable del proceso de gamificación además de entrevistas con los desarrolladores del juego / entrenamiento. Los resultados de la investigación mostraron las motivaciones de la elección de esta dinámica y cómo la empresa se preparó para la implementación del proyecto. Palabras clave: carrera sin fronteras, gamificación, gestión de personas, entrenamiento. Dados para contato: Matheus Matsuda Marangoni Escola Superior de Propaganda e Marketing. Rua Dr. Álvaro Alvim, 123 - Vila Mariana, 04018-010, São Paulo, SP, Brasil. URL da Homepage: https://www espm br/ Recebido em: 06/02/2017 Aprovado em: 14/12/2017 Matheus Matsuda Marangoni, Mauro Miguel Rodrigues Berimbau Keywords: career without borders, gamification, people management, training. Keywords: career without borders, gamification, people management, training. 1 TREINAMENTO, CARREIRA E APRENDIZAGEM A gestão de carreira nas últimas décadas passa por grandes mudanças, conforme Dutra (2010) nos apresenta em um panorâmica histórico das trajetórias de carreira desde os anos 1980. O papel da gestão de carreira que antes voltado mais para as preocupações do indivíduo dentro das organi- zações, passa no final dos anos 1990 e início dos anos 2000, a ser compartilhado entre as motiva- ções pessoais do indivíduo. Uma vez que, as mudanças no cenário do mercado de trabalho e nos anseios pessoais e culturais dos profissionais, faz com que, o planejamento individual ganhe maior destaque nesse processo. Planejamento que deverá ter o desenvolvimento pessoal e profissional fortemente alinhados. Conforme podemos ver na afirmação de Dutra (2010, p. 64): A carreira não deve ser entendida como um caminho rígido a ser seguido ela pessoa e sim como uma sequencia de posições e de trabalhos por ela realizada. Essa sequencia, articulada de forma a conciliar o desenvolvimento das pessoas como o desenvolvimento da empresa… Assim, vemos que os profissionais irão passar por diversas empresas, funções e até mesmo por projetos ao longo de sua vida profissional. E com a constante e crescente quantidade de informa- ReCaPe | Vol. 8 | Nº. 3 | Ano 2018 | set./dez. | p. 432 GAMIFICAÇÃO E GESTÃO DE PESSOAS: UM ESTUDO DE CASO SOBRE TREINAMENTO E AMBIENTE DE DIVERSIDADE CULTURAL ções e conhecimento que a sociedade contemporânea produz, entendemos que o aprendizado toma cada vez mais importância no processo de desenvolvimento da trajetória das carreiras. Trajetórias que, segundo Dutra (2010), se caracterizam por atribuições e responsabilidades de uma mesma na- tureza, de um conjunto de conhecimentos e habilidades inter-relacionados. De forma complementar, segundo Veloso, Dutra e Lima (2016), a definição de carreira con- templa uma sequencia de experiências mais do que uma linearidade hierárquica dentro das orga- nizações. A carreira passou a ser uma questão mais integrada com a vida pessoal dos indivíduos e menos dependente de visão apenas da firma, visto que “… às pessoas, cabe a gestão de seu de- senvolvimento, de sua competitividade profissional e de sua carreira.” (VELOSO, DUTRA, NAKATA. 2016, p. 90) E para o desenvolvimento de sua carreira, é exigido do trabalhador, segundo Dutra (2010), uma série de competências individuais e de acordo com as necessidades da organização. Essas competências, muitas vezes, são exigidas como requisito para a contratação de um funcionário. 1 TREINAMENTO, CARREIRA E APRENDIZAGEM Mas, podemos ver que, em muitos casos parte destas competências são desenvolvidas também pelo pro- cesso de treinamento oferecido pelas empresas. E dependendo da posição do indivíduo dentro da organização, necessidades diferentes de conhecimento e competências são necessárias. Entendendo as características das novas formas de carreiras na sociedade contemporânea e a necessidade de constante desenvolvimento pessoal e profissional por parte dos indivíduos, será necessário olhar as teorias de carreira proteana e carreira sem fronteiras na ótica da aprendizagem. O conceito de carreira proteana trazida por Bendassolli (apud SALA e TREVISAN, 2014, p.159), que nos indica apresentar como principais características: Mudança como dado de realidade; varie- dade de experiências; adaptabilidade e resiliência; identidade como âncora. Vemos desta forma, que um fato importante e comum é a necessidade de desenvolvimento constante. Na visão da carreira proteana, segundo Veloso e Dutra (2010), os indivíduos são impelidos a um processo de aprendizagem contínua, uma vez que o trabalhador precisa desenvolver a habilida- de de aprender, adquirir novas competências e de lidar com as novas demandas do ambiente. Mes- mo que esse aprendizado não seja oferecido pelas organizações. As pessoas precisam aprender “…o que e como aprender sobre elas mesmas, sobre as outras pessoas, sobre o trabalho e sobre o ambiente…” (HALL, 2002 apud DUTRA 2010). Assim, no contexto da sociedade contemporânea com maiores incertezas na estabilidade de emprego e rapidez com que os trabalhos mudam, a habilidade de aprender constantemente trás uma maior segurança para o indivíduo. Já a teoria de carreiras sem fronteiras (ARTHUR; INKSON; PRINGLE, 1999 APUD DUTRA 2010) oferece perspectiva de carreira onde não há clara divisão entre o ambiente corporativo e a vida pessoal de seus funcionários. A carreira sem fronteiras é uma resposta orientada para a intera- ReCaPe | Vol. 8 | Nº. 3 | Ano 2018 | set./dez. | p. 433 Matheus Matsuda Marangoni, Mauro Miguel Rodrigues Berimbau ção entre a firma e as mudanças sociais da sociedade industrial para a nova economia. Dentre as diversas características desta nova visão de carreira, nos parece importante destacar neste artigo a perspectiva de que as pessoas são responsáveis por suas carreiras futuras através de, entre outros fatores, a constante busca por conhecimento. 1 TREINAMENTO, CARREIRA E APRENDIZAGEM Contudo, a partir de Peiperl e Arthur (apud Dutra 2010), entendemos que o processo de apren- dizagem acontece mais internamente às organizações do que fora delas, uma vez que, nas orga- nizações temos oportunidades para a transferência interfuncional de conhecimento. É importante, também, destacar que Arthur, Inkson e Pringle (apud. Dutra 2010, p.29), nos oferecem a visão de que nas carreiras sem fronteiras as competências são “…obtidas através de educação, trabalho e experiência de vida…”. Segundo tais conceitos, podemos entender que, o processo de aprendizagem hoje é de res- ponsabilidade tanto das organizações quando dos próprios indivíduos. Há uma insegurança maior no desenvolvimento e manutenção da carreira por parte dos indivíduos, que necessitam constante- mente busca desenvolvimento pessoal e profissional. Contudo, o processo de aprendizagem ofere- ce desafios tanto para a firma quanto para seus funcionários, uma vez que, quais conhecimentos e competências são necessárias muitas vezes não estão claras para ambas as partes. O acesso a informação e as técnicas de treinamento são muitas, porém, vemos que o engajamento com tais processos é um dos maiores desafios para o processo de aprendizagem. E a partir desta discussão, propomos neste artigo uma visão mais aprofundada da aplicação da gamificação como uma dinâ- mica de aprendizagem que vem oferecendo novas formas de treinamento e desenvolvimento dos indivíduos dentro das corporações. 2 GAMIFICAÇÃO E APRENDIZAGEM O emprego de mecânicas de games nas empresas, seja para inventivo de vendas ou para treinamento, não é necessariamente uma novidade. Há muito tempo as empresas buscam o en- gajamento de seus funcionários na educação e treinamento corporativo. O que podemos indicar, neste momento, é o uso mais completo das dinâmicas gamificadas. Muito desta evolução se dá pelo desenvolvimento tecnológico dos últimos anos. A partir desde cenário, buscamos neste artigo, enten- der mais sobre como o uso estruturado de ferramentas e modelos gamificados são uma estratégia coerente no treinamento em um ambiente com diversos públicos em níveis diferentes dentro de uma firma. A gamificação apresenta como conceito básico, a aplicação de mecânicas de games em si- tuações de não game, ou seja, neste caso a construção de um game voltado para o processo de ReCaPe | Vol. 8 | Nº. 3 | Ano 2018 | set./dez. | p. 434 GAMIFICAÇÃO E GESTÃO DE PESSOAS: UM ESTUDO DE CASO SOBRE TREINAMENTO E AMBIENTE DE DIVERSIDADE CULTURAL aprendizado. Nos parece importante trazer alguns autores para discussão mais aprofundada sobre a conceituação de gamificação relacionada à aprendizagem. É importante destacar que, a gamificação como teoria ainda está em seu estágio inicial. Muitas das discussões a respeito do tema oferecem abordagens diferentes. Não buscamos aqui, esgotar o assunto, uma vez que, não se trata de nosso objetivo neste artigo. Contudo, ao buscarmos referências no principais autores do momento, enten- demos que é possível fechar a questão necessária para analisar o estudo de caso proposto. Começamos com a definição trazida por Paharia (2013), segundo a qual a gamificação é en- tendida enquanto ferramenta para os negócios, como uma forma de gerar lealdade, seja em públicos internos ou externos. Paharia (2013, p. 65) temos que: …a gamificação emprega técnicas motivacionais que os designers de videogames têm usado por anos, para motivar os jogadores, e aplica-as em contextos que não são jogos. Essas téc- nicas incluem (mas não se limitam a): concepção de objetivos a serem alcançados, reconhe- cimento por badges, engajamento via competição, encorajamento à colaboração em equipe, caracterização de status por ranking e estímulo ao acúmulo de pontos. …a gamificação emprega técnicas motivacionais que os designers de videogames têm usado por anos, para motivar os jogadores, e aplica-as em contextos que não são jogos. 2 GAMIFICAÇÃO E APRENDIZAGEM Essas téc- nicas incluem (mas não se limitam a): concepção de objetivos a serem alcançados, reconhe- cimento por badges, engajamento via competição, encorajamento à colaboração em equipe, caracterização de status por ranking e estímulo ao acúmulo de pontos. Podemos entender que a motivação, ao utilizar técnicas de gamificação, busca a associação com o engajamento através do apelo lúdico e descontraído, como se fosse mais uma forma de entretenimento e do que um treinamento entediante. Segundo Frasca (2003), o entretenimento, a partir de mecânicas gamificadas, se coloca como um facilitador das relações entre a máquina de aprendizagem digital e os indivíduos que com ela interagem, fazendo com que, esses mantenham-se engajados, ou seja, mais presentes e efetivamente participantes do processo. Aprendemos muito quando nos divertimos. Quem não gosta de assistir a um filme, ler um qua- drinho e jogar um bom game? São experiências memoráveis, que algumas vezes nos lembramos por décadas a seguir. Por que essas mídias, de onde originam tais conteúdos, não podem então nos ensinar outras coisas mais interessantes? Quem trabalha com formação de pessoas costuma se indagar desta forma, e procura as mais diferentes técnicas para transformar um conteúdo denso, trabalhoso e complicado em algo mais palatável, acessível e agradável. É através dessas preocupa- ções que surgem estudos e aplicações de elementos de jogos em situações diversas, incluindo na sala de aula. Para entender a gamificação além do contexto do entretenimento, precisamos nos libertar da observação meramente ferramental e estética do jogo (pontos, vidas, personagens animados, músi- ca feliz, etc.) e entender a sua estrutura fundamental (estabelecimento de regras, liberdade de ação, construção de experiência para o jogador, etc.). Além disso, é preciso estudar formas de planejamen- to, mensuração e aplicação, sem se esquecer sobre a proposta educativa. Assim, podemos entender que, o processo de design de um jogo voltado para a educação está segmentado em quatro cama- das (WINN, 2009): aprendizagem, storytelling, gameplay e experiência do usuário. ReCaPe | Vol. 8 | Nº. 3 | Ano 2018 | set./dez. | p. 435 Matheus Matsuda Marangoni, Mauro Miguel Rodrigues Berimbau Como aprendizagem no contexto da gamificação, Winn (2009) nos trás a necessidade de um alinhamento de toda a estrutura do game para com os objetivos do aprendizado em uma estrutra de eixos. Primeiro é necessário ensinar o jogador como participar do processo, ou seja, como jogar o game. 2 GAMIFICAÇÃO E APRENDIZAGEM Segundo Frasca (2007), todo jogo nos ensina alguma coisa, mesmo que seja apenas sobre eles mesmos: a necessidade e significado de apertar botões, o que pode ser feito dentro dos limites do jogo e quais seus objetivos. Os games voltados para educação buscam também transmitir uma mensagem, reforçar um comportamento e nos ensinar algo. Como camada de storytelling podemos entender a forma como o game produz sentido. É um elemento fundamental, mesmo que não obrigatório, para facilitar o processo educativo, uma vez que, gera contexto e ajuda no engajamento. Temos que entender a necessidade que o game tem de gerar uma experiência o mais agradável possível. Um jogo de mecânica simples, como perguntas de múl- tipla escolha após um conteúdo técnico demonstrado pode engajar seus participantes o suficiente para fixar o aprendizado. Ao tornar a narrativa do game mais lúdica, é possível gerar maior interação do jogador e, assim, maiores chances de fixação do conteúdo oferecido. O gameplay é a camada que define o jogo em si, uma vez que, é a responsável pela mecânica e regulação do game. Para termos um jogo é fundamental que tenhamos regras. O que o jogador pode ou não fazer em um jogo e quais as dinâmicas que conduzem o jogador pelo tabuleiro ou tela. É de responsabilidade do gameplay a construção da experiência e de tornar a atividade desafiadora e equilibrada. Um jogo precisa apresentar desafios, caso contrário não prenderá a atenção do jogador, mas tais desafios precisam ser equilibrados e alinhados com os objetivos de aprendizagem para que gerem o engajamento necessário. Segundo Sicart (2008), as mecânicas de jogos são um conjunto de métodos invocados por agentes, projetados para interagir com o estado do jogo. Ou seja, é o conjunto de regras colocadas para os jogadores. Essas regras determinam todas as possíveis ações dos jogadores, quais são as condições de vitórias, os objetivos do jogo, as recompensas, etc. Tudo o que é necessário para o funcionamento correto do game. Por exemplo, a obrigatoriedade de assistir a um vídeo ou a neces- sidade de cumprir uma tarefa. O processo de uma sala de aula utiliza, de certa forma, dinâmicas de gamificação. Os pro- fessores ofereceriam as regras, os desafios e a forma como o jogador irá atingir os objetivos esta- belecidos. 2 GAMIFICAÇÃO E APRENDIZAGEM Entendemos, contudo, que a maioria dos professores se limitem ao modelo clássico de aprendizagem baseado em pontos (notas), missões (lições de casa) e conquistas (estrelas e outros métodos). Sendo assim, o papel da gamificação aplicada à aprendizagem deverá trabalhar com mecânicas para o repensar o balanceamento de ações, pontuações, permissões, objetivos e outros ReCaPe | Vol. 8 | Nº. 3 | Ano 2018 | set./dez. | p. 436 AMIFICAÇÃO E GESTÃO DE PESSOAS: UM ESTUDO DE CASO SOBRE TREINAMENTO E AMBIENTE DE DIVERSIDADE CULTURAL elementos mecânicos para equilibrar a experiência de jogo com o aprendizado. É neste eixo da me- todologia que as teorias do game design tem mais a contribuir. A experiência do usuário (User Experience, em inglês ou simplesmente UX) é fundamental nos jogos digitais. É através da interface que o jogo se materializa para o jogador, e tornar o sistema didático e acessível é uma estrutura que merece uma atenção própria. Nos jogos analógicos ou de interpretação, onde se enquadrariam hoje os modelos tradicionais de aula, essa estrutura apresenta preocupações com tutoriais (ensinar sobre o próprio jogo) e sistemas de feedback (resultados rápi- dos para as ações realizadas), para que o jogador tenha menos atenção em como jogar e mais no gameplay, storytelling e na aprendizagem. Em outras palavras, não podemos deixar o jogador se perguntar “mas como eu jogo isto?”. A tecnologia, finalmente, é um elemento viabilizador, o apoio do processo. Simulações comple- xas podem exigir um apoio tecnológico maior, mas tudo depende dos objetivos de aprendizagem e dos recursos disponíveis ao designer. Não deveríamos considerar apenas o suporte dos equipamen- tos e programas digitais aqui, mas toda a estrutura que permite e facilita a aplicação dessas técnicas como os softwares de desenvolvimento e o hardware utilizado para consumir o jogo. Essa estrutura aplicada de forma estruturada e alinhada com os objetivos de aprendizagem, convergindo com técnicas de ensino e em um ambiente lúdico, faz com que a utilização de gamifica- ção em processos de treinamento corporativo possam atingir melhores resultados, como pretende- mos explorar mais a seguir na análise do estudo de caso. ReCaPe | Vol. 8 | Nº. 3 | Ano 2018 | set./dez. | p. 437 3 ESTUDO DE CASO A entrevista seguiu um roteiro semi-estruturado com o seguinte formato: • Conceito: Foram entrevistadas em uma única reunião, com duração de pouco mais de uma hora, as duas colaboradoras da empresa. A entrevista seguiu um roteiro semi-estruturado com o seguinte formato: • Conceito: • O que é gamificação? • Que necessidades corporativas a gamificação está atendendo hoje? Processo de contratação e desenvolvimento: • Processo de contratação e desenvolvimento: • Qual grau de envolvimento que a área de treinamento terá com os públicos de interesse envolvidos na gamificação? • Qual é a expectativa que a empresa tem do resultado da gamificação? • Continuidade de projetos em gamificação: 3 ESTUDO DE CASO O caso escolhido para o entendimento de como a gamificação nos processos de treinamento corporativo pode gerar melhores resultados não levará em conta resultados numéricos, uma vez que, se trata de um projeto piloto e, portanto, não temos autorização para divulgação do nome da empresa e de seus contatos. Contudo, não vemos essa limitação como problema para esse artigo, visto que, nosso objetivo é demonstrar quais as motivações e o entendimento que levam as empresas na busca por processos gamificados para o desenvolvimento de seu quadro de colaboradores. Os profissionais alvo do projeto piloto estão ligados à área de obras da empresa em questão. Contemplando desde engenheiros até cargos mais baixos da hierarquia, como ajudantes encarrega- dos de “cavar buracos”. Entendendo que, os colaboradores de grau técnico mais elevado na hierar- quia eram os responsáveis pelo conteúdo do treinamento. ReCaPe | Vol. 8 | Nº. 3 | Ano 2018 | set./dez. | p. 437 Matheus Matsuda Marangoni, Mauro Miguel Rodrigues Berimbau Podemos, no entanto, citar a empresa responsável pelo desenvolvimento do projeto piloto. Tra- ta-se da Sioux Game Brasil , uma empresa que se intitula como agência multidisciplinar que combina tecnologia, comunicação e gamificação para seus clientes. O levantamento de dados utilizou como método um levantamento qualitativo através de entre- vistas em profundidade com profissionais da Sioux, em especial os game designers responsáveis pelo piloto, e duas profissionais da área de treinamento da empresa que forma as sponsors do pro- jeto perante a diretoria da empresa em questão. As entrevistas aconteceram após o desenvolvimento do piloto e, desta forma, nos ajuda a trabalhar e analisar esse processo como fechado. Mesmos sem a necessidade de resultados es- pecíficos ligados ao piloto, é importante avaliar a percepção de todas as partes envolvidas em um processo encerrado. Primeiramente iremos trabalhar com a entrevista realizada com a empresa, para assim, de- monstrar o entendimento destes profissionais para com o processo de gamificação, quais suas ex- pectativas iniciais e como veem o produto apresentado como solução. A seguir, iremos aprofundar nossa análise junto a um dos profissionais envolvidos no desenvolvimento da gamificação, enten- dendo como o processo foi conduzido e quais as dificuldades encontradas. Para, por fim, concluir- mos o estudo com a análise dos dados coletados para com a teoria apresentada anteriormente nesse artigo. Foram entrevistadas em uma única reunião, com duração de pouco mais de uma hora, as duas colaboradoras da empresa. GAMIFICAÇÃO E GESTÃO DE PESSOAS: UM ESTUDO DE CASO SOBRE TREINAMENTO E AMBIENTE DE DIVERSIDADE CULTURAL Inicialmente questionamos qual o entendimento das profissionais com relação ao que seria gamificação, para que pudéssemos avaliar o conceito entendido e assim, validar a percepção quanto ao seu valor como treinamento. De forma geral, as duas entrevistadas nos colocaram que, entediam a gamificação apenas como um jogo, ou seja, como um programa de computador ou celular, que po- deria ser construído para fins de treinamento. Não havia, antes de iniciarem o projeto, clareza e maior entendimento do que a gamificação é e do que ela é capaz. Conceitos como competição e recom- pensa foram os mais destacados pelas entrevistadas como entendimento de um jogo no começo. Contudo, uma das entrevistadas relatou ter conhecimento do termo gamificação há alguns anos. Seu contato inicial foi por contato com uma empresa de recrutamento. Ela relatou que, seu entendimento de gamificação nesse primeiro contato, levavam ainda em conta a questão da competitividade como destaque principal. Em suas palavras: “A competitividade está na essência do ser humano. É ele buscar estar no topo diante de vá- rios elementos. É gerar uma série de elementos para trabalhar uma competitividade, no caso sadia para a organização, no processo de aprendizagem. Porque é lúdico. E uma vez que é lúdico, você aprende bem mais fácil.” Outro fator de destaque indicado para a escolha pelo uso da gamificação em processos de trei- namento, foi a questão tecnológica. Segundo as entrevistadas, a tecnologia favorece a otimização do tempo. Fazendo com que o treinamento não precise ser presencial e, portanto, evitando longos pe- ríodos de pausa no trabalho dos colaboradores. Assim como, a facilidade de oferecer o treinamento para diversas unidades da empresa, que não necessariamente estão localizadas próximas da central ou até mesmo reduzindo os gastos com deslocamento e estadia de professores/ instrutores para fora de São Paulo. Contudo, é importante destacar que, segundo uma das entrevistadas, a importância sobre o uso da tecnologia, também, está associada a um certo grau de empirismo, uma vez que, foi citada a melhor adesão com o treinamento gamificado com sua percepção de como as pessoas tem passado cada vez mais de seu tempo no celular. A questão de que a gamificação pode obter melhores resultados de engajamento/ motivação para com o treinamento, é destacada por ambas as entrevistadas. • Continuidade de projetos em gamificação: • Quando tiveram a ideia de gamificar, tinham noção do valor necessário para investimento? • Como veem a receptividade da empresa para continuidade do projeto? ReCaPe | Vol. 8 | Nº. 3 | Ano 2018 | set./dez. | p. 438 GAMIFICAÇÃO E GESTÃO DE PESSOAS: UM ESTUDO DE CASO SOBRE TREINAMENTO E AMBIENTE DE DIVERSIDADE CULTURAL GAMIFICAÇÃO E GESTÃO DE PESSOAS: UM ESTUDO DE CASO SOBRE TREINAMENTO E AMBIENTE DE DIVERSIDADE CULTURAL As duas profissionais destacam a dificuldade em motivar os colaboradores a participarem ativamente de treinamentos, que em parte dos casos, consiste em reciclagem e/ou revisão de rotinas de segurança. Uma das entrevistadas indica que em sua visão a gamificação derruba as barreiras que causam a falta de empatia com o processo de treinamento. Mais uma vez, a característica indicada foi a ludicidade do processo, que oferece o conteúdo de uma forma mais agradável para os funcionários, além de, possibilitarem uma melhor retenção do conteúdo alvo do treinamento em contraponto com práticas presenciais, em ma- teriais impressos ou em vídeo. ReCaPe | Vol. 8 | Nº. 3 | Ano 2018 | set./dez. | p. 439 Matheus Matsuda Marangoni, Mauro Miguel Rodrigues Berimbau A interatividade de um game também foi relevante para o processo de decisão pela escolha da gamificação como mecânica de treinamento. A passividade encontrada nos processos ditos mais tradicionais, como vídeo aula e aula presencial são, segundo as propositoras do projeto, outra carac- terística que as motivou na busca por entender melhor o que seria a gamificação e por solicitar um projeto piloto. A composição de teoria e prática e a necessidade de tomada de decisão por parte do público-alvo, foram destacadas na entrevista. O auto-conhecimento e o reconhecimento são fatores de destaque em um processo gamifi- cado na visão das entrevistadas. O uso de mecânicas que oferecem feedbacks mais constantes e demonstram para os pares em que nível o colaborador se encontra, seriam questões melhores resol- vidas a partir de um processo gamificado e, assim, possibilitam maior engajamento dos funcionários alvos do treinamento. Ao questionarmos os objetivos da área de treinamento para a escolha da gamificação, enten- demos que um dos principais motivos é a busca por novas formas de trabalho. Sem maior aprofun- damento, até por não ser o objetivo deste estudo de caso, as profissionais indicaram que a há neces- sidade de um melhor alinhamento de conteúdo por toda a empresa. Indicaram que, essa integração deve ser total por todas as áreas da empresa, ou seja, estão desenvolvendo conteúdo de cada uma das etapas da prestação de serviços da empresa de forma a oferecer um visão completa para todos os departamentos de forma organizada e coesa. GAMIFICAÇÃO E GESTÃO DE PESSOAS: UM ESTUDO DE CASO SOBRE TREINAMENTO E AMBIENTE DE DIVERSIDADE CULTURAL Entendem que a gamificação é uma forma interes- sante de gerar conteúdo que seja de fácil assimilação e que pode gerar uma visão integrada em uma única plataforma para todas as etapas de prestação de serviços da empresa. Uma preocupação da empresa é o alto custo da plataforma, uma vez que, o processo de ga- mificação proposto para a empresa consiste em um conjunto de conteúdos gamificados a partir do uso de um aplicativo de celular. Não tivemos acesso aos valores de tal plataforma, contudo, como ordem de grandeza, é possível usarmos como parâmetro um custo de aproximadamente 50 a 100 mil reais por conteúdo/ etapa do game. Segundo as entrevistadas, a empresa tem os recursos ne- cessários para tal investimento, mas há a necessidade uma melhor parametrização das metas que serão alcançadas com a gamificação. Contudo, o maior desafio indicado pelas profissionais, é a organização e preparação de todo o conteúdo necessário para o projeto. Elas entendem que, a sem um maior cuidado com essa etapa interna da empresa, qualquer game não atingiria resultados que justificassem tal investimento. Assim sendo, a partir da entrevista com a empresa, pudemos entender que o projeto ainda está em uma fase piloto devido a falta de cultura da empresa de forma geral em adotar novos processos de treinamento. As profissionais encarregadas pelo projeto, relatam ainda que, o alto investimento, a necessidade de estruturar melhor o conteúdo e um certo grau de ceticismo por parte da diretoria ReCaPe | Vol. 8 | Nº. 3 | Ano 2018 | set./dez. | p. 440 GAMIFICAÇÃO E GESTÃO DE PESSOAS: UM ESTUDO DE CASO SOBRE TREINAMENTO E AMBIENTE DE DIVERSIDADE CULTURAL são fatores que podem dificultar a adoção mais rápida da gamificação de forma mais ampla pelas empresas. Nossa segunda ida a campo para aprofundar o estudo de caso contou com uma entrevista junto a um dos game designers envolvidos no projeto. É importante destacar que o papel do game designer é o entendimento das necessidades ligadas à gamificação e o desenvolvimento da mecâ- nica e, neste caso, do storytelling utilizado para gerar o engajamento. Foi entrevistado o profissional ligado à Sioux. A entrevista ocorreu no escritório da empresa, localizado no bairro do Brooklin em São Paulo e contou com o questionário semi estruturado que contemplava principalmente os seguintes tópicos: Desafios. Processo. Ganhos de um treinamento gamificado. GAMIFICAÇÃO E GESTÃO DE PESSOAS: UM ESTUDO DE CASO SOBRE TREINAMENTO E AMBIENTE DE DIVERSIDADE CULTURAL Entendemos que é importante, indicarmos o processo de construção do game, que foi dividi- do em três etapas. Para melhor localizar a relevância na escolha de um game designer para nossa coleta de informações. Primeiro a equipe da Sioux recebeu o briefing da empresa e montou equipe para o desenvolvimento do projeto piloto. Tal equipe foi dividida em um time de game designers, um de designers digitais e, por fim a equipe de programação. Nosso entrevistado participou do primeiro grupo, que foi responsável por traduzir o conteúdo e formular o processo de aprendizagem ligado às mecânicas gamificadas de um jogo digital para plataforma móvel. Ao questionarmos os principais desafios com esse caso, o game designer, nos indicou não ter encontrado dificuldades diferentes de outros projetos, sejam eles maiores ou menores que nosso caso analisado. Contudo, nos foi colocado que todo processo de gamificação tem como grande de- safio o entendimento profundo do conteúdo que será gamificado. Esse conhecimento dependendo do trabalho varia de complexidade e nas formas de obtenção. Neste caso, por se tratar de uma em- presa que realiza, dentre seus serviços, obras de engenharia, foi necessário ir a campo para acom- panhar a execução do serviço escolhido para o projeto piloto. Outro fator de atenção indicado, é a necessidade de entender a linguagem da empresa e de seu público. Ou seja, analisar como os profissionais se comunicam e quais os termos técnicos funda- mentais para as atividades executadas. O game designer nos indicou que uma das principais carac- terísticas deste caso era a necessidade de trabalhar com públicos de diferentes níveis culturais. Des- de um profissional especializado em cavar buracos até o engenheiro responsável pela obra como um todo. O game deveria trabalhar uma linguagem que fosse adequada para essa diversidade cultural, mas não poderia de utilizar termos técnicos bastante específicos. Entendemos essa preocupação quando lembramos a necessidade de gerar engajamento para com o game, ou seja, o treinamento gamificado não poderia trazer o conteúdo de forma a não conseguir se comunicar adequadamente com um funcionário menos instruído, como um ajudante de pedreiro, ao mesmo tempo que precisa engajar também um engenheiro com grau superior completo. ReCaPe | Vol. 8 | Nº. 3 | Ano 2018 | set./dez. | p. GAMIFICAÇÃO E GESTÃO DE PESSOAS: UM ESTUDO DE CASO SOBRE TREINAMENTO E AMBIENTE DE DIVERSIDADE CULTURAL 441 Matheus Matsuda Marangoni, Mauro Miguel Rodrigues Berimbau O processo de desenvolvimento da gamificação foi de três meses a partir do início do projeto piloto com o briefing da empresa. A equipe de game designers, envolvida durante todo o processo, participou de três reuniões com a empresa e foi a campo em uma ocasião para acompanhar a exe- cução de um serviço de obra. Durante os três meses de produção do piloto, os game designers tra- balharam pelo menos dois terços do tempo na preparação das mecânicas e adaptação do conteúdo e um terço no acompanhamento das demais etapas de produção, o design gráfico e a programação. Desta forma, podemos entender que a mecânica de aprendizagem gamificada é a etapa do game mais trabalhosa e que mais consome tempo. Não estamos desconsiderando a identidade visual ou a programação, as quais são também indispensáveis para o game. Contudo, entendemos a partir da entrevista que é a partir do entendimento do conteúdo e da elaboração da estratégia de gamificação para o mesmo que origina o trabalho das etapas seguintes. Na visão do game designer entrevistado, um processo de treinamento gamificado de forma correta, gera maior engajamento para com o conteúdo e melhor retenção dos conceitos transmitidos no game. Desta ainda, a importância do storytelling para tornar o jogo lúdico e atrativo para os jo- gadores. Sem esquecer a possibilidade de um maior controle do processo de treinamento, uma vez que, a plataforma tecnológica possibilita a coleta de dados relevantes para metrificar os resultados, tais como: quantidade de acesso, tempo gasto com o game, número de tentativas e número de erros e acertos. Dados que ajudarão a entender se o game é relevante, para controlar a adesão ao treina- mento e para ajudar a empresa a metrificar seus resultados. Por fim, entendemos a partir da visão do game designer que a gamificação nos treinamentos corporativos tendem a crescer nos próximos anos e que com o aumento de projetos e evolução da tecnologia utilizada, o custo deve cair substancialmente, ajudando assim na melhoria do processo. 4 CONSIDERAÇÕES FINAIS Conforme indicamos anteriormente, a carreira é um conjunto de competências adquiridas ao logo da vida pessoal e profissional das pessoas. Juntando a esse fato, a definição de gamificação como aplicação de técnicas de game buscando o melhor engajamento, entendemos o emprego da gamificação como uma forma de favorecer o contínuo aprendizado. Tanto em função da ludicidade, que torna o processo mais agradável, quando do engajamento em si, buscando maior interação e retenção do que se deseja educar. O caso apresentado, nos ajuda a destacar tais questões, visto que indica a busca da firma por formas diferentes do treinamento presencial e/ ou video-aula. Formas, segundo as profissionais entrevistadas, que tomam tempo e não favorecem a retenção do conteúdo apresentado. Quando ReCaPe | Vol. 8 | Nº. 3 | Ano 2018 | set./dez. | p. 442 GAMIFICAÇÃO E GESTÃO DE PESSOAS: UM ESTUDO DE CASO SOBRE TREINAMENTO E AMBIENTE DE DIVERSIDADE CULTURAL vemos a questão do tempo, podemos destacar a dificuldade do processo dito tradicional que, requer deslocamento físico e horas de trabalho. Já na utilização de uma plataforma gamificada, há a busca da otimização do tempo. Tanto os profissionais não precisam realizar deslocamento, quanto o tempo não é o mesmo para todos e, tão pouco, necessita que todos os colaboradores estejam ligados na plataforma no mesmo momento. O game, ao ser lúdico, tende a transpassar a divisão entre a vida profissional e a vida pessoal, uma vez que, o storytelling, tão importante no processo de gamificação, buscará, de certa forma, suavizar o conteúdo do treinamento corporativo. Conceito de destaque nas teorias de carreiras sem fronteiras. Os indivíduos serão conduzidos de forma lúdica e interativa pelo game/ treinamento. Duas características colocadas como altamente relevantes pela empresa do estudo de caso, para definir pela escolha desta dinâmica. Contudo, o uso de uma plataforma tecnológica para o emprego da gamificação não apresenta apenas questões positivas. O principal ponto de atenção, segundo as entrevistas tanto com a em- presa contratante, quanto indicado pelo profissional da Sioux, é a preparação do conteúdo. As en- trevistadas da firma, indicaram a importância deste alinhamento interno e para com a Sioux. O game designer, demonstrou como maior desafio a necessidade de aprender conteúdos desconhecidos e técnicos para desenvolver o game. 4 CONSIDERAÇÕES FINAIS Por fim, devemos indicar a continuidade de novas pesquisas ligadas ao treinamento corpo- rativo gamificado, uma vez que, esse artigo inicial, objetiva abrir a discussão de como o uso de mecânicas gamificadas estão alinhadas com as perspectivas de desenvolvimento de pessoas nas firmas. Assim como, de que forma, as empresas brasileiras estão preparadas e dispostas a investir, em muitos casos altos recursos financeiros e tempo, na promessa de maior engajamento e eficácia na aprendizagem corporativa. REFERÊNCIAS BIBLIOGRÁFICAS AARSETH, E. (1997). Cybertext: Perspectives on Ergodic Literature. Maryland: The Johns Hopkins University Press. DUTRA, Joel Souza (2010). Gestão de Carreiras na Empresa Contemporânea. São Paulo. Editora Atlas. DUTRA, Joel Souza (2010). Gestão de Carreiras na Empresa Contemporânea. São Paulo. Editora Atlas. FRASCA, G. (2003). Simulation vs Narrative: Introduction to Ludology. Nova Iorque: Routledge. Disponivel em: <http://ludology.org/articles/VGT_final.pdf>. Acesso em: ago. 2018. FRASCA, G. (2003). Simulation vs Narrative: Introduction to Ludology. Nova Iorque: Routledge. Disponivel em: <http://ludology.org/articles/VGT_final.pdf>. Acesso em: ago. 2018. FRASCA, G. (2007). Play the message: play, game and videogame rethoric. IT University of Copenhagen. FULLERTON, T. (2008) Game Design Workshop: a playcentric approach to creating innovative games. Burlington: Elsevier. ReCaPe | Vol. 8 | Nº. 3 | Ano 2018 | set./dez. | p. 443 Matheus Matsuda Marangoni, Mauro Miguel Rodrigues Berimbau KOSTER, R. (2005). A theory of fun for Game Design. Arizona: Paraglyph Press. KOSTER, R. (2005). A theory of fun for Game Design. Arizona: Paraglyph Press. PAHARIA, Rajat (2013). Loyalty 3.0 - How to revolutionize customer and employee engagement with big data and gamification. Canadá: McGraw-Hill Education. SALA, Otávia Travençolo Muniz e TREVISAN, Leonardo Nelmi (2014). A construção de carreira em ambientes inovativos - um estudo nas empresas de base tecnológica. Revista de Administração e Inovação, São Paulo, v. 11, n.2, p. 154-178. SALEN, K.; ZIMMERMAN, E. (2004). Rules of Play: Game design fundamentals. Cambridge: MIT Press. SCHELL, J. (2008). The Art of Game Design: A Book of Lenses. Nova Iorque: CRC Press. VELOSO, Elza Fátima Rosa, DUTRA, Joel Souza e NAKATA, Lina Eiko (2016). Percepção sobre carreiras inteligentes: diferenças entre as gerações y, x e baby boomers. REGE - Revista de Gestão, 23, p. 88–98. YIN, Robert K. (2018). Case Study Research and Applications: Design and Methods. SAGE Publications. Kindle Edition. WERBACH, K.; HUNTER, D. (2012). For the Win: How Game Thinking Can Revolutionize Your Business. Filadélfia: Wharton Digital Press. WINN, B. (2009). The Design, Play, and Experience Framework. In: FERDIG, R. Handbook of Research on Effective Electronic Gaming in Education. 1a. ed. Nova Iorque: Information Science Reference, v. III, Cap. LVIII, p. 1010-1024. ReCaPe | Vol. 8 | Nº. 3 | Ano 2018 | set./dez. | p. 444
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Theorizations about the child's Play and Movent: implications for the pedagogical practice of Physical Education in Early Childhood Education and other problematizations
Pro-Posições
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2 References correction and bibliographic normalization services: Vera Lúcia Fator Gouvêa Bonilha - verah.bonilha@gmail.com 3 English version: Viviane Ramos - vivianeramos@gmail.com 1 Responsible editor: Carmen Lúcia Soares - https://orcid.org/0000-0002-4347-1924 (ii) Universidade Federal do Espírito Santo – UFES, Centro de Educação Física e Desportos – CEFD, Departamento de Ginástica, Vitória, ES, Brasil. http://orcid.org/0000-0002-4056-5159, fqalmeida@hotmail.com http://dx.doi.org/10.1590/1980-6248-2020-0139EN http://dx.doi.org/10.1590/1980-6248-2020-0139EN Pro-Posições | Campinas, SP | V. 33 | e20200139EN| 2022 3 English version: Viviane Ramos - vivianeramos@gmail.com 3 English version: Viviane Ramos - vivianeramos@gmail.com 2 References correction and bibliographic normalization services: Vera Lúcia Fator Gouvêa Boni verah.bonilha@gmail.com (iii) Universidad de Valparaíso, Facultad de Medicina, Escuela de Educación Parvularia, Viña del Mar, Chile. https://orcid.org/0000-0002-4277-0535, alberto.moreno@uv.cl e-ISSN 1980-6248 http://dx.doi.org/10.1590/1980-6248-2020-0139EN Abstract This article analyzes the theorization that has been undertaken about "playing and movement" and that can be considered as a possibility to support the intervention of Physical Education with Early Childhood Education. In methodological terms, it operates with some keys of reading and interpretation, attempting to understand childhood concepts; of body and movement which dialogue with "playing and moving", as well as identifying the definition of what would be the object of teaching Physical Education in Early Childhood Education and the definition of the teacher's role in this context. Then, it makes some considerations regarding the potentials, limits, and challenges of this theorization for Physical Education in Early Childhood Education. Keywords: Physical Education, Childhood Education, Play and Move e-ISSN 1980-6248 e-ISSN 1980-6248 Teorizações sobre o Brincar e o Se-movimentar da criança: implicações para a prática pedagógica da Educação Física na Educação Infantil e outras problematizações Teorizaciones sobre Jugar y Moverse de los niños y niñas: implicaciones para la práctica pedagógica de la Educación Física en la Educación Infantil y otras problematizaciones Rocha, Maria Celeste(i) Almeida, Felipe Quintão(ii) Moreno, Alberto(iii) (i) Universidade Federal do Espírito Santo – UFES, Centro de Educação Física e Desportos – CEFD, Programa de Pós-Graduação em Educação Física – PPGEF; Prefeitura Municipal de Vitória, Secretaria Municipal de Educação, Gerência de Formação e Desenvolvimento em Educação. Vitória, ES, Brasil. https://orcid.org/0000-0002-2572-9098, mcrocha@prof.edu.vitoria.es.gov.br (ii) Universidade Federal do Espírito Santo – UFES, Centro de Educação Física e Desportos – CEFD, Departamento de Ginástica, Vitória, ES, Brasil. http://orcid.org/0000-0002-4056-5159, fqalmeida@hotmail.com (iii) Universidad de Valparaíso, Facultad de Medicina, Escuela de Educación Parvularia, Viña del Mar, Chile. https://orcid.org/0000-0002-4277-0535, alberto.moreno@uv.cl (iii) Universidad de Valparaíso, Facultad de Medicina, Escuela de Educación Parvularia, Viña del Mar, Chile. https://orcid.org/0000-0002-4277-0535, alberto.moreno@uv.cl Pro-Posições | Campinas, SP | V. 33 | e20200139EN| 2022 1/26 Resumo Resumo Este artigo analisa a teorização que vem sendo empreendida sobre “brincar e se-movimentar” e que pode ser considerada como uma das possibilidades de fundamentar a intervenção da Educação Física com a Educação Infantil. Em termos metodológicos, opera com algumas chaves de leitura e interpretação, na tentativa de compreender as concepções de infância; de corpo e movimento com as quais o “brincar e se-movimentar” dialogam, assim como, identificar a definição do que seria o objeto de ensino da Educação Física na Educação Infantil e qual o papel do professor nesse contexto. Em seguida, tece algumas considerações a respeito das potencialidades, dos limites e dos desafios dessa teorização para a Educação Física na Educação Infantil. Palavras-chaves: Educação Física, Educação Infantil, Brincar e Se-movimentar e-ISSN 1980-6248 e-ISSN 1980-6248 Pro-Posições | Campinas, SP | V. 33 | e20200139EN| 2022 Resumen Este artículo analiza la teorización que se ha emprendido sobre “jugar y moverse” y que se puede considerar como una de las posibilidades para sustentar la intervención de la Educación Física con la Educación Infantil. En términos metodológicos, opera con algunas claves de lectura e interpretación, en un intento por comprender los conceptos de la infancia; de cuerpo y movimiento con los que dialogar “jugar y moverse”, así como identificar la definición de cuál sería el objeto de la enseñanza de la Educación Física en Educación Infantil y la definición del rol del docente en este contexto. Luego, hace algunas consideraciones sobre las potencialidades, límites y desafíos de esta teorización de Educación Física en Educación Infantil. Palabras Clave: Educación física, Educación infantil, Jugar y moverse Palabras Clave: Educación física, Educación infantil, Jugar y moverse Pro-Posições | Campinas, SP | V. 33 | e20200139EN| 2022 Pro-Posições | Campinas, SP | V. 33 | e20200139EN| 2022 http://dx.doi.org/10.1590/1980-6248-2020-0139EN 1. Introduction In the Brazilian context, the reflection on Physical Education (P.E.) in Childhood Education is permeated by extremely challenging questions, mainly when we consider the specificity of this educational segment. Rocha (2011) argues that we cannot ignore the existing tensions when discussing the legitimacy of a "school subject", conducted by a specialist teacher in Childhood Education. In other words, we would like to call attention to the existing implications when a curricular component such as Physical education, as a subject responsible for a piece of specific knowledge, becomes part of the curriculum of a segment of K-12 education, which is not organized by specific subjects or knowledge but by fields of experience. If, on one hand, these issues were strongly present in the academic debates in the area, on the other, the fact that P.E. is already established as a practice in many contexts of Brazilian Childhood Education, including the work of a specialist teacher, has potentialized the progress of discussions. In this direction, it is possible to follow a considerable increase of studies and publications in the area to reflect on their articulation with this educational segment. Thus, they show the need and the possibilities to build an integrated work, not fragmented, which considers children's specificities and new concepts of Childhood Education. The exercise of analyzing the academic production about P.E. in Childhood Education has indicated that some theoretic-methodological perspectives have been built to think of pedagogical interventions for children from 0 to 5 years old. Thus, in this article, we have chosen to present, discuss, and problematize the efforts of the theorization about early childhood education that has been conducted in the field of Physical education from the dialogue with the Human Self-Movement Theory (HSMT) and phenomenology itself. The option to analyze such theorization, in particular, comes from the fact that this exercise of thinking about the contributions of P.E. in Childhood education has taken place from reasoning and dialogue as knowledge accumulated and produced by the field o Physical Education itself. Its main author is Elenor Kunz, author of Abordagem Crítico-emancipatória and disseminator of HSMT in Brazil. In methodological terms, this study is a theoretical study focused on the analysis of Kunz's texts and those co-written by him with his master's and doctorate students. About this production, we highlight that there is no specific text about HSMT with a theoretical- Pro-Posições | Campinas, SP | V. e-ISSN 1980-6248 e-ISSN 1980-6248 e-ISSN 1980-6248 methodological perspective for P.E. in Childhood Education. However, there is a group of texts on issues inherent to P.E. in Childhood education, children’s play, and movement. Nevertheless, though it is not a clearly systematized proposal, it is possible to perceive the indication that HSMT works, which establishes a theorization about "Playing and Self- movement" that could eventually have a status of a propositional framework for action perspectives on P.E. teachers' work in Childhood Education. In other words, from the counter position of a certain type of intervention, the studies analyzed indicate another modus operandi for teachers, providing didactic-pedagogical elements that help teachers assume play as an element of P.E. and how to work with them. Thus, in a sense, we understand that this production indicates which formative dimension play should have in children’s lives. With that said, our first criterion to select texts was based on an analysis of the titles of bibliographic works in Kunz' Lattes curriculum. We have then selected those that mentioned in the title the terms: “play and move”, “children/childhood” and/or “childhood education”. When we had doubts, the reading of the abstract provided a second criterion. In this way, we have identified books, book chapters, articles, and complete articles published in congresses. However, we have excluded the texts from congresses because we understand that they were similar to book chapters and articles already published and which were part of the sample. In total, we selected 25 productions divided among the organization of a book, the writing of 13 book chapters, and the publication of 12 articles (see the table in the appendix). These productions were concentrated between the years 2005 and 2019, showing, therefore, that they were current publications and, in a way, recurrent in the production of the author in this time framework, corresponding to 12% of all his article production and 33% of his publication of book chapters. Besides this, these publications are mostly from collaborations with masters and Ph.D. students. Only 2 book chapters were individually published by Kunz. We have also seen that the main arguments of the co-authors’ texts derive from the authors’ individual productions, published in 2005 and 2007. 1. Introduction 33 | e20200139EN| 2022 3/26 Pro-Posições | Campinas, SP | V. 33 | e20200139EN| 2022 3/26 http://dx.doi.org/10.1590/1980-6248-2020-0139EN Pro-Posições | Campinas, SP | V. 33 | e20200139EN| 2022 4/26 e-ISSN 1980-6248 Regarding the method of analysis, we were based on Bardin (2011) to analyze content and propose the creation of categories that allow us to identify and group common elements in the analyzed texts and that, to a certain measure, can be seen in the ensemble of productions that intend to guide pedagogical practices. Therefore, we read the texts based on the identification of some important categories to understand this proposal, such as conceptions of Pro-Posições | Campinas, SP | V. 33 | e20200139EN| 2022 4/26 http://dx.doi.org/10.1590/1980-6248-2020-0139EN Pro-Posições | Campinas, SP | V. 33 | e20200139EN| 2022 5/26 Pro-Posições | Campinas, SP | V. 33 | e20200139EN| 2022 e-ISSN 1980-6248 e-ISSN 1980-6248 childhood, body, and movement with which they dialogue; the definition of the teaching object/specificity of P.E. in Childhood education; and the definition of teachers’ roles in this context. These analytical categories emerge from a double movement: first, our gaze does not start from a specific perspective or model of proposition for P.E. in Childhood Education but, as said, part of more general aspects that we believe are pertinent. Second, we consider the elements and the arguments present in the analyzed sources. Therefore, we organize our dialogue with the selected production intending to understand the effort made by the authors and create a synthesis of the practices of P.E. in Childhood Education to, later, present our analyses on these studies. The text is structured into two topics, followed by the final remarks. The first topic refers to the proposal of "playing and moving" from the previously described reading keys. After, we write some considerations on the potentials, limits, and challenges of this proposal for P.E. in Childhood Education. 2. From the Human Self-Movement Theory to “Play and Move”: fundaments for Physical Education in Childhood Education In the 1990s, based on the works of Trebels, Elenor Kunz presented HSMT as a possibility to ground pedagogical theory for Physical Education. Though the author had already been discussing the concepts of children's "play and move" since 1991, it was only in 2005 that we find publications in which he thinks specifically about the issue of P.E. in Childhood Education. Besides his publications, Kunz has been conducting studies on this topic with his master's and doctorate students. Before presenting the theorization exercise, we should conceptualize one of the key concepts of his work, also used to think about education in early childhood. Kunz defends an understanding of human movement from its dialogic potential because, for him, it manifests itself through sensibility, perception, and human intuition. Summing up, this conception considers not only the biomechanical movement of the subject but the human being that moves (Kunz, 2005). The expression “self-movement” characterizes Pro-Posições | Campinas, SP | V. 33 | e20200139EN| 2022 5/26 Pro-Posições | Campinas, SP | V. 33 | e20200139EN| 2022 5/26 5/26 http://dx.doi.org/10.1590/1980-6248-2020-0139EN e-ISSN 1980-6248 e-ISSN 1980-6248 the relation of the sense and meaning that human beings establish with the world to move (Kunz, 2005). This “self-movement” refers to an expression translated from German focusing on the "self", i.e., the subject of the movement. The pedagogical proposal developed by Kunz for P.E. is called Critical-Emancipatory. According to the author, "[...] Physical Education should contribute to developing certain competencies that cannot be summed up in the objective competence of know-how but include the social, linguistic, and creative competence" (Kunz, 2005, p.16). Therefore, Kunz considers students’ formation to transcend technical and instrumental formation. In the proposal of Self- movement Theory, grounding the pedagogical practice of Physical Education, the author operates with some concepts of phenomenology, especially the “Phenomenology of Perception” developed by Merleau-Ponty (Almeida et al., 2013). When analyzing these concepts and phenomenological ideas that compose the HSMT framework and discussing their use in Brazilian Physical education, Almeida et al. (2013) recognize its importance but problematize the fact that in his works Kunz did not give the due attention to the reviews produced by Merleau-Ponty. This means that “Kunz continues to operate, in the scope of HSMT, with theses revised by Merleau-Ponty himself” (Ghidetti et al., 2014, p.329). Some problematization on the appropriation of Merleau-Ponty’s works by Kunz in the field of P.E. can be found in the works of Ghidetti (2012), Almeida et al. (2013), and Guidetti et al. (2014). For them, by basing himself mainly on the “Phenomenology of perception”, Kunz disregards that Merleau-Ponty himself reviewed his thoughts affecting the theses presented in the 1945 book. From the perspective of these authors, it means saying that HSMT is "stuck" to some ambiguities of Merleau-Ponty’s thought. This can be seen by the fact that HSMT affirms that “the production of senses and meanings without explaining the connection between perception and language” (Ghidetti et al., 2014, p.329). In other words, contrary to what was problematized by Merleau-Ponty himself, HSMT still operates with the idea of a pre-reflexive contact of the perceptive awareness with oneself as previous to language. As an example, we can cite Kunz's focus on sensitiveness, spontaneity, and childhood as possibilities to contrapose the instrumental logic of play and human movement. Pro-Posições | Campinas, SP | V. 33 | e20200139EN| 2022 http://dx.doi.org/10.1590/1980-6248-2020-0139EN e-ISSN 1980-6248 The child before thinking and perceiving the world around perceives exactly this world before being reflected by our thought (…) this process that starts in the world previous to reflection (pre-reflexive world) is what provides the arguments for us to understand the process of awareness. (Surdi & Kunz, 2010, pp. 264-265) The authors also highlight that the body, understood as a body-subject, moves itself intending to perceive things by living them. We could say that this living and free movement is an expressive act, meaningful, and unique. The expression can be enacted through our bodies. It can reveal the meaning of our pure experiences (Surdi & Kunz, 2010, p. 274) As argued by Ghidetti et al. (2014), it is not clear how the meaning of our pure experiences articulates with language that, in this case, would continue to be conceived as a result of an original awareness. In other words, the passage from the perceptive scope to the meaning referring to language, from behavior to thematization, is not clear in HSMT framework. We highlight these questions because we identify that, in the scope of Physical Education in Childhood Education, HSMT has also been used as an important foundation using these same phenomenological concepts and ideas that were problematized in the field of Physical Education. However, besides HSMT and Merleau-Ponty’s Phenomenology, we could perceive that the theorization about early childhood education proposed by Kunz and collaborators has interfaces, in practically all their texts, with the references of Humberto Maturana and Gerda Verden-Zöller (2004), mainly the work Amar e brincar: fundamentos esquecidos do humano, as well as Violet Oaklander (1980) and Honoré (2009). In the more recent texts, mainly after 2016, we have also seen a dialogue with childhood phenomenology, disseminated and discussed by Machado (2010, 2013). Thus, regarding HSMT and Physical Education in Childhood Education, we have perceived that Kunz has incorporated in the concept of self-movement other discussions and presented the “Playing and Moving” as a theoretical perspective that can ground the work with small children. Reading the texts written by Kunz and his collaborators allows us to say that, in general, these theories have been thematized as “playing and moving”, articulating it to the discussions about being-child, the importance of playing and moving freely and spontaneously, and the questions of time, creativity, art, and imagination. We have also noticed that the proposition of “playing and moving” continues to be grounded in some criticisms of science and rationalization, the logic of schooling and formal education, the didactic play, the adult- centric perspective, and the impositions and the controls to which children are submitted. Kunz 7/26 http://dx.doi.org/10.1590/1980-6248-2020-0139EN 4 For more details, see Kunz (2007). e-ISSN 1980-6248 e-ISSN 1980-6248 (2007), before presenting the concepts of childhood with which he dialogues, criticizes the studies that treat childhood as a scientific category in social, anthropological, and educational sciences, arguing that these studies say little about children as a subject. Therefore, grounded on the work Child Psychology and Pedagogy: The Sorbonne Lectures 1949-1952 by Merleau-Ponty (2006), the author writes about the complexes children go through, emphasizing those he considers important to be considered in the context of Childhood Education, such as the Weaning Complex or Disruption, as well as talking about Perception in Children4. Considering this, we can affirm that the concept of childhood present in the theorization of "play and self-movement" takes place through a phenomenological perspective. The child is understood as a being-in-the-world and recognized by what she is, by how she expresses and presents herself in the world. In other words, children are considered by their childhood nature and as a subject that have world perceptions different than the ones of adults, thus, one should value imagination, fantasy, and emotions (Stavisk et al., 2013). Though we have not identified a punctual definition of the concepts of body and movement with which they operate, it is possible to infer from his writings about "Playing and Moving" a dialogue with the ideas of the body itself and the relational body of Merleau-Ponty. In this case, there is an understanding of the body as a child that experiences it or the understanding of the body through its expressivity and totality. Such a way to understand the body is directly associated with the dialogic concept of movement (Costa et al., 2018; Gomes- Da-Silva, 2007). In this perspective, playing and moving freely and spontaneously are emphasized as what characterizes the specificity of Physical Education in Childhood Education. In our opinion, considering propositional terms, they can be characterized as a teaching object of Physical Education for early childhood. Besides this, according to Costa et al. (2016): Physical Education should open and broaden a valuable and ample field of teaching and research and could, maybe in the future, become one of the most important and valued areas in the field of Education because it teaches children, i.e., the human being in its early age, 'the art of knowing how to live better' [original highlight] (p. 51) 8/26 Pro-Posições | Campinas, SP | V. 33 | e20200139EN| 2022 8/26 e-ISSN 1980-6248 e-ISSN 1980-6248 Amidst the defense of the importance of children’s free, autonomous, and creative play and movement, Kunz (2007) distinguishes spontaneous play and didactic play. With assertive arguments, he affirms that play is spontaneous by nature, however, adults, aiming to prepare children for the future, have given it a didactic dimension. For the author, the latter would represent a play with specific educational ends, which is interested in the future of the child from the perspective and gaze of the adult and has been greatly used in Childhood Education. Kunz (2007) argues that didactic playing, much covered in the literature even with elements of interpretation and practical application, has been transforming the "play and moving" into activities empty of meaning for the children and full of adult impositions with activities recognized and accepted by the latter. As an example, the author states that, in literature, this type of play is represented under different names, such as motor-skill learning, psychomotricity, motricity, and game, among others, concluding that the didactic play is more concerned with content and the use of play than with the child who plays. As in the theories of Human Movement in Physical education which focus more on the possibilities of copying and imitating already created movements than on the child, the being that moves. (p.20) Though established the relationship between the world of life and the experience of free, autonomous, and spontaneous movement, when taken as a teaching object of Physical Education in Childhood Education, and the ways they are approached in the texts, the concept of free and spontaneous play and movement deserves more attention and explanation from the authors, on how they are daily supported in educational institutions, at the risk of being confused with a certain spontaneity. We have observed that, by emphasizing this free and spontaneous play and movement of children with no clear articulation of its meaning with the pedagogical proposals and practices that compose Childhood Education, the texts leave doubts about the role of teachers in this process. This can be seen in a citation present in the texts of Costa et al. (2016) and Surdi et al. (2016) affirming that "children, in general, develop normally, we do not have to do anything special for this. We simply have to like them, what happens effortlessly most of the time" (retrieved from Verden-Zöller, 2004, p. 237). Pro-Posições | Campinas, SP | V. e-ISSN 1980-6248 http://dx.doi.org/10.1590/1980-6248-2020-0139EN e-ISSN 1980-6248 e-ISSN 1980-6248 Though we do not find a clear definition of what would be this “art of living better”, when analyzing the texts written by Kunz and his students, we perceive a certain connection between this expression with the possibility of playing freely and spontaneously. By understanding children in their "being-in-the-world" and their need to fully live the present, the authors, dialoguing with Oaklander (1980) and Maturana and Verden-Zoller (2004), consider playing a valid activity in itself, manifested in children’s spontaneous action, in what they are, feel, and experience during its enactment, that is, in the present and with no foreign purposes and interference (Costa et al., 2016). With that said, we have identified that free and spontaneous playing and movement, understood as an object of teaching or specificity of Physical Education in Childhood Education, comes from the understanding that “playing is established as a type of communication and dialogue of children with the world and with themselves” (Kunz, 2015, p.10) and that it only happens through movement. However, given the centrality that the concepts "free and spontaneous" have in the texts analyzed, we still have doubts about their meaning in the context of pedagogical practice in Childhood Education, because the lack of a greater explanation gives space for some problematizations, mainly, about teacher’s role in this play that, as stated, should not suffer any external influence. We could see that Kunz (2005) uses the concept of 'life world' to criticize the use of rationality/science in the references to the movement presented to the children, as well as to reaffirm the importance of building references to movement and experiences that are inherent to the subject, in these cases, free and spontaneous. In this direction, the author continues "the sensibility, the perceptions, and human intuition develop more openly and intensely, the greater are the degrees and life opportunities and experiences with activities established by a spontaneous, autonomous and free self-movement" (p.20). Thus, based on these concepts, Kunz (2007) understands that the specialization of knowledge can fragment children’s experiences in Childhood Education. In Kunz's (2005) perspective, the experiences provided to children should not always start from an external reference to themselves or be used without their respective authorization because, to him, all that is external hinders children to discover the world, others, and themselves through their resources and conditions. Pro-Posições | Campinas, SP | V. e-ISSN 1980-6248 33 | e20200139EN| 2022 http://dx.doi.org/10.1590/1980-6248-2020-0139EN 5 In her dissertation, Gomes-da-Silva (2010) deepens the theme and reflects on teachers' role from the semiotics of Charles Pierce; however, in the article co-written with Kunz, these elements are just mentioned. e-ISSN 1980-6248 33 | e20200139EN| 2022 10/26 10/26 http://dx.doi.org/10.1 wishes and recognizing thei teachers not to deny them on of time and attention t but chosen by them to dialog Simon & Kunz, 2014, p.38 cern for children, adding to ). These obligations include seve . (Castro & Kunz, 2015, p. ms understanding what w Childhood Education co onomy of children, seem ny pedagogical work. B http://dx.doi.org/10.1590/1980-6248-2020-0139EN e-ISSN 1980-6248 http://dx.doi.org/10.1590/1980-6248-2020-0139EN e-ISSN 1980-6248 e-ISSN 1980-6248 explain the meaning of this active help, the authors only affirm that “an active help does not mean to be a conductor or guide of the activities that child does” (p.32). Faced with this, the category that aims to understand teachers' roles, following the theories of play and self-movement, showed itself as the weakest category and even a little confusing, hard to define. From our perspective, it is not clear to say that teachers of Physical Education in Childhood Education should potentialize and help free and spontaneous play and self-movement, nor that they should help children "in the struggle to survive as children in a world that wants to quickly transform them in a miniature adult" (Kunz, 2007, p.14). In this context, though we agree that a great part of the criticisms towards adult-centric perspectives, it seems that, in the texts analyzed, they are, in some moments, exaggerated. When operating with the criticism of the process of formal schooling and extending it to Childhood Education, Kunz and Costa (2015) propose an “active and intelligent follow-up of the general and full development of the child” (p. 32). Despite not giving further explanations about what this would be, the authors argue that this follow-up is more adequate than the schooling process. Therefore, we identified that a great part of the criticism of this process is because the authors consider that in Childhood Education "children are educated with purposes and objectives formulated by adults" (p.33). It seems that adults' presence and role in the educational process of children are always considered from a problematic viewpoint. In our perspective, this can create a polarization between adults and children, as they attribute a negative characteristic to adults’ actions and, it seems to us, an excessive autonomy to children. Considering this, we wish to highlight two points. First, amidst the many criticisms made by Kunz and his collaborators towards Childhood education, few consider the specificity of this educational segment as, some of them, seem to ignore the debate that already takes place in the sphere of the education of young children and the proposals of Childhood Education curriculum documents. Besides this, we found in other texts: Besides this, we found in other texts: This way, becoming sensitive to children’s wishes and recognizing their needs, without depriving them of their right to live what they want, is a way for teachers not to deny them [our highlight]. (Staviski et al., 2013, p.123) The educational process demands dedication of time and attention to plays and variations developed by the children, non-guided activities, but chosen by them to dialogue with the world, with others, and with themselves [our highlight]. (Simon & Kunz, 2014, p.384) What we perceive nowadays is a strong concern for children, adding to that an imposition of precocious obligations (Kunz & Costa, 2015). These obligations include several activities that, often, do not result from the children's wishes [our highlight]. (Castro & Kunz, 2015, p. 48) Based on these excerpts, we have problems understanding what would be the teachers' role in the educational process of children in Childhood Education considering that these arguments, besides pointing out an excessive autonomy of children, seem to place their desires and wishes as fundamental determinants for any pedagogical work. Before continuing, we should say that we are not questioning the importance of hearing children and considering their needs and specificities in the context of pedagogical practices of Childhood Education. However, we think that this is different from “the right to live what they want”, or contemplate only activities chosen by them, that is, that answer to their wills. Or even, the fact that believing that there is no need to do anything, as children will develop normally, can lead to the idea or interpretation that teachers' role is dispensable. Nonetheless, in other texts, such as those of Gomes-da-Silva et al. (2010), Simon and Kunz (2014), and Kunz and Costa (2015), it is possible to identify arguments different from those previously described. In these texts, the authors affirm that it is up to the teacher to integrate the planning and to create adequate conditions for children's discovery and learning. The texts also preconize that the adult/teacher should be sometimes an interlocutor and not a mediator, according to Gomes-da-Silva et al. (2010)5, while in other times an active helper in the experiences of children’s lives, as said by Kunz and Costa (2015). However, even with such appointments, it is also not clear what would be the teachers’ role because, in the attempt to 11/26 11/26 11/26 Pro-Posições | Campinas, SP | V. 33 | e20200139EN| 2022 6 To better understand the discussion on these concepts, see Arendt (1990). e-ISSN 1980-6248 We understand that this might show some fragilities of these criticisms, mainly, because of the current debates on the education of young children in Brazil, as well as some assumptions of curriculum documents that have been based on studies of Childhood Sociology and Pedagogy. That is, the current perspectives of Childhood Education have indicated a refusal of the adult-centric perspective. They consider play as a central and fundamental axis in the educational process of children, defending a pedagogical practice that considers children and their interests, and aiming to overcome the understanding of children as Pro-Posições | Campinas, SP | V. 33 | e20200139EN| 2022 Pro-Posições | Campinas, SP | V. 33 | e20200139EN| 2022 12 12/26 http://dx.doi.org/10.1590/1980-6248-2020-0139EN Pro-Posições | Campinas, SP | V. 33 | e20200139EN| 2022 To better understand the discussion on these concepts, see Arendt (1990). Pro-Posições | Campinas, SP | V. 33 | e20200139EN| 2022 13/26 e-ISSN 1980-6248 e-ISSN 1980-6248 e-ISSN 1980-6248 e-ISSN 1980-6248 a "beings-to-be", to see them as a "beings-that-are" (Sarmento, 2013). Therefore, though starting from different references, when we think about the context of pedagogical practice in Childhood Education, we have identified some similarities in the discourses present in the theorization of "Play and Self-movement", which do not sustain part of the criticisms presented. The second point to be highlighted refers to the polarization between adults/teachers and children that emerge from the criticism of the adult-centric perspective, as well as the strong valuing and idealization granted to children's autonomy, even proposing that they decide about what they want to experience in the context of Childhood Education. In this case, the way they criticize adults does not seem to distinguish well authority and authoritarianism6. Therefore, we have doubts if the search for childhood autonomy can, in fact, be established from a project of emancipation and liberation of children from what is "imposed" by adults, mainly if we believe that they are beings under formation and, thus, need adults' care and protection (Arendt, 1990). Arendt (1990) consider absurd the ideas that treat children as an oppressed minority by the dictates of rationality and the “world” of adults, from which they need to free themselves. In her line of thought, abolishing adults’ authority would be leaving children to their own luck, having to deal with a greater tyranny, the one of the majority. Besides this, supposing children’s autonomy would be transferring our responsibilities to this generation (Arendt, 1990). Furthermore, based on this author, it is wrong to think children's education by emancipating them from adult authority or even to think of the existence of a specific world for children and another for adults. According to Arendt (1990), there is only a world shared by all, that is, children and adults. In this sense, we could say that it is up to the teachers to present the world to children. Therefore, in the context of P.E. in Childhood education, we see no problem in the fact that teachers organize the activities, guide the process to elaborate knowledge that circulates in the institutions, and propose experiences and plays to present children with different body practices that are part of the body movement culture. This does not mean silencing children or thinking education according to an adult-centric model! http://dx.doi.org/10.1590/1980-6248-2020-0139EN http://dx.doi.org/10.1590/1980-6248-2020-0139EN e-ISSN 1980-6248 Part of human problems, we deduce, emerges from the lack of "outlet" for creative impulses, because the rational, functional logic of our way of thinking, has a "dehumanizing effect" and trivializes the human, as it disrespects intuitive intelligence, present in childhood when playing or drawing. Intuitive intelligence, which leads to creation, originates from imagination and fantasy. Therefore, it emerges in the moments the person is completely passive or when the conditions of the environment allow a free and spontaneous expression, such as in plays and games, in general [our highlights]. If we take as a reference the reflection of Vaz (1995), we could question if this theorization about "Play and Self-movement", when considering children's education, would not be operating with a perspective that outlines a type of reduction to sensations and perceptions. As they defend sensitivity as a condition for children's freedom and, as something that precedes rationality, would they not be forgetting language mediation? Would devaluing rationality be a more promising pathway in the educational process with children? Though we understand that the dialogue with Maturana allows the Theory of Play and Self-movement to understand emotion as something that precedes reason in the sense that it gives direction or freedom and spontaneity as something that can potentialize rational development, we identify that the emphatic way they operate the criticisms to rationality seems to disregard this relation between emotion, sensibility, and reason. Highlighting only the negative aspects of rationality, or not clearly explaining how they think this relation and the issue of language in this context, could be a big gap in this theorization. Although we understand that the theme of language is not deeply developed in the analyzed texts, we could thematize here a ponderation already created by Almeida et al. (2013, p.12), about how this expressive sense, which precedes meaning, is related to language. In this direction, citing Sérgio (1987), the authors argue that the “transit from perception to concept, the zone of pre-constitution to constitution” (p.94) can be seen as something that Merleau- Ponty could not explain. Vaz (1995) believes that the passage from the sensitive to the intelligible implies the practical mediation of language and emphasizes that overcoming the dichotomy of body- thought has been one of the concerns in the field of Physical Education. 3. Playing and moving: other problematizations When analyzing the writings that outline the concept of playing and self-movement as a theoretical-methodological perspective for Physical Education in Childhood education, we have observed that some ideas and arguments are often repeated. Be it in the sense of presenting the conception of children and the concepts with which they operate, be it in the perspective of making some criticisms towards formal school, rationality, and science to reaffirm such concepts. Therefore, we can say, more precisely, that the proposal for Physical Education in Childhood Education undertaken by Kunz and his collaborators suppose rescuing play and self- movement, understood by the authors as natural elements inherent to children, counterposing what is called the "adult world". In this direction, Kunz (2007) argues that his understanding of play as a natural element comes from the understanding of human beings as beings born to be free and creative. In other words, the author says that freedom, recognized as the power and the desire to decide its accomplishments, and creativity, understood as the possibility of building senses and meanings from what happens, can only be manifested in the exercise of play (Kunz, 2007). Though we consider the contributions of the theorization “Play and Self-movement” for the work context in Physical Education with Childhood Education and that we also agree with part of the criticism of “didactic” play, we believe that some arguments are too emphatic and leave room to some questions, such as the understanding of play and self-movement as natural and inherent to children, or, play as the only form, original, and authentic of children learning. Therefore, some statements require prudence and care in their use because the way the arguments appear in the texts may show some dichotomies that can be problematic. For instance, when defending children's freedom to play and move freely, autonomously, and spontaneously, Kunz and co-authors frequently consider playing in opposition to rationality. Or, in other moments, besides establishing a hierarchical relationship, consider that the dimensions of human sensitivities, freedom and human creativity precede rationality. Thus, Costa et al. affirm in 2015 (p.27) and in 2018 (pp.204-205) that: Pro-Posições | Campinas, SP | V. 33 | e20200139EN| 2022 14/26 14/26 e-ISSN 1980-6248 e-ISSN 1980-6248 However, Vaz (1995) highlights exactly the opposite. It is through the work of the concept, that is, "through the possibility that human being to place himself as the one who understands the regularity of nature (his own and the surrounding one), and over which he can place his humanity, is how freedom can be built" (p. 21). Furthermore, “[...] the criticism to instrumental reason is necessary and pertinent, but we cannot forget that one can only reach it through the work of reason” (p.21). Despite recognizing the importance of the sensitive, ludic, and play dimension in the context of children's education, we should question the way they have been approached. We have identified that opposition between the terms 'ludic and play' against 'rationality/logic' has been discussed and problematized in the field of Physical Education on the studies about leisure (Bracht, 2019). According to Bracht (2019), it is common to use the term ludic in opposition to logic/rationality, giving ludic a subversive and/or utopic character, constantly associated with corporality. Thus, similarly to what Bracht (2019) identifies in the studies of leisure and Santin’s (1994) work, we perceive that free and spontaneous play is always understood through its eminently positive, pleasant, interesting, and autonomous characteristics. More specifically, the theorization of “Play and Self-movement”, by attributing those positive characteristics to free and spontaneous play, thematizes the ‘didactic’ play, or rationality elements present in the education of children from very negative aspects. This theorization makes explicit a notion of play in opposition to scientific rationality and rational logic. Quoting Bracht (2019), we could also say that, in Kunz’s writings, there is a connotation that assuming ludic is, in a way, denying the mechanism and the objectification of humans typical of scientific rationality, for example, in the mechanical conception of the human body that strongly grounded the intervention of Physical Education. Affirming the ludic would be affirming the humanity of men, highlighting extremely desirable characteristics, such as freedom, autonomy, creativity, and pleasure. (p. 72) However, we have doubts about if the suspension/denial of didactic play and rationality could not lead to an idea that the human dimension only takes place in play because, for Kunz and collaborators, reason “dehumanizes”. In this sense, we agree with Bracht (2019), when affirming that the victimization of ludic play, having reason as an executioner, is extremely problematic. When we think about the propositions for Physical Education in Childhood Education proposed by Kuns and collaborators, we identify that the criticism of reason and scientific rationality arises from the understanding that human reality would be better understood through sensitivity, a similar idea to Santin (1994). Pro-Posições | Campinas, SP | V. 33 | e20200139EN| 2022 15/26 Pro-Posições | Campinas, SP | V. 33 | e20200139EN| 2022 15/2 Pro-Posições | Campinas, SP | V. 33 | e20200139EN| 2022 http://dx.doi.org/10.1590/1980-6248-2020-0139EN e-ISSN 1980-6248 e-ISSN 1980-6248 rational dimension subtracted, as well as cannot dispense nature. Or better, in a certain sense, it is related to widening our understanding of rationality, understanding it in the direction of the body, the ludic, the sensitive, the intensities, and affections. rational dimension subtracted, as well as cannot dispense nature. Or better, in a certain sense, it is related to widening our understanding of rationality, understanding it in the direction of the body, the ludic, the sensitive, the intensities, and affections. It is interesting to think of this question, based on the analogy Bracht (2019) makes about aporia present in Adorno’s Aesthetic Theory (AT). When criticizing this Theory, Bracht (2019) highlights the ambiguity in the relation between arts and rationality and argues that they are not opposed but complementary. When operating a criticism towards rationality, this is not done to subtract it. Or even, based on Adorno, Bracht (2019) states that art is defined as a form of knowledge, it is also rational. In the relation between reason and body, the author argues that aporia would be connected to the fact that reason that believes to have a body, that transfers the “mimetic impulse” for the rationality that objectifies the body (or propose its total control through instrumental reason) indicates at the same time a return to nature (to game, to magic), and this is what prescribes the law of movement (of body practices)– an aporia or ambiguity that cannot be overcome. We could even question if this attachment to the nature of the body, to the mimetic moment of the game (of movement), for example, could be considered a reaction to the bad irrationality of the ration world as managed, Would that be the reason for the appeal to a (romantic) rescue of ludic in Physical Education? [original highlight]. (p.159) Therefore, Bracht (2019) speculates about the experiences of movement and play and their relation with nature. In another moment, Bracht (2000) argued that the counter position to the hegemony of instrumental reason should not take place only through the affirmation of its opposite. For the author, we should prioritize sensitivity, or play, as an attempt to return to a "primordial unit (nature/man; world/man)” (p. XVII). e-ISSN 1980-6248 According to the author, the aesthetic and sensitive dimension cannot have its Pro-Posições | Campinas, SP | V. 33 | e20200139EN| 2022 16/26 Pro-Posições | Campinas, SP | V. 33 | e20200139EN| 2022 http://dx.doi.org/10.1590/1980-6248-2020-0139EN e-ISSN 1980-6248 http://dx.doi.org/10.1590/1980-6248-2020-0139EN e-ISSN 1980-6248 e-ISSN 1980-6248 One of the justifications presented by texts about "Play and Self-Movement" in the defense of free and spontaneous play refers to the recognition of play as a natural need, as a possibility of direct dialogue with the world, as well as of human nature that is implicit in children. We believe that these texts miss some considerations on the relation between culture and nature, and the mediation of language. The emphasis given to the rescue of human nature in children's education, associated with criticisms of rationality, seems to disregard the role culture plays in the mediation between human beings and nature. Or even, they leave doubts about what that direct dialogue with the world would be if language is also established based on culture. Paraphrasing Bracht (2019), we consider the need to think about play not in the sense of original purity, as its potential should not be only in the freely and spontaneously play and the denial of what composes rationality and the didactic play proposed by the adult. Children are not only nature. They are born and inserted into a universe of culture. This is even shown by the appeal of the cultural industry for a “natural” interest of children in games and play (Bracht, 2019). The risks of denying teachers’ intervention, or some didactic plays in Childhood education, can create the false illusion of freedom and spontaneity, as in a context under the influence of cultural interest, the appeal to children’s natural interest to play might take place, as warned by Bracth (2019), from a logic that, when resignifying this understanding, consider children much more as an object than the protagonist of a greater game. Thus, the author continues, children's cultural contexts are often inserted in the universe of plays through the offering of certain fantasies, objects, and delimitations of time and space. e-ISSN 1980-6248 An overcoming of instrumental reason should take place through mediation and “through the recognition of the ambiguity of our being in the world and being the world”, therefore, not only through a “return to the original sensitivity” (p. XVII). With this, we want to emphasize that thinking about children's educational process in Childhood Education institutions is also to have in mind some ambiguities and contradictions present in the relations between free and spontaneous play and didactic play, between sensitiveness and rationality, and between the relationships of adults/teachers with children. Besides spontaneity and freedom, play can also be experienced from intentionality, that is, we cannot eliminate all the contradictions and ambiguities of the educational process but think of them based on a dialogic process. Pro-Posições | Campinas, SP | V. 33 | e20200139EN| 2022 Pro-Posições | Campinas, SP | V. 33 | e20200139EN| 2022 e-ISSN 1980-6248 e-ISSN 1980-6248 e-ISSN 1980-6248 adult-centric education and the valuing of children's creative and participative potentialities, these perspectives differ in their understanding of the body. If on one hand, as affirmed by Sarmento in an interview with Richter et al. (2015), Childhood sociology, by emphasizing the social and historic dimensions of childhood, understood children's bodies as guided only by cultural practices, ignoring that the child is also body and nature. On the other hand, it seems that Kunz and collaborators, when criticizing rationality, propose a return to this lost nature of children, to perception, and sensitiveness. Faced with this, we have identified that the dilemma nature and culture which has been broadly discussed in the field of Physical education can also encompass the discussions on childhood and its education regarding the body, the movement, and children's expressions. In the specific case of the field of Physical Education, Bracht and Almeida (2019) argue that the dilemma between nature and culture is an “articulation problem”. Thus, the process of articulation and mediation between the ‘sayable’ and the ‘unsayable’, between reason and emotion, between ‘thought’ and ‘movement’”[original highlights] (Bracht & Almeida, 2019, p.12) has been one of the main challenges in the production of knowledge in Physical Education, mainly in the sphere of critical perspectives in the area. We could also extend this issue of articulation, as a challenge to be faced, through the theorization of play and self-movement. In the direction of what was argued by Bracht and Almeida (2019), thinking this process of articulation in the scope of children's education in Childhood Education would mean softening or even filling some gaps between nature and culture, sensitiveness and rationality, play and reason, body and language, adult and child. In their discussions, Bracht and Almeida (2019) point out that we should “talk about an interaction between the lived situation, our pre-reflexive experience, our individual articulation, and the cultural repertoire of interpretative standards” (p. 10). Thus, we understand that it is exactly in this “sphere that new meanings (new understandings of movement) can be produced because they are articulated” (p. 10). Before finishing, we should highlight that, despite choosing an analytical cut that privileged elements we consider problematic in the theorization in question, the writings about "Play and Self-movement" also offered important contributions to the debate about Physical Education in Childhood Education. 4. Final remarks The theorization about "Play and Self-movement" establishes another way of thinking about the pedagogical work with the body and children's movement in Childhood Education. Thus, instead of 'didactic' plays, or activities indicated by adults, the proposal is for children's free and spontaneous play and self-movement. Besides that, even if it might have some assumptions closer to those of Childhood Sociology and Pedagogy, such as the rupture with an Pro-Posições | Campinas, SP | V. 33 | e20200139EN| 2022 18/26 Pro-Posições | Campinas, SP | V. 33 | e20200139EN| 2022 http://dx.doi.org/10.1590/1980-6248-2020-0139EN e-ISSN 1980-6248 e-ISSN 1980-6248 e-ISSN 1980-6248 In this sense, we highlight the understanding, the value, and recognition of children for what they are in the present; the idea of an education that intends to be integral and not fragmented; the thematization of the game as a central axis in the process of children’s learning and full development; the recognition of children’s protagonism; the value of knowledge produced by children; and, a conception of body and movement that, understood as language, also contemplates the possibilities of children’s communication and expression. To conclude, we end this text by reiterating that, despite the contributions identified, we cannot deny that the theorization about playing and moving can risk certain inflections regarding the relationship between body, rationality, and language in children's educational process. Thus, we should ask if there is not something positive in reason, in the process of humanization, or the proposal of educational activities proposed by adults in Childhood Education. We also stress the importance and the need for the theory of "Play and Self- movement" to dialogue more with the specificity and assumptions of Childhood education, showing how the concepts presented are supported in the scope of the professional intervention of Physical Education in Childhood Education. e-ISSN 1980-6248 The keys of reading/interpretation with which we operated show some fundamental aspects of the context of Childhood Education, contemplated by such theorization. Pro-Posições | Campinas, SP | V. 33 | e20200139EN| 2022 19/26 Pro-Posições | Campinas, SP | V. 33 | e20200139EN| 2022 19/26 http://dx.doi.org/10.1590/1980-6248-2020-0139EN References Almeida, F. Q., Ghidetti, F. F., & Bracht, V. (2013). A presença da fenomenologia na/da Teoria do Se-movimentar Humano (TSMH) brasileira. Pensar a Prática, 16(3), 886-902. https://www.revistas.ufg.br/fef/article/download/19554/15257/ Doi: 10.5216/rpp.v16i3.19554 Doi: Arendt, H. (1990). Entre o passado e o futuro. (M. W. Barbosa, Trans.). Perspectiva, 1990. Bardin, L. (2020). Análise de conteúdo. 5a ed. São Paulo: Edições 70. Bracht, V. (2019). A Educação Física Escolar no Brasil o que ela vem sendo e o que pode ser: Elementos de uma teoria pedagógica para a Educação Física. Ed. Unijuí. Bracht, V. (2000). Esporte na escola e esporte de rendimento. Revista Movimento, I(12), XIV- XXIV. Pro-Posições | Campinas, SP | V. 33 | e20200139EN| 2022 20/26 Pro-Posições | Campinas, SP | V. 33 | e20200139EN| 2022 20/26 20/26 http://dx.doi.org/10.1590/1980-6248-2020-0139EN e-ISSN 1980-6248 Bracht, V., & Almeida, F. Q. (2019). Pedagogia Crítica da Educação Física: dilemas e desafios na atualidade. Movimento, 25(25001), 1-15. https://doi.org/10.22456/1982-8918.96196 Bracht, V., & Almeida, F. Q. (2019). Pedagogia Crítica da Educação Física: dilemas e desafios na atualidade. Movimento, 25(25001), 1-15. https://doi.org/10.22456/1982-8918.96196 Castro, F. B., & Kunz, E. (2015) O controle da subjetividade e das experiências corporais sensíveis: implicações para o brincar e se-movimentar da criança. Motrivivência, 27(45), 44-57. https://doi.org/10.5007/2175-8042.2015v27n45p44 Costa, A. R., Barros, T. E. S., & Kunz, E. (2018). O brincar como construção racional nas aulas de Educação Física. Motrivivência, 30(53), 196-208. http://dx.doi.org/10.5007/2175- 8042.2018v30n53p196. Costa, A. R., Souza, M. F., Miranda, D., & Kunz, E. (2016) Brincar e se-movimentar da criança: a imprescindível necessidade humana em extinção? Corpoconsciência, 19(3), 45-52. https://periodicoscientificos.ufmt.br/ojs/index.php/corpoconsciencia/article/view/4 148 Gomes-da-Silva, E. (2007). Educação (Física) Infantil: se-movimentar e significação. [Master’s dissertation, Centro de Desportos]. Universidade Federal de Santa Catarina, Florianópolis, SC. https://repositorio.ufsc.br/xmlui/bitstream/handle/123456789/90344/240545.pdf?s equence=1&isAllowed=y Gomes-da-Silva, E., Kunz, E., & Sant´Agostino, L. H. F. (2010). Educação (física) infantil: território de relações comunicativas. Revista Brasileira de Ciências do Esporte, 32(2-4), 29- 42. Ghidetti, F. F. (2012). A teoria do “se-movimentar” humano (TSMH) em questão: limites e possibilidades para uma teoria da educação física. [Master’s dissertation in Physical Education, Programa de Pós-Graduação em Educação Física]. Universidade Federal do Espírito- Santo, Vitória. Ghidetti, F. F., Almeida, F. Q., & Bracht, V. (2014, jul./dez.). Merleau-Ponty, Linguagem e Fenomenologia na Educação Física. P O I É S I S – Revista do Programa de Pós-Graduação em Educação, 8(14), 318-333. http://www.portaldeperiodicos.unisul.br/index.php/Poiesis/article/view/2316 Doi http://dx.doi.org/10.19177/prppge.v8e142014318-333 Pro-Posições | Campinas, SP | V. 33 | e20200139EN| 2022 21/26 21/26 http://dx.doi.org/10.1590/1980-6248-2020-0139EN Kunz, E. (Org.). (2005). Didática da Educação Física 2 (3a ed.). Ed. Unijuí. Kunz, E. (Org.). (2005). Didática da Educação Física 2 (3a ed.). Ed. Unijuí. Kunz, E. (2007). Educação Física a questão da Educação Infantil. In Grunennvaldt, J.T., Schneider, O., Kuhn, R., & Ribeiro, S. D. D. (Eds.), Educação Física, Esporte e Sociedade: temas emergentes (Vol. 1, pp. p. 105-117). Universidade Federal de Sergipe, Departamento de Educação Física. Kunz, E. (Org). (2015). Brincar & Se-Movimentar: tempos e espaços de vida da criança. Unijuí. Kunz, E., & Costa, A. R. (2015). A imprescindível e vital necessidade da criança: brincar e se- movimentar. In E. Kunz (Ed.), Brincar & Se-Movimentar: Tempos e Espaços de vida da criança (pp. 13-37). Unijuí, Machado, M. M. (2010). Merleau-Ponty & a Educação. Autêntica. Machado, M. M. (2013). Fenomenologia e Infância: o direito da criança a ser o que ela é. Revista Educação Pública, 22(49), 249-264. https://periodicoscientificos.ufmt.br/ojs/index.php/educacaopublica/article/view/9 13 . Doi: 10.29286/rep.v22i49/1.913 Maturana, H., & Verden-Zoller, G. (2004). Amar e brincar: fundamentos esquecidos do humano. Palas Athena. Oaklander, V. (1980). Descobrindo crianças: a abordagem gestáltica com crianças e adolescentes (5a ed.). Summus. Richter, A. C., Bassani, J., & Vaz, A. F. (2015). Entrevista com Manuel Jacinto Sarmento: infância, corpo e educação física. Florianópolis, Cadernos de Formação da RBCE, 6(2),11- 37. http://revista.cbce.org.br/index.php/cadernos/article/view/2182 Rocha, M. C. (2011). Forma escolar, educação física e educação infantil: (im)pertinência. [Master’s dissertation, Centro de Educação Física e Desportos]. Universidade Federal do Espírito Santo, Vitória, ES. http://www.educacaofisica.ufes.br/pt-br/pos- graduacao/PPGEF/detalhes-da-tese?id=5117 Santin, S. (1994). Educação física: da alegria do lúdico à opressão do rendimento. EST/ESEF-UFRGS. Pro-Posições | Campinas, SP | V. 33 | e20200139EN| 2022 22/26 Pro-Posições | Campinas, SP | V. 33 | e20200139EN| 2022 22/26 22/26 http://dx.doi.org/10.1590/1980-6248-2020-0139EN e-ISSN 1980-6248 Sarmento, M. J. (2013). A sociologia da infância e a sociedade contemporânea: desafios conceituais e praxeológicos. In R. T. Ens, & M. C. Garanhani (Orgs.), Sociologia da infância e a formação de professores (pp. 13-46). Champagnat. Sarmento, M. J. (2013). A sociologia da infância e a sociedade contemporânea: desafios conceituais e praxeológicos. In R. T. Ens, & M. C. Garanhani (Orgs.), Sociologia da infância e a formação de professores (pp. 13-46). Champagnat. Simon, H. S., & Kunz, E. (2014). O brincar como diálogo/Pergunta e não como resposta à prática pedagógica. Movimento, 20(1), 75-94. https://seer.ufrgs.br/Movimento/article/view/39749, https://doi.org/10.22456/1982-8918.39749 Stavisk, G., Surdi, A., & Kunz, E. (2013). Sem tempo de ser criança: a pressa no contexto da educação de crianças e implicações nas aulas de Educação Física. Revista Brasileira de Ciências do Esporte, 35(1), 113-128. https://www.scielo.br/scielo.php?script=sci_arttext&pid=S0101-32892013000100010 Surdi, A. G., & Kunz, E. (2010). Fenomenologia, movimento humano e a educação física. Revista Movimento, Porto Alegre, 16(4), 263-290. https://doi.org/10.22456/1982-8918.16075 Surdi, A. C., Melo, J. P., & Kunz, E. (2016). O brincar e o se-movimentar da criança como manifestação artística. Licere, 19(3), 225-252. http://docs.bvsalud.org/biblioref/2020/05/847529/1293-texto-do-artigo-4522-1-10- 20160824.pdf Vaz, A. F. (1995). A filosofia na educação física: soltando as amarras e a capacidade de ser negatividade. In A. Ferreira Neto, S. V. Goelnner, & V. Bracht. (Orgs.), As Ciências do Esporte no Brasil (pp. 165-191). Autores Associados. Pro-Posições | Campinas, SP | V. 33 | e20200139EN| 2022 23/26 23/26 http://dx.doi.org/10.1590/1980-6248-2020-0139EN Appendix Table 1: Studies that are part of the theorization exercise on “Play and move” Titles Authors Type of production Year Práticas didáticas para um “conhecimento de si” de crianças e jovens na Educação Física Kunz, E. Book chapter: Didática da Educação Física 2 2005 Educação Física a questão da Educação Infantil Kunz, E. Book chapter: Educação Física, Esporte e Sociedade: temas emergentes – Vol. 1. 2007 Brincar e se-movimentar. Kunz, E.; Santos, L.M.E Book chapter: Física: Conhecimento e Saber Escolar 2009 A liberdade no brincar e se- movimentar da criança com uma perspectiva teórica para a educação (física) infantil Kunz, E.; Kuhn, R.; Santos, V. B. Book chapter: Educação Física, Esporte e Sociedade: Temas Emergentes – Vol. 3 2009, Educação (Física) Infantil: Território de Relações Comunicativas. Gomes-da- Silva, E.; Kunz, E.; Sant'agostinho, L. H. Article: RBCE, v. 32, pp. 29-41. 2010 Fenomenologia, movimento humano e a educação física. 2010. KUNZ, E; SURDI, A. G. Article: Movimento, v. 16, n. 4, p. 263- 290. 2010 Crianças não são adultos em Miniatura Kunz, E.; Müller, U.; Costa, A.R. Book chapter: Didática da Educação Física 2 2012 Sem tempo de ser criança: a pressa no contexto da educação de crianças e implicações nas aulas de Educação Física Stavisk, G.; Surdi, A. C.; Kunz, E. Article: RBCE, v. 35, pp. 113-128. 2013 Relações Comunicativas como Processo Pedagógico na Educação (Física) Infantil Gomes-da- Silva, E.; Kunz, E.; Santagostinho, L. H. F. Book chapter: Educação Física Escolar: Pesquisas e Reflexões 2014 O brincar como diálogo/Pergunta e não como resposta à prática pedagógica Simon, H. S.; Kunz, E. Article: Movimento, v. 20, pp. 375-394. 2014 Apresentação e organização do Livro Kunz, E. Book: Brincar & Se-Movimentar: Tempos e Espaços de vida da criança 2015 A imprescindível e vital necessidade da criança: brincar e se-movimentar Kunz, E.; Costa, A. R. Book chapter: Brincar & Se-Movimentar: Tempos e Espaços de vida da criança 2015 Table 1: Studies that are part of the theorization exercise on “Play and move” http://dx.doi.org/10.1590/1980-6248-2020-0139EN Corresponding author: Maria Celeste Rocha - Rua Dr. Arlíndo Sodré, 512 – Itararé, Vitória, ES, 29047- 500, Brasil. e-ISSN 1980-6248 O brincar como construção racional nas aulas de Educação Física Costa, A. R.; Barros, T.E.S.; Kunz, E. Article: Motrivivência, v. 30, pp. 196-208. 2018 Educação e sensibilidade: o brincar e o se-movimentar da criança pequena na escola Surdi, A. C.; Rodrigues, W. C. M. F.; Freire, E. J.S.M.; Kunz, E. Article: Motrivivência, v. 31, pp. 1-22. 2019 Submission data: Submitted to evaluation November 9, 2020; revised May 17, 2021; approved for publication October 13, 2021. e-ISSN 1980-6248 Sem tempo de Ser Criança Stavisk, G.; Kunz, E. Book chapter: Brincar & Se-Movimentar: Tempos e Espaços de vida da criança 2015 Criança e o brincar como obra de arte: O sentido de um esclarecimento Cunha, A. C.; Kunz, E. Book chapter: Brincar & Se-Movimentar: Tempos e Espaços de vida da criança 2015 A sensibilidade na Educação Infantil. Surdi, A. C.; Pereira, D. A.; Kunz, E. Book chapter: Brincar & Se-Movimentar: Tempos e Espaços de vida da criança 2015 A Curiosidade da criança: quem fomenta? Souza, C. A.; Kunz, E. Book chapter: Brincar & Se-Movimentar: Tempos e Espaços de vida da criança 2015 O fazer da experiência do ser- criança: entre o estímulo e a descoberta Castro, F. B.; Kunz, E. Book chapter: Brincar & Se-Movimentar: Tempos e Espaços de vida da criança 2015 Brincar e se-movimentar da criança: a imprescindível necessidade humana em extinção? Costa, A.R.; Souza, M. F; Miranda, D.; Kunz, E. Article: Corpoconsciência, v. 19, pp. 45-52, 2015. 2015 O controle da subjetividade e das experiências corporais sensíveis: implicações para o brincar e se- movimentar da criança. Castro, F.B.; Kunz, E. Article: Motrivivência, v. 27, pp. 44-57. 2015 Autonomia nas Aulas de Educação Física: para pensar a prática pedagógica com crianças Souza, C. A.; Kunz, E. Book chapter: Iniciação à Docência, Reflexões e Produção do Conhecimento: PIBID Educação Física no CEFD/UFSM 2016 O brincar e o se-movimentar da criança como manifestação artística Surdi, A.C.; Melo, J. P.; Kunz, E. Article: Licere, v. 19, pp. 225-252. 2016 O brincar e o se-movimentar nas aulas de educação física infantil: realidades e possibilidades Surdi, A.C.; Melo, J.P.; Kunz, E. Article: Movimento, v. 22, pp. 1-16. 2016 Sobre como tolhemos a curiosidade das crianças De Souza, C.A.; Donadel, T. B.; Kunz, E. Article: Motrivivência, v. 29, pp. 192-204. 2017 Elementos da fenomenologia como uma das possibilidades de compreender o jogo como um movimento humano significativo Cunha, A.C.T.N; Surdi, A. C.; Marques, D.A.P; Kunz, E.; Moreira, E Article: Revista Portuguesa de Educação, v. 31, pp. 54-67. 2018 As relações do brinquedo industrializado com o brincar e se- movimentar: uma reflexão na Educação Física Burckardt, E.V.; Costa, L.C.; Kunz, E. Article: Motrivivência, v. 30, pp. 278-294. 2018 Pro-Posições | Campinas, SP | V. 33 | e20200139EN| 2022 25/26 Pro-Posições | Campinas, SP | V. 33 | e20200139EN| 2022 25/26 25/26 http://dx.doi.org/10.1590/1980-6248-2020-0139EN Authors’ contributions: Rocha, Maria Celeste: Conceptualization (Equal), Data curation (Leader), Formal Analysis (Equal), Grant demand (Equal), Investigation (Equal), Methodology (Equal, Project Management (Equal), Resources (Equal), Software (Equal), Supervision (Equal), Validation (Equal), Visualization (Equal), Writing-original draft (Leader), Writing- review and edition (Leader). Almeida, Felipe Quintão: Conceptualization (Equal), Data curation (Equal), Formal Analysis (Equal), Grant demand (Equal), Investigation (Equal), Methodology (Equal), Project Management (Equal), Resources (Equal), Software (Equal), Supervision (Equal), Validation (Equal), Visualization (Equal), Writing-original draft (Equal), Writing- review and edition (Equal). Moreno, Alberto: Conceptualization (Equal), Data curation (Support), Formal Analysis (Equal), Grant demand (Equal), Investigation (Equal), Methodology (Equal), Project Management (Equal), Resources (Equal), Software (Equal), Supervision (Equal), Validation (Equal), Visualization (Equal), Writing-original draft (Support), Writing- review and edition (Equal). Pro-Posições | Campinas, SP | V. 33 | e20200139EN| 2022 26/26 Pro-Posições | Campinas, SP | V. 33 | e20200139EN| 2022 26/26 Pro-Posições | Campinas, SP | V. 33 | e20200139EN| 2022
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Small angle x-ray scattering with edge-illumination
Scientific reports
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Small angle x-ray scattering with edge-illumination Peter Modregger1, Tiziana P. Cremona2, Charaf Benarafa3, Johannes C. Schittny2, Alessandro Olivo1 & Marco Endrizzi1 received: 14 April 2016 accepted: 11 July 2016 Published: 05 August 2016 Sensitivity to sub-pixel sample features has been demonstrated as a valuable capability of phase contrast x-ray imaging. Here, we report on a method to obtain angular-resolved small angle x-ray scattering distributions with edge-illumination- based imaging utilizing incoherent illumination from an x-ray tube. Our approach provides both the three established image modalities (absorption, differential phase and scatter strength), plus a number of additional contrasts related to unresolved sample features. The complementarity of these contrasts is experimentally validated by using different materials in powder form. As a significant application example we show that the extended complementary contrasts could allow the diagnosis of pulmonary emphysema in a murine model. In support of this, we demonstrate that the properties of the retrieved scattering distributions are consistent with the expectation of increased feature sizes related to pulmonary emphysema. Combined with the simplicity of implementation of edge-illumination, these findings suggest a high potential for exploiting extended sub-pixel contrasts in the diagnosis of lung diseases and beyond. In small angle x-ray scattering sensitivity to sub-pixel sample features provides access to information on micro- scopic scales with macroscopic pixel sizes. In addition to complementary information about the sample this offers the opportunity for faster scans and/or dose reduction by exploiting larger pixel sizes. The ability to obtain con- trasts related to sub-pixel sample structures was demonstrated with analyzer-based imaging1,2, grating interfer- ometry (GI)3,4 and edge-illumination (EI)5,6. Currently, the potential of phase sensitive x-ray imaging for clinical application is investigated in the fields of mammography7–9, cartilage imaging10, bone structure determination11, imaging the airway surface liquid12 and the diagnosis of pulmonary emphysema13,14. Sub-pixel information is typically accessed through the broadening of the scattering distribution underlying the signal within a pixel, and is usually referred to as scatter strength. Scatter strength can be regarded as a measure for sample inhomogeneity within a detector pixel and its relation to sub-pixel sample morphology is the subject of ongoing research15–19. In this study, we applied a deconvolution-based data analysis approach previously used in GI20,21 to EI, which increases the number of complementary contrasts related to sub-pixel sample features. EI is a non-interferometric and incoherent phase sensitive x-ray imaging technique22 utilizing a set of two apertured masks (Fig. 1). www.nature.com/scientificreports www.nature.com/scientificreports www.nature.com/scientificreports Small angle x-ray scattering with edge-illumination The pre-sample mask shapes the incident radiation into an array of well defined beamlets, which are distorted by the sample. These distortions are transformed into detectable intensity variations by the detector mask, which covers approximately half of each pixel. Since the optical elements are achromatic, EI is readily compatible with inco- herent x-ray tubes, where the full spectrum of the source contributes to the signal at the detector23,24. The com- paratively large structure sizes of the apertured masks (typically tens of microns) render EI easily scalable, robust against environmental vibrations and thermal stress. Established scan and data procedures simultaneously yield absorption contrast, differential phase contrast25,26 with nano-radiant sensitivity27 and scatter contrast6. Recently, EI was also successfully combined with tomography28,29. Extended Number of Contrasts with EI-SAXS In the experiment, illumination curves (IC) were obtained by recording the detected intensity for each pixel while the sample mask was laterally scanned in M steps separated by a fraction of the mask pitch. This was performed with and without a sample present yielding the sample IC (s) and the flat IC (f), respectively. We express the ICs in terms of the scatter angle α (i.e., s(α) and f(α)), which is related to the lateral offset Δ​x of the sample mask and 1Department of Medical Physics and Bioengineering, University College London, Gower Street, WC1E 6BT London, United Kingdom. 2Institute of Anatomy, University of Berne, Baltzerstrasse 2, 3012 Bern, Switzerland. 3Theodor Kocher Institute, University of Berne, Freiestrasse 1, 3012 Bern, Switzerland. Correspondence and requests for materials should be addressed to P.M. (p.modregger@ucl.ac.uk) Scientific Reports | 6:30940 | DOI: 10.1038/srep30940 www.nature.com/scientificreports/ Figure 1. Sketch of the experimental set-up for EI. Figure 1. Sketch of the experimental set-up for EI. the sample to detector distance z by α =​ Δ​x/z. In analogy with the procedure described in20, we assumed that the distortion of the sample IC can be modelled by the sample to detector distance z by α =​ Δ​x/z. In analogy with the procedure described in20, we assumed that the distortion of the sample IC can be modelled by α α α = ⊗ s f g ( ) ( ) ( ) (1) (1) with the small-angle x-ray scattering (SAXS) distribution g(α) being determined by the sub-pixel structure of the sample. Therefore, g(α) can be retrieved by deconvolving the sample IC s with the flat IC f. In order to distinguish this analysis method from others we will refer to it as EI-SAXS.l with the small-angle x-ray scattering (SAXS) distribution g(α) being determined by the sub-pixel structure of the sample. Therefore, g(α) can be retrieved by deconvolving the sample IC s with the flat IC f. In order to distinguish this analysis method from others we will refer to it as EI-SAXS.l For this proof of concept experiment, we used a dragon fly as a biological sample expected to provide a variety of differently shaped scattering distributions. During the experimental scan M =​ 10 images were acquired on equally spaced positions of the IC. Each image consisted of 5 frames with 5 s exposure time to ensure sufficient signal to noise ratio. Extended Number of Contrasts with EI-SAXS In order to reduce possible aliasing effects due to partial illumination of the sample within one period of the sample mask, the sample was dithered 8 times (i.e., additional translations of the sample by a fraction of the sample mask pitch between the dithering steps were performed), and the corresponding images averaged prior to data analysis. g p y Data analysis was performed by deconvolving s(α) with f(α) using iterative Lucy-Richardson deconvolution30,31, where the k-th iteration is performed by computing = ⋅    ⊗ ⊗    + g g s g f f (2) k k k 1 (2) and f denotes f mirrored at the origin. In practice the occurring convolutions are computed utilizing fast Fourier transform. Lucy-Richardson deconvolution has an implicit positive constraint, converges to the maximum likeli- hood solution in the case of Poisson statistics, and is known to deliver stable results in the presence of photon shot noise. The starting value was chosen to be α α α = ∑ ∆ α g s f ( )/( ( ) ) 0 , which provides the correct normalisation of the retrieved scattering distribution g. The number of iteration steps was 1000 in order to ensure convergence. The deconvolution provided the scattering distribution g(α) on the half-open interval α α α ∈[ , [ min max . For the sub- sequent moment analysis (see below) it was convenient to symmetrize the scattering distribution (i.e., α α = g g ( ) ( ) max min ), which is justified by the periodicity of the discrete Fourier transform employed by Lucy-Richardson deconvolution. and f denotes f mirrored at the origin. In practice the occurring convolutions are computed utilizing fast Fourier transform. Lucy-Richardson deconvolution has an implicit positive constraint, converges to the maximum likeli- hood solution in the case of Poisson statistics, and is known to deliver stable results in the presence of photon shot noise. The starting value was chosen to be α α α = ∑ ∆ α g s f ( )/( ( ) ) 0 , which provides the correct normalisation of the retrieved scattering distribution g. The number of iteration steps was 1000 in order to ensure convergence. The deconvolution provided the scattering distribution g(α) on the half-open interval α α α ∈[ , [ min max . Scientific Reports | 6:30940 | DOI: 10.1038/srep30940 Extended Number of Contrasts with EI-SAXS For the sub- sequent moment analysis (see below) it was convenient to symmetrize the scattering distribution (i.e., α α = g g ( ) ( ) max min ), which is justified by the periodicity of the discrete Fourier transform employed by Lucy-Richardson deconvolution. y Figure 2 provides examples of the utilized ICs and of the retrieved scattering distributions inside and outside the sample. As expected, the scattering distribution outside the sample shows an approximate δ-function shape, where the finite width is due to finite sampling with respect to the scatter angle. The distribution inside of the sample is both shifted and broadened, which are both in accordance with expectations. Since the deconvolution procedure yields a scattering distribution g(α) for each pixel, scatter images (see Supplementary Figure 1) are available for each scattering angle α. This demonstrates an extension of the number of contrast from the previ- ous 3 to 10 for EI. Ultimately, the number of contrast modalities is limited by the stability of the imaging system and by the resolution of the motor used for scanning the sample mask. Later in this study, we will utilize M =​ 32 sample points. In GI, the retrieval of angular-resolved scattering distributions involves the deconvolution of noisy, periodic signals with suppressed even harmonics20, which is intrinsically a challenging task32. Moreover, owing to the coherence intrinsic to the contrast formation process in GI, parts of the broad spectrum provided by an x-ray tube contribute substantially less to the signal collected by a detector that integrates over photon energies33. In contrast, the ICs in EI are approximately Gaussian-shaped, and the achromaticity of the optical elements significantly improves the signal to noise ratio24. Thus, the deconvolution procedure is simpler for EI. However, due to the geometry of experimental set-ups angular sampling of the scattering distribution is about an order of magnitude larger for EI (Δ​α =​ 25 μrad here) than for GI (Δ​α =​ 1.7 μrad in20). Therefore, the two methods retrieve the scat- tering distribution on essentially complementary, and possibly expanding scales. For EI, finer angular sampling can be achieved by either increasing the distance between sample and detector or by increasing the number of acquired sample points M on the IC. The latter will be ultimately limited by the stability of the set-up and the resolution of utilized scanning motors. Scientific Reports | 6:30940 | DOI: 10.1038/srep30940 2 www.nature.com/scientificreports/ Figure 2. Extended Number of Contrasts with EI-SAXS Recovery of scattering distributions. (a) Flat IC and sample ICs for a pixel outside and inside of the sample, respectively. (b) SAXS distribution as retrieved by deconvolution of the ICs shown in (a). As expected the retrieved distribution outside of the sample is approximately δ-shaped, while the distribution inside of the sample is shifted and broadened. Figure 2. Recovery of scattering distributions. (a) Flat IC and sample ICs for a pixel outside and inside of the sample, respectively. (b) SAXS distribution as retrieved by deconvolution of the ICs shown in (a). As expected the retrieved distribution outside of the sample is approximately δ-shaped, while the distribution inside of the sample is shifted and broadened. Complementarity of contrasts provided by EI-SAXS. Based on the assumption that the flat and the sample IC can be expressed as Gaussian functions, it was demonstrated that three contrast modalities (i.e., trans- mission t, refraction Δ​xR and scatter strength σ) can be retrieved from three experimental images6. In the fol- lowing, we will use this established data analysis procedure for comparison. Please refer to the reference for more details.h The three contrast modalities can be regarded as parameters of the shape of the scattering distribution and can be retrieved from the deconvolved scattering distributions by calculating the moments Mn (with n ∈​ N being the order of the moment) according to34 ∫α α = M d g ( ) (3) 0 ∫αα α = M d g M ( )/ (4) 1 0 (3) ∫αα α = M d g M ( )/ 1 0 ∫αα α = M d g M ( )/ 1 0 (4) ∫α α α = − ≥ . Extended Number of Contrasts with EI-SAXS M d M g M n ( ) ( )/ for 2 (5) n n 1 0 (5) While M0, M1 and M2 correspond to absorption, differential phase and scatter strength contrasts, respectively, higher order moments (n >​ 2) constitute additional descriptive parameters for the scattering distribution and, thus, provide increased structural information about the sample on a sub-pixel scale.f While M0, M1 and M2 correspond to absorption, differential phase and scatter strength contrasts, respectively, higher order moments (n >​ 2) constitute additional descriptive parameters for the scattering distribution and, thus, provide increased structural information about the sample on a sub-pixel scale.f p p p Figure 3 shows a comparison of different contrast modalities as retrieved by the established data analysis to the corresponding moments of deconvolved scattering distributions at the example of a dragon fly. We found an excellent agreement for M0 and the transmission t (correlation coefficient: 0.999) as well as for M1 and the refrac- tion contrast Δ​xR (correlation coefficient: 0.954). M2 and the scatter strength σ show the same sample morphol- ogy (correlation coefficient: 0.926), while they differ only in the magnitude of the retrieved scatter strength. This deviation can attributed to the fact that the shape of the sample and flat IC differ from a Gaussian, which leads the established data analysis to wrongly estimate the scatter strength.h y g y g This establishes EI-SAXS as a viable alternative to the data analysis methods previously used in imaging with edge-illumination. It should be noted that, while previously reported data methods made assumptions about the underlying scattering distribution (e.g., negligible scattering in25,26), the proposed deconvolution method does not assume a specific shape of the involved IC and, thus, produces reliable results for arbitrarily shaped scatter distributions. In order to investigate the information content provided by higher order moments we investigated the scatter- ing properties of three household powders: salt, coffee and sugar. The same parameters for the scan and analysis procedures as above were used, except for the lack of dithering (i.e, the sample was not moved). Figure 4(a,b) show the 2nd and 4th moment of the scattering distributions, respectively retrieved by deconvolution. The comple- mentarity of M2 and M4 is clearly demonstrated by the corresponding pixel-wise scatter plot, shown in Fig. 4(c). Extended Number of Contrasts with EI-SAXS Therefore, EI-SAXS provides the opportunity to exploit extended sub-pixel contrasts, which could be useful in areas as diverse as biomedical research, materials science and security screening. As an example of a significant application, we will take a first step into exploring the potential of EI-SAXS for the diagnosis of lung diseases. EI-SAXS for the diagnosis of pulmonary emphysema. Chronic obstructive pulmonary disease (COPD) is characterized by severe changes in lung morphology and is a major and increasing cause of death associated with cigarette smoking35. The micro-structure of lungs is defined by alveolar walls and distal airspaces, which have typical sizes on the micrometer scale36. Pulmonary emphysema is a frequent constituent of COPD and a condition of the lung “characterized by abnormal, permanent enlargement of air spaces distal to the terminal bronchiole, accompanied by destruction of their walls, and without obvious fibrosis”37. Scientific Reports | 6:30940 | DOI: 10.1038/srep30940 3 www.nature.com/scientificreports/ Figure 3. Comparison of an established data analysis procedure with the proposed deconvolution-based method for a dragon fly. The top row shows the transmission t (a), refraction μ (c) and scatter strength σ (e) retrieved according to6. The bottom row shows the first three moments of deconvolved scattering distribution: (b) M0 corresponds to transmission, (d) M1 to refraction and (f) M2 to scatter strength, respectively. Scale bars are 1 cm. An excellent agreement between M0 and M1 and their corresponding contrasts retrieved from the established data analysis was found. M2 and σ show the same sample morphology while slightly differing in the magnitude of retrieved values (factor of ≈​1.5). The deviation is attributed to a violation of the strict assumptions of the previously established data analysis. Figure 3. Comparison of an established data analysis procedure with the proposed deconvolution-baselh Figure 3. Comparison of an established data analysis procedure with the proposed deconvolution-based method for a dragon fly. The top row shows the transmission t (a), refraction μ (c) and scatter strength σ (e) retrieved according to6. The bottom row shows the first three moments of deconvolved scattering distribution: (b) M0 corresponds to transmission, (d) M1 to refraction and (f) M2 to scatter strength, respectively. Scale bars are 1 cm. An excellent agreement between M0 and M1 and their corresponding contrasts retrieved from the established data analysis was found. M2 and σ show the same sample morphology while slightly differing in the magnitude of retrieved values (factor of ≈​1.5). Extended Number of Contrasts with EI-SAXS Based upon this observation, the ratio of the 4th and the 2nd moment (i.e., M4/M2), which appears as the slope of lines connecting the plotted points to the origin, was used as an illustration model for the potential diagnos- tic power of EI-SAXS. The physical interpretation of this ratio is as follows. For a constant M2, larger values of M4 indicate stronger tails of the scattering distribution (see equation 5). In turn, stronger tails indicate a larger contribution of large scattering angles to the scattering distribution compared to small scattering angles. Since large scattering angles are associated with smaller sample features, high values of the M4/M2-ratio are related to larger relative contributions of smaller sample features. Potentially, the other moments (i.e., M3,M5,…) could carry additional information about the sub-pixel sample morphology. However a detailed analysis of their possible contributions lies outside the scope of this preliminary study and will be the subject of future investigations. In the following pilot experiment with a mouse model of cigarette smoke exposure-induced emphysema, we demonstrate that EI-SAXS can detect this increase in feature sizes by exploiting the complementarity of the second and fourth moment of deconvolved scattering distributions. Two murine lungs, one control (N =​ 1) and one emphysematous (N =​ 1) were imaged. The same scan parameters and data analysis procedures as described above were used except for the number of sample points on the IC (M =​ 32) and the fact that dithering was not performed. Images for the M0, M2 and M4 can be found in the Supplementary material (supplementary Figure 2).h p g 0, 2 4 pp y ( pp y g ) The scatter plot of M2 and M4 (Fig. 5a) reveals a clear distinction between control and emphysematous lung. Due to the small number of samples, we can only consider this as preliminary evidence for the utility of higher order moments provided by EI-SAXS in the context of pulmonary emphysema diagnosis. Further, for each lung sample an approximate linear relationship between M2 and M4 was found (also visible for the powders in Fig. 4c). Based upon this observation, the ratio of the 4th and the 2nd moment (i.e., M4/M2), which appears as the slope of lines connecting the plotted points to the origin, was used as an illustration model for the potential diagnos- tic power of EI-SAXS. The physical interpretation of this ratio is as follows. Extended Number of Contrasts with EI-SAXS The deviation is attributed to a violation of the strict assumptions of the previously established data analysis. Figure 4. Complementary of moments extracted from different powders with EI-SAXS. M2 (a) and M4 (b) for salt (blue rectangle), coffee (yellow rectangle) and sugar (red rectangle). Scale bars are 5 mm. The pixel-wise scatter plot of M2 and M4 (c) demonstrates the complementarity of those moments. Figure 4. Complementary of moments extracted from different powders with EI-SAXS. M2 (a) and M4 (b) for salt (blue rectangle), coffee (yellow rectangle) and sugar (red rectangle). Scale bars are 5 mm. The pixel-wise scatter plot of M2 and M4 (c) demonstrates the complementarity of those moments. Scientific Reports | 6:30940 | DOI: 10.1038/srep30940 4 www.nature.com/scientificreports/ Figure 5. Diagnosis of lung emphysema with EI-SAXS. A clear separation of the control and the emphysematous lung in the M2–M4 scatter plot (a) is indicated by arrows. The ratio M4/M2 of the control (b) is larger than that of the emphysematous lung (c), which is most noticeable in the emphysematous left lung. Scale bars are 1 cm and the background was masked by thresholding. Comparison between scattering distributions (d) confirm that the emphysematous lung provides features smaller tails than that of the control one, which is consistent with increased average feature sizes in the former. Figure 5. Diagnosis of lung emphysema with EI-SAXS. A clear separation of the control and the emphysematous lung in the M2–M4 scatter plot (a) is indicated by arrows. The ratio M4/M2 of the control (b) is larger than that of the emphysematous lung (c), which is most noticeable in the emphysematous left lung. Scale bars are 1 cm and the background was masked by thresholding. Comparison between scattering distributions (d) confirm that the emphysematous lung provides features smaller tails than that of the control one, which is consistent with increased average feature sizes in the former. In the following pilot experiment with a mouse model of cigarette smoke exposure-induced emphysema, we demonstrate that EI-SAXS can detect this increase in feature sizes by exploiting the complementarity of the second and fourth moment of deconvolved scattering distributions. Two murine lungs, one control (N =​ 1) and one emphysematous (N =​ 1) were imaged. Scientific Reports | 6:30940 | DOI: 10.1038/srep30940 Extended Number of Contrasts with EI-SAXS The same scan parameters and data analysis procedures as described above were used except for the number of sample points on the IC (M =​ 32) and the fact that dithering was not performed. Images for the M0, M2 and M4 can be found in the Supplementary material (supplementary Figure 2). The scatter plot of M2 and M4 (Fig. 5a) reveals a clear distinction between control and emphysematous lung. Due to the small number of samples, we can only consider this as preliminary evidence for the utility of higher order moments provided by EI-SAXS in the context of pulmonary emphysema diagnosis. Further, for each lung sample an approximate linear relationship between M2 and M4 was found (also visible for the powders in Fig. 4c). Based upon this observation, the ratio of the 4th and the 2nd moment (i.e., M4/M2), which appears as the slope of lines connecting the plotted points to the origin, was used as an illustration model for the potential diagnos- ti f EI SAXS Th h i l i t t ti f thi ti i f ll F t t M l l f In the following pilot experiment with a mouse model of cigarette smoke exposure-induced emphysema, we demonstrate that EI-SAXS can detect this increase in feature sizes by exploiting the complementarity of the second and fourth moment of deconvolved scattering distributions. Two murine lungs, one control (N =​ 1) and one emphysematous (N =​ 1) were imaged. The same scan parameters and data analysis procedures as described above were used except for the number of sample points on the IC (M =​ 32) and the fact that dithering was not performed. Images for the M0, M2 and M4 can be found in the Supplementary material (supplementary Figure 2). The scatter plot of M2 and M4 (Fig. 5a) reveals a clear distinction between control and emphysematous lung. Due to the small number of samples, we can only consider this as preliminary evidence for the utility of higher order moments provided by EI-SAXS in the context of pulmonary emphysema diagnosis. Further, for each lung sample an approximate linear relationship between M2 and M4 was found (also visible for the powders in Fig. 4c). Extended Number of Contrasts with EI-SAXS For a constant M2, larger values of M4 indicate stronger tails of the scattering distribution (see equation 5). In turn, stronger tails indicate a larger contribution of large scattering angles to the scattering distribution compared to small scattering angles. Since large scattering angles are associated with smaller sample features, high values of the M4/M2-ratio are related to larger relative contributions of smaller sample features. Potentially, the other moments (i.e., M3,M5,…) could carry additional information about the sub-pixel sample morphology. However a detailed analysis of their possible contributions lies outside the scope of this preliminary study and will be the subject of future investigations. Scientific Reports | 6:30940 | DOI: 10.1038/srep30940 5 www.nature.com/scientificreports/ Figure 6. CI separation (ΔCI) of M4/M2 values for the control and the emphysematous lung as a function of delivered dose. The dashed line represents a linear extrapolation. Figure 6. CI separation (ΔCI) of M4/M2 values for the control and the emphysematous lung as a function of delivered dose. The dashed line represents a linear extrapolation. The images of M4/M2-ratios for the control and the emphysematous lung (Fig. 5b,c) appear generally flat and the emphysematous lung has noticeably darker appearance–especially in the left lung. Faint horizontal stripes are residuals from bridges in the mask design that stabilize the structure38. These minor artefacts can be easily removed by e.g. improved mask design or data analysis. The flatness of the M4/M2-contrasts is remarkable for two reasons. First, due to the opening in the sample mask only 10 μm/79 μm ≈​ 12.7% of the sample was illuminated within a pixel (no dithering was used in this case) indicating the robustness of M4/M2-ratio. Second, sample movement does affect but not destroy a quasi-homogeneous contrast modality as it was shown with breathing during dark-field imaging of mouse lungs13. Since M4/M2 is homogeneous we speculate that breathing during exposure should only have a minor impact on EI-SAXS, as has been observed in the case of GI. p y p The mean M4/M2-ratios for the two lungs, determined over the entire sample, were = M M ( / )control 4 2 . ± . ⋅ − (5 535 0 006 10 ) rad 9 2 and = . ± . ⋅ − M M ( / ) (5 128 0 004 10 ) rad emphysematous 4 2 9 2, where the uncertainties are given at 0.68 confidence interval (CI). Extended Number of Contrasts with EI-SAXS Thus, the two ratios were separated by more than 40 CIs. While the specific values of M4/M2 could potentially be affected by variations in experimental parameters, this very signifi- cant separation suggests a high potential of EI-SAXS for reliable diagnosis of emphysema at different stages of development.hi The scattering distributions in Fig. 5(d) confirmed that the tails of the control were stronger than the tails of the emphysematous lung. Since smaller scattering angles are associated with larger sample features, the smaller tails of the diseased lung are consistent with increased average feature sizes due to emphysema. Since the higher order moments correspond to scattering of sub-pixel sample features, the pixel size could be increased without losing access to this information. In this proof of concept study no special attention was paid to dose optimization. Using an ion chamber the dose rate was estimated as 0.2 mGy/s entrance air kerma. This implies a delivered dose of 400 mGy for the dragon fly (Fig. 3), 50 mGy for the powders (Fig. 4) and 160 mGy for the lungs (Fig. 5). However, the dose can be signifi- cantly reduced by optimizing aperture sizes8, adequate filtering of low energy photons29 and increasing pixel sizes. In order to demonstrate this potential, we simulated experiments with decreased dose by using 1, 2, 3, 4 or 5 out of the 5 accumulations that were acquired for the murine lungs as the input for data analysis. Figure 6 shows the resulting separation of the M4/M2-ratio for the control and the emphysematous lung in terms of CIs as a function of simulated dose. It is obvious that the separability of healthy and emphysematous lungs is preserved if the dose is reduced significantly. If a separation of 5 CIs is taken as sufficient, then a rough linear extrapolation (dashed line in Fig. 6) implies a dose of only 6.3 mGy with this un-optimized set-up. Thus, EI-SAXS offers the potential to diagnose pulmonary emphysema at low dose. Methods Sample preparation. The dragon fly was air-dried over several days prior to scanning in order to ensure stable sample features and fixed at its tail for imaging. The three household powders were put on top of each other in a rectangular plastic holder to ensure consistent sample thicknesses. Wild-type 129S6/SvEv/Tac mice were exposed to air or to cigarette smoke 5 hours/day, 5 days/week for 4 months. Smoke of 3R4F research ciga- rettes (University of Kentucky, Lexington, KY) was generated by a TE10z smoking machine (Teague Enterprises, Woodland, CA) connected to whole-body exposure chambers as described previously39. All animal studies were approved by the Cantonal Veterinary Office of Bern, Switzerland and carried out in accordance with relevant guidelines. Lungs were instilled with 1.5% paraformaldehyde-1.5% glutaraldehyde in 0.15 M HEPES pH 7.35 through a tracheal cannula at a constant pressure of 20 cm H2O. The trachea was ligated and lungs were placed in fresh fixative for at least 24 hours to complete fixation. Following super-critical drying, samples were placed in the x-ray system using sample holder with membranes for imaging. Instrumentationh The experiment was carried out with a laboratory-based set-up at University College London (London, UK). The source was a Rigaku MM007 microfocus rotating anode with a Mo target operated at 25 mA current and 40 kVp voltage. A Hamamatsu C9732DK flat panel sensor was used as a detector featuring an isotropic pixel size of 50 μm. Both the sample and detector mask were manufactured by Creatv Microtech (Potomac, MD) and consisted of a series of Au lines on a graphite substrate. The sample mask featured a pitch of 79 μm and an opening of 10 μm, while the detector mask had a pitch of 98 μm and an opening of 17 μm to account for beam divergence. In order to compensate for the comparatively large cross-pixel talk of the x-ray camera (≈​40% for nearest neighbours), both masks had a column-skipped design38 and the raw images were binned 2 times for subsequent data analysis (effective pixel size 100 μm). The sample to detector distance was z =​ 0.32 m and the total set-up length amounted to 2 m. References l 1. Rigon, L. et al. A new DEI algorithm capable of investigating sub-pixel structures. J. Phys. D. Appl. Phys. 36, A107–A112 (2003). 2. Suhonen, H., Fernández, M., Bravin, A., Keyrilainen, J. & Suortti, P. Refraction and scattering of X-rays in analyzer-based imaging J. Synchrotron. Radiat. 14, 512–521 (2007).f 3. David, C., Nöhammer, B., Solak, H. H. & Ziegler, E. Differential x-ray phase contrast imaging using a shearing interferometer. App Phys. Lett. 81, 3287–3289 (2002).fi y , ( ) 4. Pfeiffer, F. et al. Hard-X-ray dark-field imaging using a grating interferometer. Nat. Mater. 7, 134–137 (2008). 5. Endrizzi, M. & Olivo, A. Absorption, refraction and scattering retrieval with an edge-illumination-based imaging setup. J. Phys Appl. Phys. 47, 505102 (2014).i pp y , ( ) 6. Endrizzi, M. et al. Hard X-ray dark-field imaging with incoherent sample illumination. Appl. Phys. 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EI-SAXS makes no prior assumptions about the shape of the scattering distributions, and it was shown to extend the number of contrast modalities from previously three to ten and more. Using different household powders we demonstrated the complementarity of the 2nd and 4th moment of retrieved scattering distributions, which indicates access to additional information about sub-pixel sample features. We provided preliminary evidence for the possibility to exploit this complementarity in order to differentiate between healthy and emphysematous mouse lungs. We also demonstrated that the properties of experimental scattering distributions were consistent with larger average feature sizes in the emphysematous lung. Based on these observations, we introduced the ratio of the 4th and 2nd moment as an illustration model for emphysema diagnosis. Although the number of samples was small, the observed effect was very large, with the M4/M2-ratios separated by more than 40 CIs. In combi- nation with the simple translatability of edge-illumination to a commercial system, the achieved results indicate EI-SAXS’s high potential for the exploitation of additional sub-pixel contrasts in biomedical research, materials science and medical diagnostics - especially for, but not limited to, improving the detection and characterisation of pulmonary emphysema. Scientific Reports | 6:30940 | DOI: 10.1038/srep30940 6 www.nature.com/scientificreports/ References l On the origin of visibility contrast in x-ray Talbot interferometry. Opt. Express 18, 16890–16901 (2010). ( ) 16. Bech, M. et al. Quantitative x-ray dark-field computed tomography. Phys. Med. Biol. 55, 5529–5539 (2010). 17. Lynch, S. K. et al. Interpretation of dark-field contrast and particle-size selectivity in grating interferometers. Appl. Op 4310–4319 (2011). 18. Modregger, P. et al. Multiple scattering tomography. Phys. Rev. Lett. 113, 020801 (2014).i gg p g g p y y 19. Strobl, M. General solution for quantitative dark-field contrast imaging with grating interferometers. Sci. Rep. 4, 7243 (2014). General solution for quantitative dark-field contrast imaging with g . Strobl, M. General solution for quantitative dark-field contrast im i 20. Modregger, P. et al. Imaging the Ultrasmall-Angle X-Ray Scattering Distribution with Grating Interferometry. Phys. Rev. Lett. 048101 (2012).i 21. Weber, T. et al. Increasing the darkfield contrast-to-noise ratio using a deconvolution-based information retrieval algorithm in X-ray grating-based phase-contrast imaging. Opt. Express 21, 18011 (2013).i g g g g p p 22. Olivo, A. et al. An innovative digital imaging set-up allowing a low-dose approach to phase contrast applications in the medical field. Med. Phys. 28, 1610–1619 (2001). y 3. Munro, P. R. T., Ignatyev, K., Speller, R. D. & Olivo, A. Source size and temporal coherence requirements of coded aperture type x-ray phase contrast imaging systems. Opt. Express 18, 19681–19692 (2010). 4. Endrizzi, M. et al. Achromatic approach to phase-based multi-modal imaging with conventional X-ray sources. Opt. Express 23 16473 (2015). 5. Munro, P. R. T., Ignatyev, K., Speller, R. D. & Olivo, A. Phase and absorption retrieval using incoherent X-ray sources. Proc. Natl Acad. Sci. USA 109, 13922–13927 (2012). 6. Diemoz, P. C., Hagen, C. K., Endrizzi, M. & Olivo, A. Sensitivity of laboratory based implementations of edge illumination X-ray phase-contrast imaging. Appl. Phys. Lett. 103, 24–29 (2013). p g g pp y , ( ) 7. Diemoz, P. C. et al. X-Ray Phase-Contrast Imaging with Nanoradian Angular Resolution. Phys. Rev. Lett. 110, 138105 (2013). l Th d l l f f d ll h 27. Diemoz, P. C. et al. X-Ray Phase-Contrast Imaging with Nanoradian Angular Resolution. Phys. Rev. Lett. 110, 138105 (2013). 28 Hagen C K et al Theory and preliminary experimental verification of quantitative edge illumination x-ray phase contrast 8. Hagen, C. K. et al. Theory and preliminary experimental verification of quantitative edge illumination x-ray phase contras tomography. Opt. Express 22, 258–261 (2014). References l SerpinB1 deficiency is not associated with increased susceptibility to pulmonary emphysema in mice. Am. J. Physiol. Lung Cell. Mol. Physiol. 305, L981–L989 (2013). 9. Cremona, T. P., Tschanz, S. A., von Garnier, C. & Benarafa, C. SerpinB1 deficiency is not associated with increased susceptibility to pulmonary emphysema in mice. Am. J. Physiol. Lung Cell. Mol. Physiol. 305, L981–L989 (2013). Acknowledgementsh g This project has received funding from the European Union’s Horizon 2020 research and innovation programme under the Marie Skłodowska-Curie Grant Agreement No. 657621 and from the EPSRC (grant EP/I021884/1. This project was also supported by the Royal Academy of Engineering under the RAEng Research Fellowships scheme. Part of this work has been supported by the Flight Attendant Medical Research Institute (FAMRI) and the Swiss National Science Foundation (310030-127464 and 310030-149790). Author Contributionsh The experiment was devised by P.M., A.O. and M.E. and the measurements as well as the data analysis was carried out by P.M. Murine lung samples were prepared by T.P.C., C.B. and J.S. The manuscript was written by P.M. with discussion and improvements by all authors. Scientific Reports | 6:30940 | DOI: 10.1038/srep30940 References l g p y p p , ( ) 29. Hagen, C. K., Munro, P. R. T., Endrizzi, M., Diemoz, P. C. & Olivo, A. Low-dose phase contrast tomography with conventional x-ray sources. Med. Phys. 41, 070701 (2014). y ( ) 0. Richardson, W. Bayesian-based iterative method of image restoration. J. Opt. Soc. Am. 62, 55–59 (1972). Scientific Reports | 6:30940 | DOI: 10.1038/srep30940 7 www.nature.com/scientificreports/ 2. Scattarella, F. et al. Post-detection analysis for grating-based ultra-small angle X-ray scattering. Phys Medica 29, 478–486 (2013). 3. Thüring, T. et al. Energy resolved X-ray grating interferometry. Appl. Phys. Lett. 102, 191113 (2013).i , y g g g y g y , 33. Thüring, T. et al. Energy resolved X-ray grating interferometry. Appl. Phys. Lett. 102, 191113 (2013).i 34. James, F. Statistical Methods In Experimental Physics. 2nd edn (World Scientific Publishing Co. Pte. Ltd., Singapore, 2006 35. Thun, M. J. et al. 50-Year Trends in Smoking-Related Mortality in the United States. N. Engl. J. Med. 368, 351–364 (2013 h , J g y g J , ( ) 36. Haberthür, D. et al. Visualization and stereological characterization of individual rat lung acini by high-resolution X-ray tomographic microscopy. J. Appl. Physiol. 115, 1379–1387 (2013).hhi h g y g 6. Haberthür, D. et al. Visualization and stereological characterization of individual rat lung acini by high-resolution X-ra tomographic microscopy. J. Appl. Physiol. 115, 1379–1387 (2013).hhi g p py pp y 37. Snider, G., Kleinerman, J., Thurlbeck, W. & Bengali, Z. The Definition of Emphysema. Am. Rev. Repiratory Dis. 132, 182–185 (1985). 38. Ignatyev, K., Munro, P. R. T., Speller, R. D. & Olivo, A. Effects of signal diffusion on x-ray phase contrast images. Rev. Sci. Instrum. 82, 073702 (2011).i 7. Snider, G., Kleinerman, J., Thurlbeck, W. & Bengali, Z. The Definition of Emphysema. Am. Rev. Repiratory Dis. 132, 182–185 (1985) 8. Ignatyev, K., Munro, P. R. T., Speller, R. D. & Olivo, A. Effects of signal diffusion on x-ray phase contrast images. Rev. Sci. Instrum 82, 073702 (2011).i 37. Snider, G., Kleinerman, J., Thurlbeck, W. & Bengali, Z. The Definition of Emphysema. Am. Rev. Repiratory Dis. 132, 182–185 (1985). 38. Ignatyev, K., Munro, P. R. T., Speller, R. D. & Olivo, A. Effects of signal diffusion on x-ray phase contrast images. Rev. Sci. Instrum. 82, 073702 (2011).i , ( ) 39. Cremona, T. P., Tschanz, S. A., von Garnier, C. & Benarafa, C. Additional Information Supplementary information accompanies this paper at http://www.nature.com/srepi Competing financial interests: The authors declare no competing financial interests. How to cite this article: Modregger, P. et al. Small angle x-ray scattering with edge-illumination. Sci. Rep. 6, 30940; doi: 10.1038/srep30940 (2016). This work is licensed under a Creative Commons Attribution 4.0 International License. The images or other third party material in this article are included in the article’s Creative Commons license, unless indicated otherwise in the credit line; if the material is not included under the Creative Commons license, users will need to obtain permission from the license holder to reproduce the material. To view a copy of this license, visit http://creativecommons.org/licenses/by/4.0/ © The Author(s) 2016 Scientific Reports | 6:30940 | DOI: 10.1038/srep30940 8
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TGF-β is elevated in hyperuricemic individuals and mediates urate-induced hyperinflammatory phenotype in human mononuclear cells
Arthritis research & therapy
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Arthritis Research & Therapy Arthritis Research & Therapy Klück et al. Arthritis Research & Therapy (2023) 25:30 https://doi.org/10.1186/s13075-023-03001-1 Open Access Abstract Background  Soluble urate leads to a pro-inflammatory phenotype in human monocytes characterized by increased production of IL-1β and downregulation of IL-1 receptor antagonist, the mechanism of which remains to be fully elucidated. Previous transcriptomic data identified differential expression of genes in the transforming growth factor (TGF)-β pathway in monocytes exposed to urate in vitro. In this study, we explore the role of TGF-β in urate-induced hyperinflammation in peripheral blood mononuclear cells (PBMCs). Methods  TGF-β mRNA in unstimulated PBMCs and protein levels in plasma were measured in individuals with nor- mouricemia, hyperuricemia and gout. For in vitro validation, PBMCs of healthy volunteers were isolated and treated with a dose ranging concentration of urate for assessment of mRNA and pSMAD2. Urate and TGF-β priming experi- ments were performed with three inhibitors of TGF-β signalling: SB-505124, 5Z-7-oxozeaenol and a blocking antibody against TGF-β receptor II. Results  TGF-β mRNA levels were elevated in gout patients compared to healthy controls. TGF-β-LAP levels in serum were significantly higher in individuals with hyperuricemia compared to controls. In both cases, TGF-β correlated posi- tively to serum urate levels. In vitro, urate exposure of PBMCs did not directly induce TGF-β but did enhance SMAD2 phosphorylation. The urate-induced pro-inflammatory phenotype of monocytes was partly reversed by blocking TGF-β. Conclusions  TGF-β is elevated in individuals with hyperuricemia and correlated to serum urate concentrations. In addition, the urate-induced pro-inflammatory phenotype in human monocytes is mediated by TGF-β signalling. Future studies are warranted to explore the intracellular pathways involved and to assess the clinical significance of urate-TGF-β relation. Keywords  TGF-β, Hyperuricemia, Inflammation, Mononuclear leukocytes 2 Radboud Institute for Molecular Life Sciences (RIMLS), Nijmegen, The Netherlands 3 Department of Medical Genetics, “Iuliu Haţieganu” University of Medicine and Pharmacy, Cluj Napoca, Romania 4 Departement of Rheumatology, Radboud UMC, Nijmegen, The Netherlands TGF‑β is elevated in hyperuricemic individuals and mediates urate‑induced hyperinflammatory phenotype in human mononuclear cells Viola Klück1,2†, Georgiana Cabău3†, Linda Mies1, Femke Bukkems4, Liesbeth van Emst1, René Bakker4, Arjan van Caam4, HINT consortium, Tania O. Crişan3 and Leo A. B. Joosten1,2,3* †Viola Klück and Georgiana Cabău share first authorship. *Correspondence: Leo A. B. Joosten Leo.Joosten@radboudumc.nl 1 Department of Internal Medicine, Radboud UMC, Nijmegen, The Netherlands *Correspondence: Leo A. B. Joosten Leo.Joosten@radboudumc.nl 1 Department of Internal Medicine, Radboud UMC, Nijmegen, The Netherlands *Correspondence: Leo A. B. Joosten Leo.Joosten@radboudumc.nl 1 Department of Internal Medicine, Radboud UMC, Nijmegen, The Netherlands @ 1 Department of Internal Medicine, Radboud UMC, Nijmegen, The Netherlands Background When TGF-β is secreted, it is in inactive form bound to its latency-associated peptide (LAP) of which it can get separated via ROS, metalloproteinases and integrins [20]. The active form of TGF-β can subsequently bind to TGF-β receptor II (TGF-βRII). Upon binding of TGF- β, two TGFβRII receptors form a heterotetramer with TGFβRI, forming a signalling-competent complex able to induce the C-terminal phosphorylation of receptor-acti- vated SMADs [19]. The activated SMADs form a com- plex with SMAD4 and translocate to the nucleus, where they regulate the expression of a large number of genes involved in e.g. fibrosis and immune signalling. Indepen- dently of SMADs, TGF-β can also signal via, e.g. TAK1, ERK and the PI3K-Akt pathway, of which the latter was also shown to be involved in urate priming [15]. g Hyperuricemia, defined as elevated serum urate levels above the saturation threshold, is the major risk factor for gout [1]. Supersaturated serum urate precipitates into monosodium urate (MSU) crystals which deposit within joints leading to recurrent inflammatory arthritis. These gout flares are initiated by interleukin (IL)-1β produc- tion by macrophages within the synovium. In these cells, stimulation of a Toll-like receptor (TLR), by free fatty acids for instance, results in the synthesis of pro-IL-1β, while MSU crystals activate the NLRP3 inflammasome leading to active caspase-1, which processes this pro- IL-1β to mature IL-1β [2]. Based on this finding, several therapies targeting IL-1β demonstrated efficacy in treat- ing gout flares [3].l In myeloid cells, the function of TGF-β depends on the specific nature of the activating conditions. Gener- ally, TGF-β stimulates cells at the resting state, whereas activated cells are inhibited [21]: in activated monocytes, TGF-β inhibits MyD88-dependent TLR- and IL-1R sig- nalling pathways by promoting MyD88 degradation [22]. However, TGF-β alone can induce gene expression of IL-1 in peripheral blood monocytes [23–25].l l Although presenting as an intermittent flaring condi- tion, gout is a chronic disease [4]. It affects about 2.5– 3.9% of the Western population and has become more prevalent in the last decades [5]. In addition, hyperurice- mia is associated with higher incidence of comorbidities such as cardiovascular disease, type 2 diabetes, metabolic syndrome, chronic kidney disease, cancer and premature ageing [6–9]. Moreover, gout patients have an increased mortality rate of 2.21 compared with the total popula- tion, and this increase is associated with high urate levels [10]. Background Therefore, elucidating the mechanisms responsible for this enhanced risk to develop comorbidities caused by hyperuricemia is crucial. Taken together, hyperuricemia has pro-inflammatory effects in human monocytes and is a risk factor for gout and its associated comorbidities. Previous findings sug- gest TGF-β pathway might be a relevant target to assess in relationship to the inflammation elicited by urate. Therefore, in this study, we explore the role of TGF-β in the context of hyperuricemia and urate induced repro- gramming of myeloid cells. Crişan et  al. previously demonstrated that soluble urate leads to a pro-inflammatory phenotype in primary human monocytes characterized by increased produc- tion of IL-1β, a classical pro-inflammatory cytokine, and downregulation of IL-1 receptor antagonist (IL-1Ra), the natural inhibitor of IL-1 [11–13]. Features of proinflam- matory reprogramming after urate exposure persisted for up to 6 days in PBMCs in vitro and were associated with epigenetic changes [14]. Previously published tran- scriptomic assessment revealed several differentially enriched pathways in primary monocytes treated with urate for 20 hours, including the TGF-β signalling path- way [15]. In line with this, several genetic variants in genes encoding activin receptors and inhibins belonging to the TGF-β superfamily were found to be associated with serum urate concentrations [16]. In addition, IL-37, an anti-inflammatory cytokine with an important role in gout, also functions via an interaction with SMAD3, a major intracellular signalling effector of TGF-β, further enforcing the importance of this signalling pathway in gout [17, 18].l © The Author(s) 2023. Open Access This article is licensed under a Creative Commons Attribution 4.0 International License, which permits use, sharing, adaptation, distribution and reproduction in any medium or format, as long as you give appropriate credit to the original author(s) and the source, provide a link to the Creative Commons licence, and indicate if changes were made. The images or other third party material in this article are included in the article’s Creative Commons licence, unless indicated otherwise in a credit line to the material. If material is not included in the article’s Creative Commons licence and your intended use is not permitted by statutory regulation or exceeds the permitted use, you will need to obtain permission directly from the copyright holder. To view a copy of this licence, visit http://​creat​iveco​mmons.​org/​licen​ses/​by/4.​0/. The Creative Commons Public Domain Dedication waiver (http://​creat​iveco​ mmons.​org/​publi​cdoma​in/​zero/1.​0/) applies to the data made available in this article, unless otherwise stated in a credit line to the data. Klück et al. Arthritis Research & Therapy (2023) 25:30 Klück et al. Arthritis Research & Therapy (2023) 25:30 Page 2 of 12 Page 2 of 12 Materials and methods Volunteers For the discovery cohort, blood from 9 gout patients (8 male, 1 female, mean age 66.2 years old) was used for identification experiments. Blood from 7 healthy volun- teers was used as a control (6 male, 1 female, mean age 60.4 years old). All volunteers gave informed consent to use leftover blood for research purposes. Blood draw from healthy volunteers were approved by the Ethical Committee of the Radboud University Medical Center (no. NL32357.091.10 and no. NL42561.091.12) Our validation cohort consisted of 197 individuals with normouricemia, 179 individuals with hyperuricemia without gout, and 195 patients with gout. All study par- ticipants in the gout group were included if they corre- sponded to the ACR/EULAR 2015 classification criteria with a score of 8 or higher. Thirty-six patients presented to the rheumatologist with acute flares. For gout man- agement, allopurinol was used either alone or in com- bination with NSAIDs and/or colchicine. Volunteers in the hyperuricemia group were included based on serum TGF-β is generally considered an anti-inflammatory cytokine with pro-fibrotic properties which can be secreted by most immune cells [19]. It consists of three paralogs of which paralog 1 is expressed in monocytes. Klück et al. Arthritis Research & Therapy (2023) 25:30 Page 3 of 12 for Western blotting. Protein concentrations were deter- mined using Pierce BCA Protein Assay Kit (ThermoFis- cher; Cat#23227) following manufacturer’s instructions, and equal amounts of protein were loaded in Laemmli sample buffer and separated on a 10% SDS/PAGE gel for 2  h at 120V. After running the gel, the proteins transferred to a 0.45-μM nitrocellulose membrane (GE Healthcare; Cat#10600002) using wet transfer in Tow- bin buffer on ice. The membrane was blocked for unspe- cific binding with 5% BSA-TBST followed by incubation with the primary antibody (Table S2). After overnight at 4 °C, incubation blots were washed and incubated with the secondary antibody for 30 min at RT (Table S3). After another washing step, the blots were developed using the Odyssey CLX Infrared imaging system (Licor). Quan- titative assessment of band intensity was performed by Image Lab software (Bio-Rad). urate levels of 7 mg/dl or higher and negative history of gout flares. None of the individuals with hyperuricemia were treated with urate-lowering therapies. Ex vivo mRNA expression experimentsh The PBMCs from patients and matched healthy controls were seeded on flat-bottom 96-well plates at a density of 0.5 × ­106 cells per well and incubated at 37  °C with 5% CO2 for an hour. Subsequently, non-adhering cells were washed away using pre-warmed PBS and the adherent monocytes were incubated with RPMI for 4 h before cells were stored in TRIzol reagent. RNA purification was per- formed according to manufacturer’s instructions. Sub- sequently, RNA concentrations were determined using NanoDrop software and cDNA was synthesized using iScript cDNA Synthesis Kit. The SYBR Green method was used to determine the mRNA expression of TGFB1, TGFBR1, TGFBR2, MMP9, ITGAV and SMAD7 relative to reference gene B2M (primer sequences Table S1) To inhibit TGF-β receptor signalling, three inhibitors were used. The ALK4/5/7-kinase inhibitor SB-505124 (Sigma) in a concentration of 5 μM with DMSO as sol- vent control, 5Z-7-oxozeaenol (100 nM) dissolved in DMSO (Tocris Bioscience) and a blocking antibody against TGF-β receptor II (AF-241-NA, R&D systems) with mouse IgG1 as the isotype control (10 μg/mL). Cells were pre-incubated with the inhibitor for 0.5 h before adding urate. Materials and methods Volunteers All partici- pants were included in Cluj-Napoca, Romania, as part of the HINT project (supported by the Romanian Minis- try of European Funds; P_37_762, MySMIS 103587), and both clinical characteristics and blood were collected for analysis. For in  vitro experiments, buffy coats from healthy donors were obtained after written informed consent (Sanquin blood bank, Nijmegen, the Netherlands). Urate priming experiments For urate priming experiments, adherent monocytes were primed for 24 h in RPMI supplemented with 10% human pool serum with or without urate (Sigma, 69-93- 2) and recombinant TGF-β1 (R&D Systems, Catalogue number 7754-BH-005). After 24 h, cells were restimu- lated with 10 ng/mL ultra-pure E. coli LPS (InVivogen, Catalogue number tlrl-pelps). Subsequently, cell-free supernatants were collected. Secretion of cytokines was measured in supernatants using ELISA kits for IL-1β, IL-6, IL-1Ra and TGF-β (R&D Systems, Catalogue num- ber DY201, DY206, DY280 and DY240 respectively). In vitro TGF‑β1 signalling experiments For mRNA expression, adherent monocytes from healthy volunteers were treated with dose-ranging concentra- tions of urate for 24 h. Subsequently, cells were stored in TRIzol and RNA isolation and qPCR were performed as described above. Cell isolation Peripheral blood mononuclear cells (PBMCs) were iso- lated using Ficoll-gradient from whole blood of volun- teers and were resuspended in RPMI 1640 supplemented with 50 μg/mL gentamycin, 2 mM L-glutamine and 1 mM pyruvate medium. Monocytes were further enriched by either adherence for 1 hour followed by washout of non-adherent lymphocytes or using Percoll gradient. Proteomics Serum samples from controls (N = 196), hyperurice- mic (N = 173) and gout patients (N = 213) collected and stored at – 80 °C were used for commercial targeted serum proteomics analysis (Olink, Uppsala, Sweden). Olink Target 96 Inflammation panel measures 92 protein biomarkers and four internal control samples, using 1 μl serum sample, by multiplex proximity extension assays [26]. The method uses two specific DNA-labelled anti- bodies for each protein that upon target binding come in close proximity to each other and allow the formation of a PCR reporter sequence that is quantified by real-time PCR (qPCR). Results are generated from cycle threshold For the pSMAD2 assessment, Percoll monocytes iso- lated from healthy volunteers were seeded into a 12-wells plate (1 × ­106 cells/well) and treated overnight with urate and, subsequently, TGF-β1 was added for the last hour. For collection of cell lysates, cells were kept on ice and lysed with lysis buffer (Cell signalling; Cat#9803) contain- ing 1x Complete Protease Inhibitor Cocktail (PIC; Roche Diagnostics, #11697498001). Lysates were centrifuged at 25.000×g for 15 min at 4 °C, and supernatants were taken Klück et al. Arthritis Research & Therapy (2023) 25:30 Klück et al. Arthritis Research & Therapy (2023) 25:30 Page 4 of 12 Page 4 of 12 RNA. Fragmented target RNA was reverse transcribed to cDNA using random N6 primers followed by end- repair and A tailing for adaptor ligation. Purified liga- tion products were enriched using PCR amplification followed by denaturation and cyclization of ssDNA by splint oligos and DNA ligase generating DNA nanoballs (DNBs). Sequencing of DNBs was performed on DNBseq platform. (Ct) values. The normalized protein expression (NPX) values are arbitrary ­(log2 scale) units in which 1 NPX dif- ference equals a two-fold change in protein abundance. Data pre-processing to minimize any technical intra- and inter-assay variation is performed using internal plate controls. Quality control was performed at both sample and protein levels and samples that did not pass QC were excluded. All proteomic data was corrected for age and gender before targeted analysis. In case of LIF where the majority of data were below the lower limit of detection, we chose to use the actual data as was recommended by Olink. Raw data was generated by removing reads mapped to rRNAs. Transcriptomics Peripheral blood mononuclear cells were isolated using whole blood from normouricemic or hyperuricemic controls and from patients with gout by density gra- dient centrifugation using Ficoll-Paque PLUS (Sigma Aldrich). Freshly isolated cells were kept in TRIzol rea- gent (Invitrogen), stored at – 80 °C and were later used for commercial RNA-Seq analysis (Beijing Genomics Institute, BGI, Beijing, China). The integrity of extracted RNA was assessed using Agilent 2100 Bio. Oligo dT magnetic beads were used to capture mRNA from total Read counts were normalized using DESeq2 (Version: DESeq2_1.34.0) median of ratios method using R pack- age (Version: R4.0.4.) and were used for downstream tar- geted gene expression statistical analysis. Proteomics Clean reads were generated using SOAPnuke software (version:v1.5.2) by removing reads with adap- tors, reads with unknown bases > 10% and low-quality reads, defined as reads with a quality score less than 15 in over 50% bases. Clean reads were mapped to human transcriptome assembly GRCh37 (hg19) using bowtie2. Statistical analysis In ex  vivo experiments, Mann-Whitney U test and Welch ANOVA were performed to compare means between groups. For correction of multiple comparisons, Fig. 1  TGF-β mRNA is upregulated in gout patients and correlates to serum urate levels in the discovery cohort. PBMCs from patients with gout and matched healthy controls (HC) were isolated and adhered to a flat-bottom plate, mRNA was isolated and compared to the mean dCT of healthy controls by Mann-Whitney U tests *p < 0.05 (A, B). Serum urate levels were correlated to relative mRNA expression levels and analysed by Spearman’s correlation (C, D) Fig. 1  TGF-β mRNA is upregulated in gout patients and correlates to serum urate levels in the discovery cohort. PBMCs from patients with gout and matched healthy controls (HC) were isolated and adhered to a flat-bottom plate, mRNA was isolated and compared to the mean dCT of healthy controls by Mann-Whitney U tests *p < 0.05 (A, B). Serum urate levels were correlated to relative mRNA expression levels and analysed by Spearman’s correlation (C, D) Klück et al. Arthritis Research & Therapy (2023) 25:30 Page 5 of 12 Klück et al. Arthritis Research & Therapy (2023) 25:30 Klück et al. Arthritis Research & Therapy Games-Howell correction was employed for proteomic data (n > 50 within each group) and Tamhane T2 for tran- scriptomics. Spearman analyses were used for correla- tions. For in in vitro experiments, Wilcoxon signed rank tests were employed to compare means. Differences with adjusted p-value < 0.05 were considered statistically sig- nificant. All analyses were done in GraphPad Prism 5. unstimulated PBMCs from individuals with normour- icemia, hyperuricemia and patients with gout (HINT study). Within the group of gout patients we observed no differences between intercritical and flaring patients. We observed no significant difference in receptor expression or TGFB1, ITGAV and SMAD7, but the downstream tar- get MMP9 was increased in patients with gout compared to controls (Fig. 2A-D). For further assessment on a protein level, serum TGF- β-LAP and two downstream targets LIF [27] and VEGFA [28] levels were determined in the same individuals with normouricemia, hyperuricemia and gout. All were signifi- cantly higher in hyperuricemic individuals compared to controls (Fig. 3A–C). Serum LIF was significantly higher in gout patients during gout flare compared to intercriti- cal gout. Statistical analysis However, for TGF-β-LAP no differences were observed between controls and patients with gout, suggest- ing they are more related to high urate levels than to gout. To test for this hypothesis, TGF-β-LAP was correlated with serum urate levels. In line with the results observed in the discovery experiments, serum urate levels were positively correlated with serum TGF-β-LAP in all cohorts combined (Pearson’s correlation = 0,19; p < 0,0001; Fig. 3D). TGF‑β is elevated in hyperuricemic individuals and correlates positively to serum urate levels TGF‑β is elevated in hyperuricemic individuals and correlates positively to serum urate levels As a first exploration, mRNA levels of TGFB1, TGFBRI, TGFBRII and three TGF-β target genes ITGAV, MMP9 and SMAD7 were compared between untreated adher- ent monocytes of patients with gout and age and sex matched healthy controls. Expression levels of TGFB1 were increased in individuals with gout compared to controls, and ITGAV mirrored this expression of TGFB1 (Fig. 1A, B). We identified no change for TGFBRI, TGF- BRII, MMP9 and SMAD7. Moreover, serum urate lev- els correlated positively to TGFB1 mRNA expression in patients with gout (Fig. 1C).i To validate these findings in a larger cohort, mRNA expression of the same genes (TGFB1, TGFBRI, TGF- BRII, ITGAV, MMP9 and SMAD7) was assessed in Fig. 2  mRNA expression of TGF-β and downstream targets in PBMCs from individuals with hyperuricemia or gout. PBMCs from individuals with normoruricemia (n = 110), hyperuricemia (n = 22) and gout (n = 72) of which 15 flaring (marked in red) were isolated and transcriptomics were analysed. Relative mRNA expression of TGFB1 (A), MMP9 (B), ITGAV (C) and SMAD7 (D) are shown. Lines represent means with SD. Means were compared by Welch ANOVA with Tamhane’s T2 multiple comparisons test. **p < 0.01 Fig. 2  mRNA expression of TGF-β and downstream targets in PBMCs from individuals with hyperuricemia or gout. PBMCs from individuals with normoruricemia (n = 110), hyperuricemia (n = 22) and gout (n = 72) of which 15 flaring (marked in red) were isolated and transcriptomics were analysed. Relative mRNA expression of TGFB1 (A), MMP9 (B), ITGAV (C) and SMAD7 (D) are shown. Lines represent means with SD. Means were compared by Welch ANOVA with Tamhane’s T2 multiple comparisons test. **p < 0.01 Page 6 of 12 (2023) 25:30 Klück et al. Arthritis Research & Therapy (2023) 25:30 Klück et al. Arthritis Research & Therapy Fig. 3  Serum TGF-β-LAP, LIF and VEGF-A levels are elevated in individuals with hyperuricemia and TGF-β-LAP correlates positively to serum urate. Serum proteins were analysed by Olink proteomics panel. Flaring gout patients (n = 36) are marked in red dots. NPX was shown for TGF-β-LAP (A), LIF (B) and VEGF-A (C). Means were compared by Welch ANOVA with Games-Howell’s multiple comparisons test. *p < 0.05, **p < 0.01, ***p < 0.001. Spearman correlation was used to analyse the correlation between serum TGF-β-LAP to urate levels (D). TGF‑β is elevated in hyperuricemic individuals and correlates positively to serum urate levels Serum LIF levels were significantly higher in flaring gout patients compared to intercritical gout patients (Welch’s t-test p < 0.0001) Fig. 3  Serum TGF-β-LAP, LIF and VEGF-A levels are elevated in individuals with hyperuricemia and TGF-β-LAP correlates positively to serum urate. Serum proteins were analysed by Olink proteomics panel. Flaring gout patients (n = 36) are marked in red dots. NPX was shown for TGF-β-LAP (A), LIF (B) and VEGF-A (C). Means were compared by Welch ANOVA with Games-Howell’s multiple comparisons test. *p < 0.05, **p < 0.01, ***p < 0.001. Spearman correlation was used to analyse the correlation between serum TGF-β-LAP to urate levels (D). Serum LIF levels were significantly higher in flaring gout patients compared to intercritical gout patients (Welch’s t-test p < 0.0001) Urate induces TGF‑β signalling in vitro To assess the functional consequences of enhanced TGF-β signalling, in vitro priming experiments inves- tigating the combined effects of urate and TGF-β on cytokine production were performed. Human mono- cytes from healthy volunteers were treated with urate, TGF-β or a combination of the two for 24 h, washed and subsequently stimulated with LPS. Cytokine release was measured in supernatant. Both TGF-β and urate priming increased the release of IL-1β and IL-6. Whereas urate lowered IL-1Ra release, TGF-β had no effect on the production of IL-1Ra. Interestingly, we observed a small additive effect, but no synergistic effect between TGF-β and urate (Fig. 5).h To explore whether urate may drive TGF-β expression or production, further in vitro studies were performed. Human primary monocytes isolated from healthy vol- unteers treated with urate showed no elevated TGFB1 mRNA or TGF-β1 protein production after 24  h as assessed by qPCR, ELISA and luciferase bioassay (Sup- plemental Figures 1, 2, 3). Interestingly, in monocytes treated with urate, mRNA expression of MMP9, which can activate latent TGF-β to its active form, was upreg- ulated. Moreover, the expression of SMAD7, a nega- tive regulator of TGF-β signalling, was significantly downregulated compared to RPMI control condition (Supplemental Figure  1). To further assess intracel- lular TGF-β signalling, C-terminally phosphorylated SMAD2 was measured in urate and/or TGF-β treated monocytes. Strikingly, both urate and TGF-β induced C-terminal phosphorylation of SMAD2 showing that intracellularly TGF-β signalling was more active (Fig. 4). The lack of synergy between TGF-β and urate prim- ing led us to hypothesize that the urate-induced inflam- matory phenotype of the monocytes is mediated via TGF-β. Therefore, we primed human monocytes with urate in the presence of an antibody against the TGF-β receptor II. Blocking the TGF-β receptor II partly Page 7 of 12 Klück et al. Arthritis Research & Therapy (2023) 25:30 Page 7 of 12 Klück et al. Arthritis Research & Therapy (2023) 25:30 Fig. 4  Protein expression of pSMAD2 (Ser465/467) in urate primed monocytes. The monocytes were primed overnight with no, 6.25 or 12.5 mg/dL urate followed with a stimulation of 1 ng/mL TGFβ for 1 h. Cell lysates were used for western blotting. Relative pSMAD2 expression (n = 7) (A) and a representative blot (B) are shown. Wilcoxon signed-rank test was used to compare means. *p < 0.05 Fig. 4  Protein expression of pSMAD2 (Ser465/467) in urate primed monocytes. Urate induces TGF‑β signalling in vitro The monocytes were urate followed with a stimulation of 1 ng/mL TGFβ for 1 h. Cell lysates were used for western blotting representative blot (B) are shown. Wilcoxon signed-rank test was used to compare means. *p < 0.05 Fig. 4  Protein expression of pSMAD2 (Ser465/467) in urate primed monocytes. The monocytes were primed overnight with no, 6.25 or 12.5 mg/dL urate followed with a stimulation of 1 ng/mL TGFβ for 1 h. Cell lysates were used for western blotting. Relative pSMAD2 expression (n = 7) (A) and a representative blot (B) are shown. Wilcoxon signed-rank test was used to compare means. *p < 0.05 induced elevated IL-1β production can be partly reversed by blocking TGF-β and several TGF-β receptor blockers ameliorate the urate-induced reduction of the monocyte IL-1Ra production. reversed the urate induced phenotype. This was shown by the fact that IL-1β production was greatly reduced and that IL-1Ra levels were partly restored (Fig. 6A). Also, SB-505124, a kinase inhibitor of TGF-β I recep- tors ALK 4, 5, and 7, inhibited urate induced IL-1β and restored IL-1Ra (Fig. 6B). In addition, 5Z-7-oxozeaenol, a TGF-β-activated kinase 1 inhibitor (TAK1), reduced urate-induced IL-1β, but did not affect IL-1Ra (Fig. 6C). Together, these findings pinpoint TGF-β as a potential mediator in urate-induced pro-inflammatory pheno- type of human primary monocytes. In this study, we explored the expression levels of TGFB1, its two receptors TGFBRI and TGFBRII and three TGF-β target genes ITGAV, MMP9 and SMAD7, the latter being a negative regulator of TGF-β signalling. In two different groups, we  observed either an increased expression of TGF-β itself in gout patients or an increase in TGF-β-LAP protein in hyperuricemic individuals. Also, the expression of the downstream targets ITGAV and MMP9 were  increased in gout patients. Discussion In our in  vitro  setting,  we observed no change to TGFB1 expression, a decrease in TGFBRI and SMAD7 expression, while MMP9 expression was again increased after treatment of PBMC with soluble urate for 24  h. Although both findings point towards enhanced TGF-β signalling pathway, prolonged exposure of PBMCs to ele- vated urate levels in vivo results to different TGF-β sig- nalling kinetics compared to 24 h urate priming in vitro. By showing SMAD2 phosphorylation and blocking In this study, we assessed TGF-β in the context of hyper- uricemia  and gout and found that there is a role for TGF-β in urate-induced pro-inflammatory status of monocytes. In two different populations, TGF-β was elevated in subjects with hyperuricemia or gout and cor- related to serum urate concentrations. In  vitro, urate exposure did not directly induce TGF-β transcription or protein release in human monocytes but did induce C-terminally phosphorylated SMAD2. Moreover, urate Page 8 of 12 Page 8 of 12 Klück et al. Arthritis Research & Therapy Klück et al. Arthritis Research & Therapy (2023) 25:30 (2023) 25:30 Fig. 5  Both TGF-β and urate demonstrate pro-inflammatory effects in a priming model without a synergistic effect. Adherent monocytes isolated from healthy volunteers (n = 6) were treated with dose-ranging concentrations of recombinant TGF-β and/or urate (50 mg/dL) for 24 h after which cells were washed and stimulated with LPS (10 ng/mL) for 24 h. IL-1β (A), IL-6 (B) and IL-Ra (C) were measured in the supernatant after 48 h culture flares. In contrast, we observed no differences in TGFB1 expression or serum TGF-β-LAP within our gout cohort between patients during gout flare and intercritical gout patients. Possibly, TGF-β has a local effect at the site of arthritis but does not result in changes in serum protein or transcripts in circulating PBMCs. However, serum LIF protein was significantly elevated in gout patients dur- ing flare. Apart from TGF-β, LIF can also be induced by IL-1β during arthritis [33, 34], which could account for these differences. In  vitro, we found that co-incubation with LPS, MSU crystals and TGF-β reduces pro-inflam- matory cytokines (data not shown). However, priming monocytes with TGF-β before stimulation with LPS has pro-inflammatory effects similar to urate priming. A pro-inflammatory role for TGF-β has previously been described in adaptive immunity where TGF-β is a key regulator of T helper 17 differentiation. Discussion f Confirming our in  vivo findings, a positive correla- tion between serum TGF-β and urate was also observed in patients with coronary artery disease [35]. This raises the question what the functional consequences are of elevated urate and TGF-β in humans. In mice, hyper- uricemia induces TGF-β in renal tubular tissue [36, 37]. In parallel, allopurinol withdrawal in patients with chronic kidney disease leads to worsening of hyperten- sion, acceleration of the rate of loss of kidney function and an increase in the urinary excretion of TGF-β [38]. The observed increase in fibrosis by urate is not limited to renal disorders. Serum urate has been described as being predictive of pulmonary arterial hypertension, a severe complication in patients with systemic sclerosis [39]. Moreover, Febuxostat, a urate-lowering drug, was shown to suppress angiotensin II-induced aortic fibrosis in mice [40]. We observed that the pro-inflammatory effects of urate were partly mediated by TGF-β. Treating monocytes with both urate and TGF-β had no obvious synergistic effect on cytokine production. Potentially, this could be explained by the presence of human serum in the culture medium which accounts for 3–4 ng/mL TGF-β already. Blocking the TGF-β pathway with several inhibitors reduced the production of IL-1β in human monocytes after stimulation with LPS. Combined with the observed SMAD phosphorylation, this suggests urate activates TGF-β signalling. One of the possible underlying mecha- nisms is that urate activates the TGF-β activated kinase 1 (TAK1), an important kinase in the TGF-β pathway. Uric acid molecules are capable of arresting TAK1 in an active-state conformation, resulting in sustained TAK1 kinase activation [41]. Both TGF-β induced SMAD2/3 and SMAD1/5 phosphorylation are mediated by TAK1 kinase activity. By using different inhibitors with each slightly different targets in the TGF-β signalling cascade, Fig. 5  Both TGF-β and urate demonstrate pro-inflammatory Fig. 5  Both TGF-β and urate demonstrate pro-inflammatory effects in a priming model without a synergistic effect. Adherent monocytes isolated from healthy volunteers (n = 6) were treated with dose-ranging concentrations of recombinant TGF-β and/or urate (50 mg/dL) for 24 h after which cells were washed and stimulated with LPS (10 ng/mL) for 24 h. IL-1β (A), IL-6 (B) and IL-Ra (C) were measured in the supernatant after 48 h culture TGF-β in vitro, we were still able to demonstrate its rel- evance in urate-induced inflammatory phenotype. TGF-β in vitro, we were still able to demonstrate its rel- evance in urate-induced inflammatory phenotype. Discussion Previously, TGF-β was studied in the context of gout. MSU crystals induce TGF-β in macrophages [29] and exogenous TGF-β inhibits MSU-crystal induced inflam- mation in  vivo [30]. In synovial fluid, TGF-β1 is sig- nificantly elevated in acute gouty arthritis compared to osteoarthritis [31] and increases during duration of gout flare [32]. These data combined suggests an anti-inflam- matory role for TGF-β in the resolution phase of gout l Previously, TGF-β was studied in the context of gout. MSU crystals induce TGF-β in macrophages [29] and exogenous TGF-β inhibits MSU-crystal induced inflam- mation in  vivo [30]. In synovial fluid, TGF-β1 is sig- nificantly elevated in acute gouty arthritis compared to osteoarthritis [31] and increases during duration of gout flare [32]. These data combined suggests an anti-inflam- matory role for TGF-β in the resolution phase of gout Page 9 of 12 Klück et al. Arthritis Research & Therapy Klück et al. Arthritis Research & Therapy (2023) 25:30 Page 9 of 12 Klück et al. Arthritis Research & Therapy (2023) 25:30 we could potentially pinpoint the pathways effected by urate. SB-505124, targeting ALK5, inhibits gene expres- sion downstream of both pSMAD2/3 and pSMAD1/5, whereas (5Z)-7-Oxozeaenol, a TAK1 inhibitor, does so [42]. In our in  vitro urate priming models, SB-505124 both reduced IL-1β and restored IL-1Ra, whereas (5Z)- 7-Oxozeaenol only inhibited IL-1β without affecting IL-1Ra. Possibly the difference in target gene expression Fig. 6  Blocking TGF-β signalling pathway partly reverses urate priming effects. Adherent monocytes isolated from healthy volunteers (An = 10; B-C n = 6) were treated with dose-ranging concentrations of urate (50 mg/dL) in the presence or absence of a TGF-β inhibitor for 24 h after which cells were washed and stimulated with LPS (10 ng/mL) for 24 h. IL-1β and IL-Ra were measured in the supernatant after 48 h culture. TGF-β inhibitors: a blocking antibody against TGF-β receptor II with mouse IgG1 as the isotype control (10 μg/mL), SB-505124 (5 μM) and 5Z-7-oxozeaenol (100nM) both with DMSO as solvent control. Wilcoxon signed rank test was applied to compare means. *p < 0.05 Fig. 6  Blocking TGF-β signalling pathway partly reverses urate priming effects. Adherent monocytes isolated from healthy volunteers (An = 10; B-C n = 6) were treated with dose-ranging concentrations of urate (50 mg/dL) in the presence or absence of a TGF-β inhibitor for 24 h after which cells were washed and stimulated with LPS (10 ng/mL) for 24 h. Conclusions In conclusion, TGF-β is elevated in individuals with hyperuricemia correlating to serum urate levels and the urate induced pro-inflammatory phenotype in human monocytes is mediated by TGF-β signalling. Future stud- ies are warranted to explore the intracellular pathways involved and to assess the clinical significance of the rela- tion between serum urate and TGF-β. Abbreviations TGF Transforming growth factor TAK1 TGF-β-activated kinase 1 inhibitor IL Interleukin IL-1Ra Interleukin 1 receptor antagonist PBMC Peripheral blood mononuclear cells mRNA Messenger ribonucleic acid SMAD An acronym from the fusion of Caenorhabditis elegans Sma genes and the Drosophila Mad, mothers against decapentaplegic MSU Monosodium urate TLR Toll-like receptor LAP Latency-associated peptide MMP Matrix metallopeptidase ITGAV Integrin subunit alpha V LPS Lipopolysaccharide PCR Polymerase chain reaction LIF Leukemia inhibitory factor VEGF Vascular endothelial growth factor Another unexplored mechanism of enhanced TGF-β signalling in urate treated monocytes is the regulation of integrin αvβ8. Human ­CD14+ monocytes activate TGF-β via the expression of the integrin αvβ8 and matrix metal- loproteinase 14 [44]. Since TGF-β is always secreted as a latent complex, the function of TGF-β in the regulation of immune responses is controlled by mechanisms that regulate latent TGF-β activation. Finally, the cellular responses to TGF-β have been shown to be altered by pro-inflammatory cytokines such as IL-1β. In chondrocytes, TGF-β induced SMAD7 could be reversed by IL-1β treatment [45]. Likewise, we observed a significant reduction in SMAD7 after urate treatment which is known to lower IL-1Ra expression. Potentially, not urate itself, but a reduction in IL-1Ra could modulate the cellular response to TGF-β similar to IL-1β. In our priming model, blocking TGF-β signal- ling with SB-505124 enhanced IL-1Ra release independ- ent of urate, further suggesting a link between IL-1Ra and TGF-β signalling. Supplementary Information The online version contains supplementary material available at https://​doi.​ org/​10.​1186/​s13075-​023-​03001-1. Additional file 1: Table S1. Primer sequences ex vivo experiments. Table S2. Primary antibodies for western blot. Table S3. Secondary antibodies for western blot. Figure S1. mRNA expression of genes in the TGF-β signalling pathway in adherent monocytes treated with urate in vitro. PBMCs of healthy volunteers were isolated, adhered to a flat- bottom plated and cultured in medium supplemented with 10% HPS with dose-ranging concentrations of urate. mRNA was isolated after 24h and compared to control condition by Wilcoxon matched-pairs signed rank test. *p < 0.05, **p < 0.01. Figure S2. Urate does not increase TGF-β release of human monocytes. Acknowledgements We would like to thank Dr. Sander van Leuven (Department of Rheumatology, Radboudumc) for his input in this project. Also, a special thanks to all patients and volunteers who donated blood for this study. Consortia HINT consortium: Leo A. B. Joosten, Ioan V. Pop, Radu A. Popp, Simona Rednic, Cristina Pamfil, Tania O. Crişan, Marius Farcaş†, Dragoş H. Marginean, Orsolya I. Gaal, Medeea O. Badii, Ioana Hotea, Loredana Peca, Andreea-Manuela Mirea, Georgiana Cabău, Valentin Nica, Doina Colcear, Mariana S. Pop, Ancuta Rus Discussion IL-1β and IL-Ra were measured in the supernatant after 48 h culture. TGF-β inhibitors: a blocking antibody against TGF-β receptor II with mouse IgG1 as the isotype control (10 μg/mL), SB-505124 (5 μM) and 5Z-7-oxozeaenol (100nM) both with DMSO as solvent control. Wilcoxon signed rank test was applied to compare means. *p < 0.05 [42]. In our in  vitro urate priming models, SB-505124 both reduced IL-1β and restored IL-1Ra, whereas (5Z)- 7-Oxozeaenol only inhibited IL-1β without affecting IL-1Ra. Possibly the difference in target gene expression accounts for this observed difference. we could potentially pinpoint the pathways effected by urate. SB-505124, targeting ALK5, inhibits gene expres- sion downstream of both pSMAD2/3 and pSMAD1/5, whereas (5Z)-7-Oxozeaenol, a TAK1 inhibitor, does so to a lesser extent and in a more limited number of genes Page 10 of 12 Page 10 of 12 Klück et al. Arthritis Research & Therapy (2023) 25:30 Klück et al. Arthritis Research & Therapy (2023) 25:30 Another possible intracellular mechanism involved in urate and TGF-β priming is the PI3K/Akt/mTOR path- way. Although we demonstrated pSMAD2 involvement, TGF-β can also activate PI3K resulting in phosphoryla- tion of Akt, independently of SMADs [43]. Similarly, urate priming induces pAkt in monocytes which was reversed by a PI3K inhibitor [15]. Both urate and TGF-β activate the mammalian target of rapamycin (mTOR) via PI3K/Akt pathway, thereby presumably inhibiting autophagy. Since urate induces phosphorylation of Akt within 15 min, it is uncertain whether PI3K activation is mediated via TGF-β. Further research should explore whether these shared pathways are regulated dependent or independent of each other. Conclusions PBMCs were isolated from healthy volunteers and adherent monocytes were primed for 24 hours in RPMI supplemented with 10% human pool serum with or without urate. TGF-β was measured in the supernatant by ELISA (R&D standard). Figure S3. Urate does not affect TGF-β bioactivity. PBMCs were isolated from healthy volunteers and adherent monocytes were primed for 24 hours in RPMI supplemented with 10% human pool serum with or without urate. Supernatant was used in a CAGA​12-luciferase bioassay. β The unexplored intracellular signalling is a clear limi- tation of this study. Moreover, the consequence of ele- vated TGF-β was only evaluated in vitro. Exploring the clinical relevance of urate induced changes in TGF-β signalling is not only important for gout but also for other rheumatic diseases as osteoarthritis and systemic sclerosis. In osteoarthritis, changes in in TGF-β signal- ling are known to contribute to the pathogenesis [46], and serum urate was also identified as a risk factor for symptomatic knee osteoarthritis and joint space nar- rowing [47, 48]. Elevated serum urate is also associated with increased risk for pulmonary arterial hypertension in patients with systemic sclerosis, a complex connec- tive tissue disease characterized by inflammation, vas- culopathy and excessive fibrosis, meditated by TGF-β [39, 49]. References 23. McCartney-Francis N, Mizel D, Wong H, Wahl L, Wahl S. TGF-beta regulates production of growth factors and TGF-beta by human peripheral blood monocytes. Growth Factors. 1990;4(1):27–35. 1. Shiozawa A, Szabo SM, Bolzani A, Cheung A, Choi HK. Serum uric acid and the risk of incident and recurrent gout: a systematic review. J Rheumatol. 2017;44(3):388–96. 1. Shiozawa A, Szabo SM, Bolzani A, Cheung A, Choi HK. Serum uric acid and the risk of incident and recurrent gout: a systematic review. J Rheumatol. 2017;44(3):388–96. 24. Wahl SM, Hunt DA, Wakefield LM, McCartney-Francis N, Wahl LM, Roberts AB, et al. Transforming growth factor type beta induces monocyte chemotaxis and growth factor production. Proc Natl Acad Sci U S A. 1987;84(16):5788–92. 2. Joosten LA, Netea MG, Mylona E, Koenders MI, Malireddi RK, Oost- ing M, et al. Engagement of fatty acids with Toll-like receptor 2 drives interleukin-1beta production via the ASC/caspase 1 pathway in monoso- dium urate monohydrate crystal-induced gouty arthritis. Arthritis Rheum. 2010;62(11):3237–48. 2. Joosten LA, Netea MG, Mylona E, Koenders MI, Malireddi RK, Oost- ing M, et al. Engagement of fatty acids with Toll-like receptor 2 drives interleukin-1beta production via the ASC/caspase 1 pathway in monoso- dium urate monohydrate crystal-induced gouty arthritis. Arthritis Rheum. 2010;62(11):3237–48. 25. Chantry D, Turner M, Abney E, Feldmann M. Modulation of cytokine production by transforming growth factor-beta. J Immunol. 1989;142(12):4295–300. 3. So A, Dumusc A, Nasi S. The role of IL-1 in gout: from bench to bedside. Rheumatology (Oxford). 2018;57(suppl_1):i12–i9. 26. Assarsson E, Lundberg M, Holmquist G, Bjorkesten J, Thorsen SB, Ekman D, et al. Homogenous 96-plex PEA immunoassay exhibiting high sensitiv- ity, specificity, and excellent scalability. PLoS One. 2014;9(4):e95192. 4. Dalbeth N, Gosling AL, Gaffo A, Abhishek A. Gout. Lancet. 2021;397(10287):1843–55. 4. Dalbeth N, Gosling AL, Gaffo A, Abhishek A. Gout. Lancet. 5. Kuo CF, Grainge MJ, Zhang W, Doherty M. Global epidemiology of gout: prevalence, incidence and risk factors. Nat Rev Rheumatol. 2015;11(11):649–62. 27. Humbert L, Ghozlan M, Canaff L, Tian J, Lebrun J-J. The leukemia inhibi- tory factor (LIF) and p21 mediate the TGFβ tumor suppressive effects in human cutaneous melanoma. BMC Cancer. 2015;15(1):200. 6. Athyros VG, Mikhailidis DP. Uric acid, chronic kidney disease and type 2 diabetes: a cluster of vascular risk factors. J Diabetes Complications. 2014;28(2):122–3. 6. Athyros VG, Mikhailidis DP. Uric acid, chronic kidney disease and type 2 diabetes: a cluster of vascular risk factors. J Diabetes Complications. 2014;28(2):122–3. 28. Availability of data and materials The datasets supporting the conclusions of this article are included within the article and its additional file. 16. Kottgen A, Albrecht E, Teumer A, Vitart V, Krumsiek J, Hundertmark C, et al. Genome-wide association analyses identify 18 new loci associated with serum urate concentrations. Nat Genet. 2013;45(2):145–54. Received: 12 May 2022 Accepted: 29 January 2023 22. Naiki Y, Michelsen KS, Zhang W, Chen S, Doherty TM, Arditi M. Transform- ing growth factor-β differentially inhibits MyD88-dependent, but not TRAM- and TRIF-dependent, lipopolysaccharide-induced TLR4 signaling*. J Biol Chem. 2005;280(7):5491–5. Funding This work was supported by a Competitiveness Operational Programme grant of the Romanian Ministry of European Funds (P_37_762, MySMIS 103587). VK was supported by a 2016 Radboudumc Master-PhD grant. These funding sources had no role in the design, collection, analysis, interpretation of data nor in writing the manuscript. 14. Badii M, Gaal OI, Cleophas MC, Klück V, Davar R, Habibi E, et al. Urate- induced epigenetic modifications in myeloid cells. Arthritis Res Ther. 2021;23(1):202. 15. Crisan TO, Cleophas MCP, Novakovic B, Erler K, van de Veerdonk FL, Stunnenberg HG, et al. Uric acid priming in human monocytes is driven by the AKT-PRAS40 autophagy pathway. Proc Natl Acad Sci U S A. 2017;114(21):5485–90. Consent for publication Not applicable 19. Derynck R, Zhang YE. Smad-dependent and Smad-independent path- ways in TGF-beta family signalling. Nature. 2003;425(6958):577–84. Declarations 17. Nold MF, Nold-Petry CA, Zepp JA, Palmer BE, Bufler P, Dinarello CA. IL-37 is a fundamental inhibitor of innate immunity. Nat Immunol. 2010;11(11):1014–22. Authors’ contributions VK, LM, FB, LE and AC performed the in vitro experiments. GC, the HINT consortium and TOC designed, performed and collected all the data from Page 11 of 12 Klück et al. Arthritis Research & Therapy (2023) 25:30 Klück et al. Arthritis Research & Therapy (2023) 25:30 the cohort of individuals with normouricemia, hyperuricemia and gout. RB was involved in the acquisition of patient material. VK, AC, TOC and LABJ the cohort of individuals with normouricemia, hyperuricemia and gout. RB was involved in the acquisition of patient material. VK, AC, TOC and LABJ contributed to the design of the work and interpretation of data. VK and GC drafted the manuscript. All authors revised the manuscript and approved the submitted version. the cohort of individuals with normouricemia, hyperuricemia and gout. RB was involved in the acquisition of patient material. VK, AC, TOC and LABJ cytokine production by human primary cells via inhibition of IL-1Ra. Ann Rheum Dis. 2016;75(4):755–62. 12. Cabau G, Crisan TO, Kluck V, Popp RA, Joosten LAB. Urate-induced immune programming: consequences for gouty arthritis and hyperurice- mia. Immunol Rev. 2020;294(1):92–105. contributed to the design of the work and interpretation of data. VK and GC drafted the manuscript. All authors revised the manuscript and approved the submitted version. 13. Joosten LAB, Crisan TO, Bjornstad P, Johnson RJ. Asymptomatic hyperuri- caemia: a silent activator of the innate immune system. Nat Rev Rheuma- tol. 2020;16(2):75–86. Competing interests 20. Taylor AW. Review of the activation of TGF-beta in immunity. J Leukoc Biol. 2009;85(1):29–33. The authors declare that they have no competing interests. 21. Li MO, Wan YY, Sanjabi S, Robertson A-KL, Flavell RA. Transforming growth factor-β regulation of immune responses. Annu Rev Immunol. 2006;24(1):99–146. Received: 12 May 2022 Accepted: 29 January 2023 Received: 12 May 2022 Accepted: 29 January 2023 Received: 12 May 2022 Accepted: 29 January 2023 Ethics approval and consent to participate All ethics approval and consent are described in the methods section of this manuscript. 18. Kluck V, van Deuren RC, Cavalli G, Shaukat A, Arts P, Cleophas MC, et al. Rare genetic variants in interleukin-37 link this anti-inflammatory cytokine to the pathogenesis and treatment of gout. Ann Rheum Dis. 2020;79(4):536–44. References Arthritis Rheumatol. 2017;69(6):1213–20. 49. van Caam A, Vonk M, van den Hoogen F, van Lent P, van der Kraan P. Unraveling SSc pathophysiology; the myofibroblast. Front Immunol. 2018;9:2452. 49. van Caam A, Vonk M, van den Hoogen F, van Lent P, van der Kraan P. Unraveling SSc pathophysiology; the myofibroblast. Front Immunol. 2018;9:2452. • fast, convenient online submission • thorough peer review by experienced researchers in your field • rapid publication on acceptance • support for research data, including large and complex data types • gold Open Access which fosters wider collaboration and increased citations maximum visibility for your research: over 100M website views per year • At BMC, research is always in progress. Learn more biomedcentral.com/submissions Ready to submit your research Ready to submit your research ? Choose BMC and benefit from: ? Choose BMC and benefit from: • fast, convenient online submission • thorough peer review by experienced researchers in your field • rapid publication on acceptance • support for research data, including large and complex data types • gold Open Access which fosters wider collaboration and increased citations maximum visibility for your research: over 100M website views per year • At BMC, research is always in progress. Learn more biomedcentral.com/submissions Ready to submit your research Ready to submit your research ? Choose BMC and benefit from: ? Choose BMC and benefit from: References 35. Chen C, Lei W, Chen W, Zhong J, Gao X, Li B, et al. Serum TGF-beta1 and SMAD3 levels are closely associated with coronary artery disease. BMC Cardiovasc Disord. 2014;14:18. 36. Alghamdi YS, Soliman MM, Nassan MA. Impact of lesinurad and allopuri- nol on experimental hyperuricemia in mice: biochemical, molecular and immunohistochemical study. BMC Pharmacol Toxicol. 2020;21(1):10. y 37. Kim SM, Choi YW, Seok HY, Jeong KH, Lee SH, Lee TW, et al. Reduc- ing serum uric acid attenuates TGF-beta1-induced profibrogenic progression in type 2 diabetic nephropathy. Nephron Exp Nephrol. 2012;121(3-4):e109–21. 38. Talaat KM, el-Sheikh AR. The effect of mild hyperuricemia on urinary transforming growth factor beta and the progression of chronic kidney disease. Am J Nephrol. 2007;27(5):435–40. p 39. Coghlan JG, Denton CP, Grunig E, Bonderman D, Distler O, Khanna D, et al. Evidence-based detection of pulmonary arterial hypertension in systemic sclerosis: the DETECT study. Ann Rheum Dis. 2014;73(7):1340–9. y 40. Kondo M, Imanishi M, Fukushima K, Ikuto R, Murai Y, Horinouchi Y, et al. Xanthine oxidase inhibition by febuxostat in macrophages suppresses angiotensin II-induced aortic fibrosis. Am J Hypertens. 2019;32(3):249–56. 41. Singh AK, Haque M, O’Sullivan K, Chourasia M, Ouseph MM, Ahmed S. Suppression of monosodium urate crystal-induced inflammation by inhibiting TGF-beta-activated kinase 1-dependent signaling: role of the ubiquitin proteasome system. Cell Mol Immunol. 2021;18(1):162–70. 42. van Caam A, Madej W, Garcia de Vinuesa A, Goumans MJ, Ten Dijke P, Blaney Davidson E, et al. TGFβ1-induced SMAD2/3 and SMAD1/5 phos- phorylation are both ALK5-kinase-dependent in primary chondrocytes and mediated by TAK1 kinase activity. Arthritis Res Ther. 2017;19(1):112. 43. Zhang YE. Non-Smad pathways in TGF-beta signaling. Cell Res. 2009;19(1):128–39. 44. Kelly A, Gunaltay S, McEntee CP, Shuttleworth EE, Smedley C, Houston SA, et al. Human monocytes and macrophages regulate immune tolerance via integrin αvβ8-mediated TGFβ activation. J Exp Med. 2018;215(11):2725–36. 45. Roman-Blas JA, Stokes DG, Jimenez SA. Modulation of TGF-β signaling by proinflammatory cytokines in articular chondrocytes. Osteoarthr Cartil. 2007;15(12):1367–77. 46. Thielen NGM, van der Kraan PM, van Caam APM. TGFβ/BMP signaling pathway in cartilage homeostasis. Cells. 2019;8(9):969. 47. Wang S, Pillinger MH, Krasnokutsky S, Barbour KE. The association between asymptomatic hyperuricemia and knee osteoarthritis: data from the third National Health and Nutrition Examination Survey. Osteoarthr Cartil. 2019;27(9):1301–8. 48. Krasnokutsky S, Oshinsky C, Attur M, Ma S, Zhou H, Zheng F, et al. Serum urate levels predict joint space narrowing in non-gout patients with medial knee osteoarthritis. References Shi X, Guo LW, Seedial SM, Si Y, Wang B, Takayama T, et al. TGF-β/Smad3 inhibit vascular smooth muscle cell apoptosis through an autocrine signaling mechanism involving VEGF-A. Cell Death Dis. 2014;5(7):e1317-e. 7. Kim SY, Guevara JP, Kim KM, Choi HK, Heitjan DF, Albert DA. Hyperurice- mia and coronary heart disease: a systematic review and meta-analysis. Arthritis Care Res (Hoboken). 2010;62(2):170–80. 29. Yagnik DR, Evans BJ, Florey O, Mason JC, Landis RC, Haskard DO. Mac- rophage release of transforming growth factor beta1 during resolution of monosodium urate monohydrate crystal-induced inflammation. Arthritis Rheum. 2004;50(7):2273–80. 8. Chaudhary K, Malhotra K, Sowers J, Aroor A. Uric acid - key ingredient in the recipe for cardiorenal metabolic syndrome. Cardiorenal Med. 2013;3(3):208–20. 30. Liote F, Prudhommeaux F, Schiltz C, Champy R, Herbelin A, Ortiz-Bravo E, et al. Inhibition and prevention of monosodium urate monohydrate crystal-induced acute inflammation in vivo by transforming growth fac- tor beta1. Arthritis Rheum. 1996;39(7):1192–8. 9. Feldman N, Rotter-Maskowitz A, Okun E. DAMPs as mediators of sterile inflammation in aging-related pathologies. Ageing Res Rev. 2015;24(Pt A):29–39. 31. Chen Y, Huang Y, Li X, Xu M, Bi Y, Zhang Y, et al. Association of arterial stiffness with HbA1c in 1,000 type 2 diabetic patients with or without hypertension. Endocrine. 2009;36(2):262–7. 10. Disveld IJM, Zoakman S, Jansen T, Rongen GA, Kienhorst LBE, Janssens H, et al. Crystal-proven gout patients have an increased mortality due to cardiovascular diseases, cancer, and infectious diseases especially when having tophi and/or high serum uric acid levels: a prospective cohort study. Clin Rheumatol. 2019;38(5):1385–91. 32. Scanu A, Oliviero F, Ramonda R, Frallonardo P, Dayer JM, Punzi L. Cytokine levels in human synovial fluid during the different stages of acute gout: role of transforming growth factor beta1 in the resolution phase. Ann Rheum Dis. 2012;71(4):621–4. y 11. Crisan TO, Cleophas MC, Oosting M, Lemmers H, Toenhake-Dijkstra H, Netea MG, et al. Soluble uric acid primes TLR-induced proinflammatory Page 12 of 12 Klück et al. Arthritis Research & Therapy (2023) 25:30 Klück et al. Arthritis Research & Therapy (2023) 25:30 33. Enomoto H, Saito S, Yabe H, Toyama Y, Tomatu T. The levels of leukemia inhibitory factor in synovial tissues of patients with rheumatoid arthritis: inflammation and other proinflammatory cytokines. Mod Rheumatol. 2003;13(2):121–8. 34. Lotz M, Moats T, Villiger PM. Leukemia inhibitory factor is expressed in car- tilage and synovium and can contribute to the pathogenesis of arthritis. J Clin Invest. 1992;90(3):888–96. • fast, convenient online submission • thorough peer review by experienced researchers in your field • rapid publication on acceptance • support for research data, including large and complex data types • gold Open Access which fosters wider collaboration and increased citations maximum visibility for your research: over 100M website views per year • At BMC, research is always in progress. Learn more biomedcentral.com/submissions Ready to submit your research Ready to submit your research ? Choose BMC and benefit from: ? Choose BMC and benefit from: Publisher’s Note S N Springer Nature remains neutral with regard to jurisdictional claims in pub- lished maps and institutional affiliations.
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https://europepmc.org/articles/pmc4999641?pdf=render
English
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Extensive Mucocutaneous Verruca Vulgaris in a Nonimmunocompromised Patient
Jaypee's international journal of clinical pediatric dentistry
2,011
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1,736
IJCPD CASE REPORT  10.5005/jp-journals-10005-1084 Extensive Mucocutaneous Verruca Vulgaris in a Nonimmunocompromised Patient 1Vela D Desai, 2Rajeev Sharma, 3Durgesh N Bailoor 1Vela D Desai, 2Rajeev Sharma, 3Durgesh N Bailoor 1Professor and Head, Department of Oral Medicine and Radiology, Jaipur Dental College, Jaipur, Rajasthan, India 2Senior Lecturer, Department Oral Medicine and Radiology, Jaipur Dental College, Jaipur, Rajasthan, India 3Professor, Department of Oral Medicine and Radiology, Karnavati Dental College, Ahmedabad, Gujarat, India Correspondence: Vela D Desai, Professor and Head, D-302, Trimurthy Apartments, Opp. BSNL Telecom Colony, Model Town Malviya Nagar, Jaipur, Rajasthan-302017, India, Fax: +91 1426-234176, e-mail: veladesai@hotmail.com, drsunilmv@hotmail.com ABSTRACT Papilloma virus infections of the oral cavity have been long recognized with various clinical expressions characterized as verruca vulgaris, Heck’s disease, multiple papilloma and condyloma acuminata. In this paper, we are highlighting a case of verruca vulgaris involving the oral cavity with extensive skin lesions in a nonimmunocompromised 9-year-old boy. Different treatment modalities are discussed in this article. Keywords : Verruca vulgaris, Warts, Human papilloma virus. INTRODUCTION Fig. 1: A 9-year-old boy presenting with papillary growths on the forehead, inner canthus of eye, left angle of mouth and on vermilion border of lip Verruca vulgaris is a relatively uncommon oral lesion which is caused by an infectious agent HPV (human papilloma virus). Verruca vulgaris (VV) of oral mucosa is rare and is typically a childhood problem. The lesions of verruca vulgaris are circumscribed, firm, elevated papule with papillomatous hyperkeratotic surface. They occur most commonly on the dorsal aspect of the fingers and hand. They are also found on sole of feet. Lesions of verucca occur extensively in immunocompromised host. Here, we present a case of severe form of verruca in a healthy individual. CASE REPORT A 9-year-old boy reported to the Department of Oral Medicine, Radiology and Diagnosis with multiple, small, whitish growth on his legs, hands, fingers and face (Fig. 1). The first symptom of this appeared 2 year ago. Lesions were asymptomatic except for slight occasional itching. Similar type of lesion also had been noticed in his elder brother and peers, which regressed spontaneously without any treatment. Systemic review was noncontributory. Patient neither suffer from any long-term illness in the past nor was on any medications. On extraoral examination, numerous keratotic, well-defined whitish colored, papules varying in size from 2 to 4 mm were found on dorsum of hand, feet, face, chin and angle of mandible (Figs 1 to 2B). On left hand they were found in groups of more than 20 in number. Some of the lesions on left hand were in a linear fashion and exhibited Koebner phenomenon. Mild discharge of blood was noticed on scraping the lesion (Fig. 3). Fig. 1: A 9-year-old boy presenting with papillary growths on the forehead, inner canthus of eye, left angle of mouth and on vermilion border of lip Examination of the oral cavity revealed whitish solitary lesion on right buccal mucosa with finger like projection and multiple small (4-5 in number) lesions were also appreciable on lower labial mucosa and on alveolar ridge in the region of lower left lateral incisor and canine (Fig. 4). On palpation they were firm in consistency and nontender. Patient had an otherwise good oral hygiene. Examination of other systems was noncontributory. A provisional diagnosis of verruca vulgaris was made. 65 International Journal of Clinical Pediatric Dentistry, January-April 2011;4(1):65-68 8/13/2016 5:09:20 PM Art-12.indd 65 Vela D Desai et al Figs 2A and B: Multiple grayish white lesions on the hands and legs A B A B Figs 2A and B: Multiple grayish white lesions on the hands and legs The patient underwent a complete blood investigation including Elisa test, which were within normal range. Excisional biopsy was performed with the patients parents consent (as he was a minor) from two different sites, i.e. dorsum of hand and intraorally from the right buccal mucosa. Histopathological examination revealed acanthosis with proliferative, hyperkeratotic, stratified squamous epithelium showing finger like projection and elongated rate pegs converging towards the center, producing cupping effect suggestive of verruca vulgaris. There were foci of vacuoled cells referred to as koilocytotic cells. CASE REPORT Vertical tier of parakeratotic cells and foci of clumped keratohyalin granules were also seen (Figs 5A and B). Patient underwent a thorough oral prophylaxis only. Fig. 4: Intraorally small flat lesion on right buccal mucosa Fig. 4: Intraorally small flat lesion on right buccal mucosa DISCUSSION Fig. 3: Coalesced linear occurring lesions showing bleedi A variety of papillary lesions occur in the oral cavity and while many can be differentiated on the basis of histopathological features, they may closely resemble one another clinically1. Verruca vulgaris, condyloma acuminate, Heck’s disease, squamous papilloma are all local papillary lesions that share similar clinical feature yet their microscopic patterns differ.1,2 Verruca vulgaris commonly occurs in children and usually regresses spontaneously over a period of time.2,3 Papilloma virus type 2 and 4 are the most prevalent isolates from cutaneous warts, and these same viral genotypes are identifiable when verruca valgaris arise on the lip or on mucous membrane. Whereas condylomas are usually diagnosed in teenagers and young adults and are more frequently encountered in the mucosa membranes of the anogenital region, it may also be seen in the oral cavity perhaps arising therein as a consequence of oragenital sexual transmission.1 They are bigger in size and appear pink to red as a results of less keratinization. 66 JAYPEE JAYPEE J 8/13/2016 5:09:20 PM 8/13/2016 5:09:20 PM Art-12.indd 66 IJCPD Extensive Mucocutaneous Verruca Vulgaris in a Nonimmunocompromised Patient A reports.4,5 Kirchner has speculated that a certain kind of lesion is more frequently found before puberty and links its presence to a genetic defect that retards the establishment of an effective defense mechanism until puberty leaving some children more susceptible than others to the infection.6 Verruca vulgaris may not require any treatment, but is done for cosmetic purposes and to prevent their spread.7 Most people develop an immune response that causes verruca vulgaris to vanish by themselves.8 The patient’s sibling’s lesions vanished in a similar way and in the present case also, no treatment was initiated as the patient denied any treatment. Skin verruca vulgaris is treated effectively by liquid nitrogen, cryotheraphy, conservative surgical excision or curettage or topical application of keratinolytic agents containing salicylic acid and lactic acid.7,8 Oral lesions are successfully surgically excised or they may be destroyed by laser, cryotheraphy or electrosurgery. Recurrence is seen in a small proportion of treated case. Verruca vulgaris does not transform into malignancy with or without treatment and two third will disappear spontaneously within 2 years especially in children.7 Detailed genetic study should be carried out to rule out any genetic origin in all the suspected cases. Figs 5A and B: Histopathology showing the classical lesion of verucca vulgaris B CONCLUSION Due to paucity of cases of oral verruca vulgaris being reported, information regarding malignant potential is limited. In the current case despite an extensive skin lesion there was no evidence of underlying disease (AIDS/ dysplasia on histopathology). On basis of a single case, it is very difficult to infer that they may occur in nonimmuno- compromised patient and have no malignant potential. More studies need to be carried out to corroborate that they occur in immunocompromised patient. Any patient with presence of extensive verruca vulgaris lesion must be eyed with suspicion of AIDS/compromised immune system or internal malignancy and thorough blood investigation including ELISA/immunohistochemistry/in situ hybridization/ southern blot hybridization should be carried out. Fortunately in our case patient did not have any of this life threatening condition. The association of oral lesion with HPV should be extensively examined to clarify the pathogenesis of HPV infection in oral cavity. B Figs 5A and B: Histopathology showing the classical lesion of verucca vulgaris The case herein represent classical form of oral verruca vulgaris with extensive multiple skin involvement. Such type of exacerbated involvement is usually observed in immunocompromised host.4 There have been very few reported cases of verruca vulgaris in a nonimmuno­ compromised patients. Thus, presented case is unique as there was no underlying immune supression. 1. Eversole LR, Laipis PJ,. Oral squamous papillomas: Detection of HPV DNA by in situ hybridization. Oral Surg Oral Med Oral Pathol 1988 May;65(5):545-550. 2. Sharma G, Nagpal A, Veagal M, Pai K. Multiple Papilloma: A case report and review of literature. J Indian Acad Oral Med Radiol 2007;19(3):384-388. 1. Eversole LR, Laipis PJ,. Oral squamous papillomas: Detection of HPV DNA by in situ hybridization. Oral Surg Oral Med Oral Pathol 1988 May;65(5):545-550. 2. Sharma G, Nagpal A, Veagal M, Pai K. Multiple Papilloma: A case report and review of literature. J Indian Acad Oral Med Radiol 2007;19(3):384-388. International Journal of Clinical Pediatric Dentistry, January-April 2011;4(1):65-68 4. Adler-Storthz K, Newland JR, Tessin BA, Yeudall WA, Shillitoe EJ. Identification of human papilloma virus types in oral verruca vulgaris. J Oral Pathol 1986 Apr;15(4):230-233. 3. Sivapathasundharam B, Shifa S. Oral verruca vulgaris: Report of a rare case. Indian J Dent Res 2004 Jan-Mar;15(1):32-34. 5. Green TL, Eversole LR, Leider AS. Oral and labial verruca vulgaris: Clinical, histological and immunohistochemical evaluation. Oral Surg Oral Med Oral Pathol 1986 Oct:62(4):410-416. 6. Kirchner H. Immunobiology of human papillomavirus infection. Prog Med Virol 1986;33:1-41. 3. Sivapathasundharam B, Shifa S. Oral verruca vulgaris: Report of a rare case. Indian J Dent Res 2004 Jan-Mar;15(1):32-34. 4. Adler-Storthz K, Newland JR, Tessin BA, Yeudall WA, Shillitoe EJ. Identification of human papilloma virus types in oral verruca vulgaris. J Oral Pathol 1986 Apr;15(4):230-233. 5. Green TL, Eversole LR, Leider AS. Oral and labial verruca vulgaris: Clinical, histological and immunohistochemical evaluation. Oral Surg Oral Med Oral Pathol 1986 Oct:62(4):410-416. REFERENCES From clinical observation, we found that the lesions were mainly located on the buccal mucosa, lower labial mucosa, also autoinoculation from a lesion located on hand has been observed. A slightly higher incidence was encountered for OVV in children and young adults and differs from other 2. Sharma G, Nagpal A, Veagal M, Pai K. Multiple Papilloma: A case report and review of literature. J Indian Acad Oral Med Radiol 2007;19(3):384-388. 67 International Journal of Clinical Pediatric Dentistry, January-April 2011;4(1):65-68 Art-12.indd 67 8/13/2016 5:09:21 PM Vela D Desai et al 6. Kirchner H. Immunobiology of human papillomavirus infection. Prog Med Virol 1986;33:1-41. 7. Neville.; B, Damm.; DD, Allen.; CM, et al. In: Oral and maxillofacial pathology. 2nd edition. Saunders: Elsevier, 2002. p. 317-318. Chapter 8. Moondragon Health [Internet]. Available from: http://www. moondragon.org/health/disorders/warts.html 68 JAYPEE Art-12.indd 68 Art-12.indd 68 8/13/2016 5:09:21 PM
https://openalex.org/W2980238500
https://repositum.tuwien.at/bitstream/20.500.12708/20132/1/Schultis-2019-Energies-vor.pdf
English
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Behaviour of Distribution Grids with the Highest PV Share Using the Volt/Var Control Chain Strategy
Energies
2,019
cc-by
18,772
Received: 30 August 2019; Accepted: 10 October 2019; Published: 12 October 2019 Received: 30 August 2019; Accepted: 10 October 2019; Published: 12 October 2019 Abstract: The large-scale integration of rooftop PVs stalls due to the voltage limit violations they provoke, the uncontrolled reactive power flow in the superordinate grids and the information and communications technology (ICT) related challenges that arise in solving the voltage limit violation problem. This paper attempts to solve these issues using the LINK-based holistic architecture, which takes into account the behaviour of the entire power system, including customer plants. It focuses on the analysis of the behaviour of distribution grids with the highest PV share, leading to the determination of the structure of the Volt/var control chain. The voltage limit violations in low voltage grid and the ICT challenge are solved by using concentrated reactive devices at the end of low voltage feeders. Q-Autarkic customer plants relieve grids from the load-related reactive power. The optimal arrangement of the compensation devices is determined by a series of simulations. They are conducted in a common model of medium and low voltage grids. Results show that the best performance is achieved by placing compensation devices at the secondary side of the supplying transformer. The Volt/var control chain consists of two Volt/var secondary controls; one at medium voltage level (which also controls the TSO-DSO reactive power exchange), the other at the customer plant level. Keywords: volt/var control; distribution grid; photovoltaic; L(U)-control; Q-autarky; smart grid Behaviour of Distribution Grids with the Highest PV Share Using the Volt/Var Control Chain Strategy Daniel-Leon Schultis * and Albana Ilo TU Wi I tit t f E S t d El t i l D i 1040 Vi A t i lb il @t i t Daniel-Leon Schultis * and Albana Ilo TU Wien—Institute of Energy Systems and Electrical Drives, 1040 Vienna, Austria; albana.ilo@tuwien.ac.a * Correspondence: daniel-leon.schultis@tuwien.ac.at; Tel.: +43-1-58801-370132 Daniel-Leon Schultis * and Albana Ilo www.mdpi.com/journal/energies energies energies energies www.mdpi.com/journal/energies 1. Introduction Nowadays, climate change has become apparent, not only for scientists but also for everybody [1]. Reducing emissions through the use of renewable sustainable resources while maintaining a reliable and secure electricity supply is becoming increasingly imperative. In this context, the large-scale implementation of Distributed Generation (DG) holds a considerable potential [2]. European utilities supply 260 million customers, of which more than 99% are connected at the Low Voltage (LV) level [3]. The use of inverters available in DGs, e.g., rooftop photovoltaic (PV), to control the voltage in distribution networks [4] has introduced various local control strategies such as cos ϕ (P), Q(U), and so on [5–7] that provoke uncontrolled reactive power flows on the superordinate grids. Implementing PVs on the rooftop of each customer requires the coordination of millions of local controllers needed to operate the power system reliably and securely. A flood of data exchange is necessary that poses serious challenges to ICT, cyber security and data privacy [8–10]. The optimal volt/var management or control is one of the most essential processes for utilities to maintain reliable voltages and to keep the power factor close to one. Currently, they use reactive devices (RDs) to reduce the amount of reactive power flowing through transmission lines and to maintain sufficient reactive power capability in transmission systems [11]. In medium voltage (MV) grids, capacitor banks are mostly used to support the voltage [12]. The uncontrolled reactive power flow in high voltage (HV) and MV levels provoked by the large scale implementation of local control strategies in LV level upsets the current practices [13]. In the process of reactive power management are Energies 2019, 12, 3865; doi:10.3390/en12203865 2 of 23 Energies 2019, 12, 3865 already involved different actors, i.e., transmission (TSO) and distribution system operators (DSO) and customers (as owners of PV inverters). Considering the perspective of individual actors or optimizing individual functionalities may lead to suboptimal solutions and other, difficult to overcome, challenges. In contrast, the holistic architecture vision enables large-scale rollout of the new control paradigms leading to optimal solutions [14]. Volt/var control chain strategy promises the solution of the problem related to voltage control and reactive power management that is of utmost importance to utilities, as they may favor the large scale integration of distributed generation. 2. Materials and Methods This work builds on the LINK-based holistic architecture and the associated strategy of volt/var control chain, the fundamentals of which are outlined below. After that follows the model description of the considered grids and of the simulated control setups. 1. Introduction y y g g g The volt/var control chain strategy that comprises all grid levels, i.e., HV, MV, LV and Customer Plant (CP) grid, located on the vertical axis (Y-axis) of the holistic model “The energy supply chain net” [15] is introduced in [16]. It relies on the LINK-based holistic architecture [17], where the Secondary Control (SC) is the crucial instrument to realize a sustainable and reliable grid operation of the whole power system including CPs. The implementation of secondary control in CP level enables the full reactive power (Q) compensation of the customer plants, leading to Q-self-sufficient or Q-Autarkic customers [18]. The DSO owned reactive devices, e.g., L(U)-controlled coils, installed at LV feeders with voltage limit violation potential, keep the voltage within the limits [19], while providing substantial technical (reduced losses, Q-exchange and Distribution Transformer (DTR) loading [20]; and increased hosting capacity [21]) and social (preserved data privacy and avoided discrimination [20]) benefits compared to the cos ϕ (P)- and Q(U)-control strategies. Further on, the L(U)+Q-Autarky control ensemble provides substantial technical benefits compared to the L(U)-control strategy [18]. All var-local control strategies, i.e., cosϕ (P), Q(U), L(U), etc., provoke uncontrolled reactive power flows in the superordinate voltage level grids inclusive the HV grid. The reactive power margin (RPM) decreases significantly if the DG production comes close to the demand or even exceeds it [22]. The power system approaches a voltage instable situation. This paper examines the effect of additional compensation devices (CDs) on the distribution grid behaviour, when the volt/var control chain strategy that implies the L(U)+Q-autarky control ensemble is used. Furthermore, the optimal link-grid size is investigated for the specific conditions. Section 2 gives an overview of the fundamentals of the volt/var control chain strategy, the description of the customer plant and distribution grid model, and the definition of the simulated control setups. Section 3 presents the investigation results concerning the distribution grid behaviour and the effect of CD placement. Furthermore, a grid-link setup for practical implementation is presented. Finally, in Section 4, some concluding remarks are summarized. 2.1. Fundamentals of Volt/Var Control Chain Strategy SC is the base instrument to realize the volt/var control chain strategy. It is one of the fundamental components of each grid-link [17]. It refers to control actions that are calculated based on the grid-link control area. It fulfils a predefined objective function by respecting the dynamic grid constraints on the grid-link boundaries and the static constraints of electrical appliances (PQ-diagrams of generators, transformer rating, etc.). Dynamic grid constraints are the reactive and active power exchange at the grid-link boundaries that are agreed from the corresponding grid-link operators. The grid-link contains SC-loops for both major entities of power systems—frequency and voltage. This paper deals with the volt/var secondary control (VVSC) schemas. The generalized form of volt/var control chain strategy [16] in the Y-axis of the holistic model “The energy supply chain net” that implies the L(U)+Q-autarky control ensemble [18] is shown in Figure 1. All relevant links are drawn in gold-coloured solid lines, while the neighbour grid-links are indicated by gold-dashed lines. Grid-links 3 of 23 Energies 2019, 12, 3865 are set upon three classical levels: CP, LV and MV level. The automation and communication path is blue, while the power flow path is black. Energies 2019, 12, x FOR PEER REVIEW 3 of 23 Figure 1. The generalized form of the volt/var control chain strategy implying the L(U)+Q-autarky o t ol e e ble Figure 1. The generalized form of the volt/var control chain strategy implying the L(U)+Q-autarky control ensemble. Figure 1. The generalized form of the volt/var control chain strategy implying the L(U)+Q-autarky t l bl Figure 1. The generalized form of the volt/var control chain strategy implying the L(U)+Q-autarky control ensemble. RDs are used in power systems for two purposes: for voltage control (voltage control reactive device – VCRD) or reactive power compensation (compensation device—CD). The corresponding varPCs receive the set points U* and Q* as in Table 1 RDs are used in power systems for two purposes: for voltage control (voltage control reactive device—VCRD) or reactive power compensation (compensation device—CD). The corresponding varPCs receive the set-points U* and Q* as in Table 1. T bl 1 D i id d f lt t l d ti Table 1. Devices considered for voltage-control and var-compensation. Table 1. Devices considered for voltage-control and var-compensation. Device Purpose Set-Point for varPC RD VCRD Voltage control U* CD Var compensation Q* Table 1. Devices considered for voltage-control and var-compensation. 2.1. Fundamentals of Volt/Var Control Chain Strategy Device Purpose Set-Point for varPC RD VCRD Voltage control U* CD Var compensation Q* The main reason to introduce inductive devices on the low voltage grid is to keep the voltage within the limits over the all-time horizon. L(U)s are VCRD, e.g., coils, connected at the end of LV feeders, which may violate the upper voltage limit. The positioning of L(U)s at the LV feeder end shows high effectiveness due to the prevalent high voltage sensitivity ( ∂U ∂Q ⁄ ) [23]. Q-autarky is a special mode of operation of the volt/var secondary control set up in the CP level (VVSCCP ). Neither Q (𝑄஼௉ ௅௏= 0 ) nor Q-data exchange with the LV grid is required when operating the distribution grid with high PV-share. CPs are Q-self-sufficient. The main reason to introduce inductive devices on the low voltage grid is to keep the voltage within the limits over the all-time horizon. L(U)s are VCRD, e.g., coils, connected at the end of LV feeders, which may violate the upper voltage limit. The positioning of L(U)s at the LV feeder end shows high effectiveness due to the prevalent high voltage sensitivity (∂U/∂Q) [23]. Q-autarky is a special mode of operation of the volt/var secondary control set up in the CP level (VVSCCP). Neither Q (QLV CP = 0) nor Q-data exchange with the LV grid is required when operating the distribution grid with high PV-share. CPs are Q-self-sufficient. 2.2. Model Description The customer plant model and the distribution grid model comprising MV_ and LV_link-grids that are used to analyse the behaviour of distribution grids with different control setups are presented below. 2.1. Fundamentals of Volt/Var Control Chain Strategy VVSCLV calculates in real time: (d) the var set-points for the Volt/var secondary controls VVSCMV,LV ngb of all neighbour MV_ or LV_grid-links, while respecting the var constraint varCnsHV MV at the border to the HV_link-grid. VVSCLV calculates in real time: (a) the voltage and var set-points for the primary controls varPCLV RD of all RDs included in the LV_link-grid; (a) the voltage and var set-points for the primary controls varPCLV RD of all RDs included in the LV_link-grid; (b) the var set-points for the primary controls varPCLV DG/DSt of all DGs and DSts connected to the LV_link-grid; LV CP (c) the var set-points for the Volt/var secondary controls VVSCLV,CP ngb of all neighbour LV_ or CP_grid-links, while respecting the var constraint varCnsMV LV at the border to the MV_link-grid. (d) VVSCCP calculates in real time (e) the var set-point for the primary control varPCCP inv of the PV-inverter connected to CP_link-grid; while respecting the var constraint varCnsLV CP at the border to the LV_link-grid. (e) the var set-point for the primary control varPCCP inv of the PV-inverter connected to CP_link-grid; while respecting the var constraint varCnsLV CP at the border to the LV_link-grid. In the generalized form of the VVC chain discussed above, grid-links are set up based on the classical splitting method of the power system structure into HV, MV and LV levels. But, by definition (the link-grid size is variable and is defined from the area, where the secondary-control is set up), the link-grid size is variable. It may apply not only to the classical grid parts but also to a part of the grid, which may include one or more voltage levels together, e.g., MV and LV level [17]. In the following is analysed the effect of the CDs on the distribution grid behaviour, when the Volt/var control chain strategy that implies the L(U)+Q-autarky control ensemble is used to control the voltage and the reactive power flow in distribution grids. It is supposed that the distribution grid is operated by one DSO. The optimal link-grid size for these specific conditions is investigated. The basic principle is keeping the number of secondary and primary control units as low as possible to avoid complex automation schemes. 2.1. Fundamentals of Volt/Var Control Chain Strategy The following generalized equation is introduced for the first time to compactly represent the VVC chain in the Y-axis: The following generalized equation is introduced for the first time to compactly represent the VVC chain in the Y-axis: MV Y-axis = {VVSCMV൫voltPCOLTC MV , varPCRD MV, varPCDG/DSt MV , VVSCngb MV,LV, varCnsMV HV൯, CLV൫varPCRD LV, varPCDG/DSt LV , VVSCngb LV,CP, varCnsLV MV൯, VVSCCP൫varPCinv CP, varCnsCP LV = 0൯} (1) VVCY−axis= {VVSC MV  voltPCMV OLTC, varPCMV RD , varPCMV DG/DSt, VVSCMV,LV ngb , varCnsHV MV  , VVSCLV  varPCLV RD, varPCLV DG/DSt, VVSCLV,CP ngb , varCnsMV LV  , VVSCCP varPCCP inv, varCnsLV CP= 0  (1) (1)(1) where VVSC calculates in real time (a) the voltage set-point for the primary where VVSCMV calculates in real time where VVSC calculates in real time (a) the voltage set-point for the primary where VVSCMV calculates in real time where VVSC calculates in real time (a) the voltage set-point for the primary control voltPCOLTC MV of the supplying transformer and other where VVSCMV calculates in real time e VVSC calculates in real time the voltage set-point for the primary control voltPCOLTC MV of the supplying transformer and other re VVSCMV calculates in real time transformers included in the MV_link-grid (e.g., 34.5 kV/11 kV, etc.) that have On-Load-Tap- Changer (OLTC); (b) the var set-points for the primary controls varPCRD MV of all RDs included in the MV_link-grid; (c) the var set points for the primary controls varPCMV of all DGs and Distributed Storages (a) the voltage set-point for the primary control voltPCMV OLTC of the supplying transformer and other transformers included in the MV_link-grid (e.g., 34.5 kV/11 kV, etc.) that have On-Load-Tap-Changer (OLTC); transformers included in the MV_link-grid (e.g., 34.5 kV/11 kV, etc.) that have On-Load-Tap- Changer (OLTC); (b) the var set-points for the primary controls varPCRD MV of all RDs included in the MV_link-grid; (c) the var set points for the primary controls varPCMV of all DGs and Distributed Storages (a) the voltage set-point for the primary control voltPCMV OLTC of the supplying transformer and other transformers included in the MV_link-grid (e.g., 34.5 kV/11 kV, etc.) that have On-Load-Tap-Changer (OLTC); ) the var set-points for the primary controls varPCDG/DSt of all DGs and Distributed Storag (DSt) connected to the MV_link-grid; b) the var set-points for the primary controls varPCMV RD of all RDs included in the MV_link-grid var set-points for the primary controls varPCDG/DSt of all DGs and Distributed Storages St) connected to the MV_link-grid; e var set-points for the primary controls varPCMV RD of all RDs included in the MV_link-grid; 4 of 23 Energies 2019, 12, 3865 (c) the var set-points for the primary controls varPCMV DG/DSt of all DGs and Distributed Storages (DSt) connected to the MV_link-grid; (d) the var set-points for the Volt/var secondary controls VVSCMV,LV ngb of all neighbour MV_ or LV_grid-links, while respecting the var constraint varCnsHV MV at the border to the HV_link-grid. 2.2.1. Customer Plant Model The reactive power contribution PV y te i dete i ed by the a lied o t ol t ate y a de ibed i Se tio 2 3 It i where f P,load t and f Q,load t are the active and reactive power load profile factors at time-point t; f P,PV t is the active power production profile factor at time-point t; and Pload peak = 1.368 kW is the peak active l d 𝑃𝑙𝑜𝑎𝑑,𝑖,𝑡= 𝑃𝑛𝑜𝑚,𝑡· ቀ𝐶𝑡 · ൫𝑈𝑖,𝑡𝑈𝑛𝑜𝑚 ⁄ ൯+ 𝐶𝑡 · ൫𝑈𝑖,𝑡𝑈𝑛𝑜𝑚 ⁄ ൯+ 𝐶𝑡ቁ The load voltage dependency is modelled with a ZIP model according to: 𝑎𝑑,𝑖,𝑡 𝑃 = 𝑄𝑛𝑜𝑚,𝑡 𝑙𝑜𝑎𝑑· ቀ𝐶𝑡 𝑄,𝑍· ൫𝑈𝑖,𝑡𝑈𝑛𝑜𝑚 𝐿𝑉 ⁄ ൯ 2 + 𝐶𝑡 𝑄,𝐼· ൫𝑈𝑖,𝑡𝑈𝑛𝑜𝑚 𝐿𝑉 ⁄ ൯+ 𝐶𝑡 𝑄,𝑃ቁ (4b) ூ , 𝐶௧ ௉,௉ and 𝐶௧ ொ,௓ , 𝐶௧ ொ,ூ , 𝐶௧ ொ,௉ are the active and reactive power ZIP nt t. ZIP coefficients and load profiles are given for the considered 24h time load and production profiles shown in Figure 2b are sampled into ∆t = 15min PCP load,i,t = Pload nom,t·  CP,Z t ·  Ui,t/ULV nom 2 + CP,I t ·  Ui,t/ULV nom  + CP,P t  (4a) QCP load,i,t = Qload nom,t·  CQ,Z t ·  Ui,t/ULV nom 2 + CQ,I t ·  Ui,t/ULV nom  + CQ,P t  (4b) (4a) (4b) izon in [26,27]. The load and production profiles shown in Figure 2b are sampled into ∆t = 15min e-steps, resulting in N = 24h/∆t = 96 load-flow simulations per scenario. 2. Distribution Grid Models MV and LV levels are modelled and simulated in a common model. For the sake of simplicity, where CP,Z t , CP,I t , CP,P t and CQ,Z t , CQ,I t , CQ,P t are the active and reactive power ZIP coefficients at time-point t. ZIP coefficients and load profiles are given for the considered 24 h time horizon in [26,27]. The load and production profiles shown in Figure 2b are sampled into ∆t = 15 min time-steps, resulting in N = 24 h/∆t = 96 load-flow simulations per scenario. 2.2.1. Customer Plant Model The profiles determine the active and reactive power consumption of loads for nominal grid voltage (Pload nom,t and Qload nom,t) and the active power production of the PV-system, as in: ௡௢௠,௧= 𝑓௧ 𝑃௣௘௔௞ (3b) 𝑃௜௡௩,௜,௧ ஼௉ = 𝑓௧ ௉,௉௏· 𝑃௉௏,௥ ஼௉ (3c) ve and reactive power load profile factors at time-point t; le factor at time-point t; and 𝑃௣௘௔௞ ௟௢௔ௗ= 1.368 kW is the peak is calculated based on the maximum 15-minutes mean Pload nom,t = f P,load t ·Pload peak, (3a) Qload nom,t = f Q,load t ·Pload peak (3b) PCP inv,i,t = f P,PV t ·PCP PV,r (3c) (3a) (3b) (3c) p ( ௣௘௔௞ ue of the active power flow measured throughout 2016 at the secondary side of the DTR of the LV_link-grid described in Section 2.2.2 [24]) of each CP’s load. The reactive power contribution PV-systems is determined by the applied control strategy as described in Section 2.3. It is resting to note that nowadays the load has changed the behaviour in terms of reactive power. The d behaves capacitive in the evening because the residential customers have mainly turned to LED hting [26]. The load voltage dependency is modelled with a ZIP model according to: 𝑃𝑙 𝑑𝑖𝑡 𝐶𝑃 = 𝑃 𝑡 𝑙𝑜𝑎𝑑· ቀ𝐶𝑡 𝑃,𝑍· ൫𝑈𝑖𝑡𝑈𝐿𝑉 ⁄ ൯ 2 + 𝐶𝑡 𝑃,𝐼· ൫𝑈𝑖𝑡𝑈𝐿𝑉 ⁄ ൯+ 𝐶𝑡 𝑃,𝑃ቁ (4a) where f P,load t and f Q,load t are the active and reactive power load profile factors at time-point t; f P,PV t is the active power production profile factor at time-point t; and Pload peak = 1.368 kW is the peak active power demand (the value of Pload peak is calculated based on the maximum 15-minutes mean value of the active power flow measured throughout 2016 at the secondary side of the DTR of the real LV_link-grid described in Section 2.2.2 [24]) of each CP’s load. The reactive power contribution of PV-systems is determined by the applied control strategy as described in Section 2.3. It is interesting to note that nowadays the load has changed the behaviour in terms of reactive power. The load behaves capacitive in the evening because the residential customers have mainly turned to LED lighting [26]. p ௣ ue of the active power flow measured throughout 2016 at the secondary side of the DTR of the LV_link-grid described in Section 2.2.2 [24]) of each CP’s load. 2.2.1. Customer Plant Model Figure 2a shows the used customer plant structure. It has two components: the load or power consumption and the electricity production. For each customer plant i and time-point t, it is characterized by the active and reactive power consumption and production of the internal loads (PCP load,i,t and QCP load,i,t) and the PV-system ( PCP inv,i,t and QCP inv,i,t), respectively. The active PLV CP,i,t and reactive power QLV CP,i,t flows from the CP i to LV_link-grid at time-point t are given by: PLV CP,i,t = PCP inv,i,t −PCP load,i,t, (2a) QLV CP,i,t = QCP inv,i,t −QCP load,i,t (2b) (2a) (2b) 5 of 23 23 Energies 2019, 12, 3865 i 2019 12 FOR PEER (a) (b) Figure 2. Customer plant model: (a) structure; (b) load and production profiles. Figure 2. Customer plant model: (a) structure; (b) load and production profiles. (b) (a) (b) (a) ure 2. Customer plant model: (a) structure; (b) load and production profiles. Figure 2. Customer plant model: (a) structure; (b) load and production profiles. Figure 2b shows the load and production profiles represented by solid and dashed lines, pectively. Active and reactive power are coloured red and blue, respectively. The critical time- nt tcrit, where the maximal PV production occurs, is marked as a black vertical line. The profiles Each CP is connected to a boundary link node (BLiN) of the corresponding LV_link-grid with the actual Ui,t and nominal voltage ULV nom, and includes a PV-system with a module-rating of PCP PV,r = 5 kW [24] and an inverter-rating of SCP inv,r = PCP PV,r/0.9 [25]. ermine the active and reactive power consumption of loads for nominal grid voltage (𝑃𝑛𝑜𝑚,𝑡 𝑙𝑜𝑎𝑑 and 𝑚,𝑡 𝑑 ) and the active power production of the PV-system, as in: 𝑃𝑛𝑜𝑚,𝑡 𝑙𝑜𝑎𝑑= 𝑓𝑡 𝑃,𝑙𝑜𝑎𝑑· 𝑃𝑝𝑒𝑎𝑘 𝑙𝑜𝑎𝑑, (3a) ௟௢௔ௗ 𝑓ொ௟௢௔ௗ ௟௢௔ௗ b , Figure 2b shows the load and production profiles represented by solid and dashed lines, respectively. Active and reactive power are coloured red and blue, respectively. The critical time-point tcrit, where the maximal PV production occurs, is marked as a black vertical line. y are described separately below. 2.2.2. Distribution Grid Models Low Voltage Grid Figure 3 shows a simplified one-line diagram of the used LV_link-grid model. MV and LV levels are modelled and simulated in a common model. For the sake of simplicity, they are described separately below. 6 of 23 Energies 2019, 12, 3865 (A) Low Voltage Grid E i 2019 12 FOR PEER REVIEW Figure 3 shows a simplified one-line diagram of the used LV_link-grid model. nergies 2019, 12, x FOR PEER REVIEW Energies 2019, 12, x FOR PEER REVIEW e 3 shows a simplified one-line diagram of the used LV_link-grid model. 12, x FOR PEER REVIEW , 12, x FOR PEER REVIEW Figure 3. Simplified one-line diagram of the LV_link-grid model. Figure 3. Simplified one-line diagram of the LV_link-grid model. Figure 3. Simplified one-line diagram of the LV_link-grid model. Figure 3. Simplified one-line diagram of the LV_link-grid model. Figure 3. Simplified one-line diagram of the LV_link-grid model. Figure 3. Simplified one-line diagram of the LV_link-grid model. It is a real rural grid with four feeders with a minimum and maximum length of 565 m and 1.63 km, respectively. In this link-grid with a cable share of about 59% and a nominal voltage of 𝑈௡௢௠ ௅௏ = 0.4 kV are connected 61 residential customers. It is connected to the MV_link-grid through a 21 kV/0.42 kV, 400 kVA DTR with its tap changer fixed in mid-position. The detailed LV_link-grid model data (instead of the 160 kVA DTR given in the mentioned reference, a 400 kVA one with a rated primary and secondary voltage of 21 kV and 0.42 kV, respectively, and a short circuit voltage of 3.7% with a resistive part of 1% is used, because of the high PV share. The tap changer is fixed in its mid-position) is given in [28]. Figure 3 shows with red crosses the connection points of the L(U)s. It is a real rural grid with four feeders with a minimum and maximum length of 565 m and 1.63 km, respectively. In this link-grid with a cable share of about 59% and a nominal voltage of ULV nom = 0.4 kV are connected 61 residential customers. It is connected to the MV_link-grid through a 21 kV/0.42 kV, 400 kVA DTR with its tap changer fixed in mid-position. (B) Medium Voltage Grid (B) Medium Voltage Grid (B) Medium Voltage Grid Fi 4 h i Figure 4 shows a simplified one-line diagrams of the used MV_link-grids. Both MV_link-grids are identical, except for the conductor type: Figures 4a,b represent the cases with cable and overhead conductors in MV level, respectively. To each MV_link-grid are connected 32 LV_link-grids and two PV-systems, each with a module and inverter rating of PPV,r MV = 1 MW and Sinv,r MV = 1 MVA, respectively. The active power production 𝑃𝑖𝑛𝑣,𝑡 ெ௏ of each PV-system connected to the MV_link-grid at time-point t is determined by: Figure 4 shows a simplified one-line diagrams of the used MV_link-grids. Both MV_link-grids are identical, except for the conductor type: Figure 4a,b represent the cases with cable and overhead conductors in MV level, respectively. To each MV_link-grid are connected 32 LV_link-grids and two PV-systems, each with a module and inverter rating of PMV PV,r= 1 MW and SMV inv,r= 1 MVA, respectively. The active power production PMV inv,t of each PV-system connected to the MV_link-grid at time-point t is determined by: Figure 4 shows a simplified one-line diagrams of the used MV_link-grids. Both MV_link-grids are identical, except for the conductor type: Figures 4a,b represent the cases with cable and overhead conductors in MV level, respectively. To each MV_link-grid are connected 32 LV_link-grids and two PV-systems, each with a module and inverter rating of PPV,r MV = 1 MW and Sinv,r MV = 1 MVA, respectively. The active power production 𝑃𝑖𝑛𝑣,𝑡 ெ௏ of each PV-system connected to the MV_link-grid at time-point t is determined by: 𝑃௜௡௩,௧ ெ௏= 𝑓௧ ௉,௉௏· 𝑃௉௏,௥ ெ௏ (5) PMV inv,t = f P,PV t ·PMV PV,r (5) 𝑃௜௡௩,௧ ெ௏= 𝑓௧ ௉,௉௏· 𝑃௉௏,௥ ெ௏ (5) (5) (5) (5) ௜௡௩,௧ 𝑓௧ ௉௏,௥ ( ) while the reactive power contribution is determined by the applied control strategy as described in Section 2.3. The MV feeder length is 24 km and slack voltage is set to the nominal value of 110 kV. The detailed data of the MV link-grid models is given in Appendix A while the reactive power contribution is determined by the applied control strategy as described in Section 2.3. The MV feeder length is 24 km and slack voltage is set to the nominal value of 110 kV. The detailed data of the MV_link-grid models is given in Appendix A. while the reactive power contribution is determined by the applied control strategy as described in Section 2.3. (B) Medium Voltage Grid (B) Medium Voltage Grid (B) Medium Voltage Grid Fi 4 h i The MV feeder length is 24 km and slack voltage is set to the nominal value of 110 kV. The detailed data of the MV_link-grid models is given in Appendix A. (a) (b) Figure 4. Simplified one-line diagrams of the MV_link-grid models with different conductor types: (a) cable; (b) overhead line. (a) (b) Figure 4. Simplified one-line diagrams of the MV_link-grid models with different conductor types: (a) cable; (b) overhead line. Figure 4. Simplified one-line diagrams of the MV_link-grid models with different conductor types: (a) cable; (b) overhead line. (a) (a) (a) (b) (b) (b) (b) (b) Figure 4. Simplified one-line diagrams of the MV_link-grid models with different conductor types: (a) cable; (b) overhead line Figure 4. Simplified one-line diagrams of the MV_link-grid models with different conductor types: (a) cable; (b) overhead line. Figure 4. Simplified one-line diagrams of the MV_link-grid models with different conductor types: (a) cable; (b) overhead line. (b) Figure 4. Simplified one-line diagrams of the MV_link-grid models with different conductor types: (a) cable; (b) overhead line Figure 4. Simplified one-line diagrams of the MV_link-grid models with different conductor types: (a) cable; (b) overhead line. Figure 4. Simplified one-line diagrams of the MV_link-grid models with different conductor types: (a) cable; (b) overhead line. (b) Figure 4. Simplified one-line diagrams of the MV_link-grid models with different conductor types: (a) cable; (b) overhead line Figure 4. Simplified one-line diagrams of the MV_link-grid models with different conductor types: (a) cable; (b) overhead line. Figure 4. Simplified one-line diagrams of the MV_link-grid models with different conductor types: (a) cable; (b) overhead line. y are described separately below. 2.2.2. Distribution Grid Models The detailed LV_link-grid model data (instead of the 160 kVA DTR given in the mentioned reference, a 400 kVA one with a rated primary and secondary voltage of 21 kV and 0.42 kV, respectively, and a short circuit voltage of 3.7% with a resistive part of 1% is used, because of the high PV share. The tap changer is fixed in its mid-position) is given in [28]. Figure 3 shows with red crosses the connection points of the L(U)s. It is a real rural grid with four feeders with a minimum and maximum length of 565 m and 1.63 km, respectively. In this link-grid with a cable share of about 59% and a nominal voltage of 𝑈௡௢௠ ௅௏ = 0.4 kV are connected 61 residential customers. It is connected to the MV_link-grid through a 21 kV/0.42 kV, 400 kVA DTR with its tap changer fixed in mid-position. The detailed LV_link-grid model data (instead of the 160 kVA DTR given in the mentioned reference, a 400 kVA one with a rated primary and secondary voltage of 21 kV and 0.42 kV, respectively, and a short circuit voltage of 3.7% with a resistive part of 1% is used, because of the high PV share. The tap changer is fixed in its mid-position) is given in [28]. Figure 3 shows with red crosses the connection points of the L(U)s. (B) M di V lt G id (a) cable; (b) overhead line. Si l d C l S 2.3. Simulated Control Setups 2.3. Simulated Control Setups 2.3. Simulated Control Setups The use of the reactive power to control the voltage in LV grids leads to an uncontrolled reactive power flow up to the HV grid reducing its RPM This uncontrolled reactive power flow can be The use of the reactive power to control the voltage in LV grids leads to an uncontrolled reactive power flow up to the HV grid, reducing its RPM. This uncontrolled reactive power flow can be The use of the reactive power to control the voltage in LV grids leads to an uncontrolled reactive power flow up to the HV grid, reducing its RPM. This uncontrolled reactive power flow can be 7 of 23 ration Here, Energies 2019, 12, 3865 coloured dotted lin balancing. The latter practically compensated by CDs connected at different points of the grid. Therefore, a series of simulations is performed to investigate the grid behaviour for various placements of CDs when the VVC chain strategy is used. The grid-link setups are set assuming that the same DSO owns and operates the MV and LV grids. They are derived from the generalized VVC chain strategy shown in Figure 1. The five identified cases are depicted in Figures 5–9. For each case is derived a specific equation from the generalized form presented in Equation (1) that describes all elements involved in the VVC chain, Equations (6)–(10). It is supposed that neither DSts nor DGs are connected to the LV_link-grids. One of the basic principles in setting up the grid-links is the minimization of the number of secondary and primary control units to keep the CapEx and OpEx as low as possible. Therefore, no VVSC is provided for the LV_grid-link, since only L(U) local controls (varLCLV L(U)) are connected at the end of some laterals: coordination is not relevant. The LV_grid-link is shown in gold-coloured dotted lines because its existence must be discussed also in terms of load-generation balancing. The latter is not within the scope of this paper. A grid-link is set up in the MV level. Here, the VVSC is important to coordinate the Q-contribution of DGs, RDs and the neighbour grid-links with the voltage at the secondary side or OLTC position of the supplying transformer (STR) while respecting all constraints and optimizing the network performance at the same time. (a) cable; (b) overhead line. Si l d C l S 2.3. Simulated Control Setups 2.3. Simulated Control Setups with the voltage at the secondary side or OLTC position of the supplying transformer (STR) while respecting all constraints and optimizing the network performance at the same time. All simulations are performed using both distribution grid models. The STR tap is fixed in its mid-position so that the impact of CD placement on distribution grid behaviour can be clearly analysed. 1. Control setup: Without any var control (no control) Figure 5 shows the simplified form of the VVC chain strategy representing the setup without any var control. In this form, the VVC chain in the Y-axis is presented by the following generalized equation: VVCY-axis= {VVSCMV൫varPCDG MV൯} (6) Usually, VVSCMV sends the var set-points QDG MV* to all DGs connected to the MV_link-grid. In our simulations, all DGs, i.e., PV-systems, inject into the grid with a power factor of one, QDG MV* = 0 . PV-systems in CP level inject with a power factor of one as well. Therefore, the LV grids supply reactive power to the loads connected at the CP level. Figure 5. Simplified form of the VVC chain strategy representing the setup without any var control. In this control setup, reactive power is exchanged between all three levels: between HV_ and MV_link-grid, MV_ and LV_link-grids, and LV_link-grids and CPs. 2. Control setup: L(U)-control and CP_Q-autarky (no CDs) Figure 6 depicts the simplified form of the VVC chain strategy representing the setup with L(U)- control and CP_Q-autarky. In this form, the VVC chain in the Y-axis is presented by the following equation: The VVSCMV sends the var set-points QDG MV*= 0 to all PV-systems connected to the MV_link- grid. To alleviate upper voltage limit violations, varLCL(U) LV are set at the ends of the violated LV VVCY-axis= {VVSCMV൫varPCDG MV൯, varLCL(U) LV , VVSCCP൫varPCinv CP, varCnsCP LV= 0൯} (7) Figure 5. Simplified form of the VVC chain strategy representing the setup without any var control. Energies 2019, 12, x FOR PEER REVIEW 8 of 23 feeders (see Figure 3). Each VVSCCP sends the required var set-point Qinv CP* to the corresponding PV- system to achieve Q-autarky, i.e., full reactive power compensation in CP level, satisfying the var constraint varCnsCP LV = 0 at all times [18]. Figure 6. Simplified form of the VVC chain strategy representing the setup with L(U)-control and CP Q autarky Figure 6. Simplified form of the VVC chain strategy representing the setup with L(U)-control and CP_Q-autarky. (a) cable; (b) overhead line. Si l d C l S 2.3. Simulated Control Setups 2.3. Simulated Control Setups Therefore, the reactive power is exchanged only between HV_ and MV_link-grid. 5. Control setup: L(U)-control, CP_Q-Autarky and CDs at the DTRs’ LV-busbars ( CDLV DTR ) Here, CD positioning is moved from the MV- to the LV-bus bars of DTRs, compensating the reactive power required by LV_link-grid, Figure 9. The VVC chain in the Y-axis is presented by: VVCY-axis= {VVSCMV൫varPCDG MV, varPCCD LV, varCnsLV MV= 0൯, varPCL(U) LV , VVSCCP൫varPCinv CP, varCnsCP LV= 0൯ The VVC chain Figure 8. Simplified form of the VVC chain strategy representing the setup with L(U)-control, CP_Q- k d h b b Figure 8. Simplified form of the VVC chain strategy representing the setup with L(U)-control, CP_Q-autarky and CDs at the DTRs’ MV-busbars. VCY-axis= {VVSCMV൫varPCDG MV, varPCCD LV, varCnsLV MV= 0൯, varPCL(U) LV , VVSCCP൫varPCinv CP, varCnsCP LV autarky and CDs at the DTRs MV busbars. Therefore, the reactive power is exchanged only between HV_ and MV_link-g 5. Control setup: L(U)-control, CP_Q-Autarky and CDs at the DTRs’ LV-busbars ( CD Here, CD positioning is moved from the MV- to the LV-bus bars of DTRs, reactive power required by LV_link-grid, Figure 9. The VVC chain in the Y-axis is VVCY-axis= {VVSCMV൫varPCDG MV, varPCCD LV, varCnsLV MV= 0൯, varPCL(U) LV , VVSCCP൫varP au a y a a e u a Therefore, the reactive power is exchanged only between HV_ and MV_link-grid. 5. Control setup: L(U)-control, CP_Q-Autarky and CDs at the DTRs’ LV-busbars ( CDLV DTR ) Here, CD positioning is moved from the MV- to the LV-bus bars of DTRs, compensating the reactive power required by LV_link-grid, Figure 9. The VVC chain in the Y-axis is presented by: VVCY-axis= {VVSCMV൫varPCDG MV, varPCCD LV, varCnsLV MV= 0൯, varPCL(U) LV , VVSCCP൫varPCinv CP, varCnsCP LV= 0൯ Figure 9. Simplified form of the VVC chain strategy representing the setup with L(U)-control, CP_Q- t k d CD t th DTR ’ LV b b Figure 9. Simplified form of the VVC chain strategy representing the setup with L(U)-control, CP_Q-autarky and CDs at the DTRs’ LV-busbars. Figure 9. Simplified form of the VVC chain strategy representing the setup with L(U)-control, CP_Q- t k d CD t th DTR ’ LV b b Figure 9. Simplified form of the VVC chain strategy representing the setup with L(U)-control, CP_Q-autarky and CDs at the DTRs’ LV-busbars. y Also in this case, the reactive power is exchanged only between HV_ and MV_link-grid. 3. (a) cable; (b) overhead line. Si l d C l S 2.3. Simulated Control Setups 2.3. Simulated Control Setups Figure 5 Simplified form of the VVC chain strategy representing the setup without any var control Figure 5. Simplified form of the VVC chain strategy representing the setup without any var control. Energies 2019, 12, x FOR PEER REVIEW 8 of 23 feeders (see Figure 3). Each VVSCCP sends the required var set-point Qinv CP* to the corresponding PV- system to achieve Q-autarky, i.e., full reactive power compensation in CP level, satisfying the var constraint varCnsCP LV = 0 at all times [18]. power compen eve Q-autarky, LV Figure 5 Simplified form of the VVC chain strategy representing the setup without any var control Figure 5. Simplified form of the VVC chain strategy representing the setup without any var control. raint varCnsCP = 0 at all times [18]. In this control setup, reactive power is exchanged between all three levels: between HV_ and MV_link-grid, MV_ and LV_link-grids, and LV_link-grids and CPs. 2. Control setup: L(U)-control and CP_Q-autarky (no CDs) Figure 6 depicts the simplified form of the VVC chain strategy representing the setup with L(U)- control and CP_Q-autarky. In this form, the VVC chain in the Y-axis is presented by the following equation: The VVSCMV sends the var set-points QDG MV*= 0 to all PV-systems connected to the MV_link- grid. To alleviate upper voltage limit violations, varLCL(U) LV are set at the ends of the violated LV VVCY-axis= {VVSCMV൫varPCDG MV൯, varLCL(U) LV , VVSCCP൫varPCinv CP, varCnsCP LV= 0൯} (7) Figure 6. Simplified form of the VVC chain strategy representing the setup with L(U)-control and CP Q-autarky Figure 6. Simplified form of the VVC chain strategy representing the setup with L(U)-control and CP_Q-autarky. . To alleviate upper voltage limit violations, varLCL(U) are set at the ends of the violated L Figure 6. Simplified form of the VVC chain strategy representing the setup with L(U)-control and CP Q autarky Figure 6. Simplified form of the VVC chain strategy representing the setup with L(U)-control and CP_Q-autarky. Energies 2019, 12, 3865 VVCY-axis= {VVSCM 8 of 23 nsCP LV= 0൯} 8 of 23 nsCP LV= 0൯} Figure 7. Simplified form of the VVC chain strategy representing the setup with L(U)-control, CP_Q- autarky and a CD at the STR MV busbar Figure 7. Simplified form of the VVC chain strategy representing the setup with L(U)-control, CP_Q-autarky and a CD at the STR MV-busbar. (a) cable; (b) overhead line. Si l d C l S 2.3. Simulated Control Setups 2.3. Simulated Control Setups Energies 2019, 12, x FOR PEER REVIEW 9 of 23 Energies 2019, 12, x FOR PEER REVIEW 9 of 23 Figure 7. Simplified form of the VVC chain strategy representing the setup with L(U)-control, CP_Q- autarky and a CD at the STR MV-busbar. For the simulations, the CD is parametrized to respect the constraint varCnsMV HV= 0 . Therefore, the reactive power is exchanged only between MV_ and LV_link-grids. 4. Control setup: L(U)-control, CP_Q-Autarky and CD at the DTRs’ MV-busbars ( CDMV DTR ) In this case, CD positioning is moved from the MV-bus bar of the STR to the MV-bus bars of the DTRs, compensating the reactive power required by LV_link-grids, Figure 8. The VVC chain in the Y-axis is presented by the following equation: VVCY-axis= {VVSCMV൫varPCDG MV, varPCCD MV, varCnsLV MV= 0൯, varLCL(U) LV , VVSCCP൫varPCinv CP, varCnsCP LV= 0൯} Figure 7. Simplified form of the VVC chain strategy representing the setup with L(U)-control, CP_Q-autarky and a CD at the STR MV-busbar. Energies 2019, 12, x FOR PEER REVIEW 9 of 23 Figure 8. Simplified form of the VVC chain strategy representing the setup with L(U)-control, CP_Q- autarky and CDs at the DTRs’ MV-busbars. Figure 8. Simplified form of the VVC chain strategy representing the setup with L(U)-control, CP_Q-autarky and CDs at the DTRs’ MV-busbars. Energies 2019, 12, x FOR PEER REVIEW 9 of 23 Figure 8. Simplified form of the VVC chain strategy representing the setup with L(U)-control, CP_Q- autarky and CDs at the DTRs’ MV-busbars. Therefore, the reactive power is exchanged only between HV_ and MV_link-grid. 5. Control setup: L(U)-control, CP_Q-Autarky and CDs at the DTRs’ LV-busbars ( CDLV DTR ) Here, CD positioning is moved from the MV- to the LV-bus bars of DTRs, compensating the reactive power required by LV_link-grid, Figure 9. The VVC chain in the Y-axis is presented by: VVCY-axis= {VVSCMV൫varPCDG MV, varPCCD LV, varCnsLV MV= 0൯, varPCL(U) LV , VVSCCP൫varPCinv CP, varCnsCP LV= 0൯} Figure 7. Simplified form of the VVC chain strategy representing the setup with L(U)-control, CP_Q- t k d CD t th STR MV b b Figure 7. Simplified form of the VVC chain strategy representing the setup with L(U)-control, CP_Q-autarky and a CD at the STR MV-busbar. gies 2019, 12, x FOR PEER REVIEW 9 o autarky and a CD at the STR MV busbar. (a) cable; (b) overhead line. Si l d C l S 2.3. Simulated Control Setups 2.3. Simulated Control Setups For the simulations, the CD is parametrized to respect the constraint varCnsMV HV= 0 . Therefore, the reactive power is exchanged only between MV_ and LV_link-grids. 4. Control setup: L(U)-control, CP_Q-Autarky and CD at the DTRs’ MV-busbars ( CDMV DTR ) In this case, CD positioning is moved from the MV-bus bar of the STR to the MV-bus bars of the DTRs, compensating the reactive power required by LV_link-grids, Figure 8. The VVC chain in the Y-axis is presented by the following equation: VVCY-axis= {VVSCMV൫varPCDG MV, varPCCD MV, varCnsLV MV= 0൯, varLCL(U) LV , VVSCCP൫varPCinv CP, varCnsCP LV= 0൯ Figure 8. Simplified form of the VVC chain strategy representing the setup with L(U)-control, CP_Q- Figure 8. Simplified form of the VVC chain strategy representing the setup with L(U)-control, CP_Q-autarky and CDs at the DTRs’ MV-busbars. Figure 8. Simplified form of the VVC chain strategy representing the setup with L(U)-control, CP_Q- autarky and CDs at the DTRs’ MV-busbars. Therefore, the reactive power is exchanged only between HV_ and MV_link-grid. 5. Control setup: L(U)-control, CP_Q-Autarky and CDs at the DTRs’ LV-busbars ( CDLV DTR ) Here, CD positioning is moved from the MV- to the LV-bus bars of DTRs, compensating the reactive power required by LV_link-grid, Figure 9. The VVC chain in the Y-axis is presented by: VVCY-axis= {VVSCMV൫varPCDG MV, varPCCD LV, varCnsLV MV= 0൯, varPCL(U) LV , VVSCCP൫varPCinv CP, varCnsCP LV= 0൯ autarky and a CD at the STR MV busbar. For the simulations, the CD is parametrized to respect the constraint varCnsMV HV= 0 . Therefore, the reactive power is exchanged only between MV_ and LV_link-grids. 4. Control setup: L(U)-control, CP_Q-Autarky and CD at the DTRs’ MV-busbars ( CDMV DTR ) In this case, CD positioning is moved from the MV-bus bar of the STR to the MV-bus bars of the DTRs, compensating the reactive power required by LV_link-grids, Figure 8. The VVC chain in the Y-axis is presented by the following equation: VVCY-axis= {VVSCMV൫varPCDG MV, varPCCD MV, varCnsLV MV= 0൯, varLCL(U) LV , VVSCCP൫varPCinv CP, varCnsCP LV= 0൯ Figure 8. Simplified form of the VVC chain strategy representing the setup with L(U)-control, CP_Q- Figure 8. Simplified form of the VVC chain strategy representing the setup with L(U)-control, CP_Q-autarky and CDs at the DTRs’ MV-busbars. Figure 8. Simplified form of the VVC chain strategy representing the setup with L(U)-control, CP_Q- autarky and CDs at the DTRs’ MV-busbars. (a) cable; (b) overhead line. Si l d C l S 2.3. Simulated Control Setups 2.3. Simulated Control Setups The VVSCMV sends the var set-points QMV∗ DG = 0 to all PV-systems connected to the MV_lin To alleviate upper voltage limit violations, varLCLV L(U) are set at the ends of the violated LV CP CP The VVSCMV sends the var set-points QMV∗ DG = 0 to all PV-systems connected to the MV_link-grid. To alleviate upper voltage limit violations, varLCLV L(U) are set at the ends of the violated LV feeders (see Figure 3). Each VVSCCP sends the required var set-point QCP∗ inv to the corresponding PV-system g L(U) (see Figure 3). Each VVSCCP sends the required var set-point QCP∗ inv to the corresponding PV-system to achieve Q-autarky, i.e., full reactive power compensation in CP level, satisfying the var constraint varCnsLV CP = 0 at all times [18]. CP In this control setup, reactive power is exchanged between two levels: between HV_ and MV_link-grid and between MV_ and LV_link-grids. 3. Control setup: L(U)-control, CP_Q-Autarky and CD at the STR MV-busbar (CDSTR MV ) This control setup is derived from the second one and supplemented with a CD connected to the STR MV-bus bar, Figure 7. The VVC chain in the Y-axis is presented as follows: VVCY−axis= {VVSC MV varPCMV CD , varPCMV DG, varCnsHV MV= 0  , varLCLV L(U), VVSCCP varPCCP inv, varCnsLV CP= 0  (8) VCY−axis= {VVSC MV varPCMV CD , varPCMV DG, varCnsHV MV= 0  , varLCLV L(U), VVSCCP varPCCP inv, varCnsLV CP= 0  (8 (8) For the simulations, the CD is parametrized to respect the constraint varCnsHV MV= 0. Therefore, the reactive power is exchanged only between MV_ and LV_link-grids. 4. Control setup: L(U)-control, CP_Q-Autarky and CD at the DTRs’ MV-busbars (CDDTR MV ) In this case, CD positioning is moved from the MV-bus bar of the STR to the MV-bus bars of the DTRs, compensating the reactive power required by LV_link-grids, Figure 8. The VVC chain in the Y-axis is presented by the following equation: VVCY−axis= {VVSC MV varPCMV DG, varPCMV CD , varCnsMV LV = 0  , varLCLV L(U), VVSCCP varPCCP inv, varCnsLV CP= 0  (9) VVCY−axis= {VVSC MV varPCMV DG, varPCMV CD , varCnsMV LV = 0  , varLCLV L(U), VVSCCP varPCCP inv, varCnsLV CP= 0  (9 (9) Therefore, the reactive power is exchanged only between HV_ and MV_link-grid. Therefore, the reactive power is exchanged only between HV_ and MV_link-grid. (a) cable; (b) overhead line. Si l d C l S 2.3. Simulated Control Setups 2.3. Simulated Control Setups Results and Discussion All simulations are performed using both distribution grid models. The STR tap is fixed in its mid-position so that the impact of CD placement on distribution grid behaviour can be clearly analysed. 1. Control setup: Without any var control (no control) 3. Results and Discussion 1. Control setup: Without any var control (no control) The behaviour of both distribution grids is described below for the simulated control setups; thereby, the effect of CD placement is analysed in detail. Based on these results, the optimal setup of Figure 5 shows the simplified form of the VVC chain strategy representing the setup without any var control. In this form, the VVC chain in the Y-axis is presented by the following generalized equation: he VVC chain strategy representing the setup with L(U)-control, CP_Q- cussed. VVCY−axis= {VVSC MV varPCMV DG o (6) he VVC chain strategy representing the set ussed. VVCY−axis= {VVSC MV varPCMV DG o (6) Energies 2019, 12, 3865 9 of 23 Usually, VVSCMV sends the var set-points QMV∗ DG to all DGs connected to the MV_link-grid. In our simulations, all DGs, i.e., PV-systems, inject into the grid with a power factor of one, QMV∗ DG = 0. PV-systems in CP level inject with a power factor of one as well. Therefore, the LV grids supply reactive power to the loads connected at the CP level. In this control setup, reactive power is exchanged between all three levels: between HV_ and MV_link-grid, MV_ and LV_link-grids, and LV_link-grids and CPs. 2. Control setup: L(U)-control and CP_Q-autarky (no CDs) . Control setup: L(U)-control and CP_Q-autarky (no CDs) 2. Control setup: L(U)-control and CP_Q-autarky (no CDs) Figure 6 depicts the simplified form of the VVC chain strategy representing the setup with L(U)-control and CP_Q-autarky. In this form, the VVC chain in the Y-axis is presented by the following equation: VVCY−axis= {VVSC MV varPCMV DG  , varLCLV L(U), VVSCCP varPCCP inv, varCnsLV CP= 0  (7) (7) The VVSCMV sends the var set-points QMV∗ DG = 0 to all PV-systems connected to the MV_link-grid. To alleviate upper voltage limit violations, varLCLV L(U) are set at the ends of the violated LV feeders (see Figure 3). Each VVSCCP sends the required var set-point QCP∗ inv to the corresponding PV-system to achieve Q-autarky, i.e., full reactive power compensation in CP level, satisfying the var constraint varCnsLV CP = 0 at all times [18]. 3.1. Behaviour of Distribution Grids As explained in Section 2.2.2, all simulations are performed in the common model of MV and LV levels. Simulation results over the 24 h time horizon are shown graphically in Figures 10 and 12 for the cable and overhead line structure, respectively. Whereas in Tables 2 and 3results at tcrit are listed. Simulations are made for different control setups; results are drown in different colours as follows: “no control“ in dashed blackline; “no CDs” in purple; “CDSTR MV ” in green; “CDDTR MV ” in ocra yellow and “CDDTR LV ” in red solid line. (a) cable; (b) overhead line. Si l d C l S 2.3. Simulated Control Setups 2.3. Simulated Control Setups Control setup L(U) control CP Q Autarky and CDs at the DTRs’ LV busbars (CDDTR) Therefore, the reactive power is exchanged only between HV_ and MV_link-grid. 5. Control setup: L(U)-control, CP_Q-Autarky and CDs at the DTRs’ LV-busbars (CDDTR LV ) 5. Control setup: L(U)-control, CP_Q-Autarky and CDs at the DTRs’ LV-busbars (CDDTR LV ) Here, CD positioning is moved from the MV- to the LV-bus bars of DTRs, compensating the reactive power required by LV_link-grid, Figure 9. The VVC chain in the Y-axis is presented by: VVCY−axis= {VVSC MV varPCMV DG, varPCLV CD, varCnsMV LV = 0  , varPCLV L(U), VVSCCP varPCCP inv, varCnsLV CP= 0  (10) VCY−axis= {VVSC MV varPCMV DG, varPCLV CD, varCnsMV LV = 0  , varPCLV L(U), VVSCCP varPCCP inv, varCnsLV CP= 0  (10 Also in this case, the reactive power is exchanged only between HV_ and MV_link-grid. Also in this case, the reactive power is exchanged only between HV_ and MV_link-grid. 10 of 23 Energies 2019, 12, 3865 10 of 23 3. Results and Discussion The behaviour of both distribution grids is described below for the simulated control setups; thereby, the effect of CD placement is analysed in detail. Based on these results, the optimal setup of the volt/var control chain is discussed. 3.1. Behaviour of Distribution Grids The behaviour of distribution grids is analysed using various parameters as: a) the total reactive power consumption of all L(U)s included in the LV_link-grids, QL(U) tot,t; (a) the total reactive power consumption of all L(U)s included in the LV_link-grids, QL(U) tot,t b) the total reactive power contribution of all CDs included in MV_ or LV_link-grids, QCD tot,t; (b) the total reactive power contribution of all CDs included in MV_ or LV_link-grids, QCD tot,t; (c) the reactive power exchange between HV_ and MV_link-grid, QHV MV,t, at the STR primary side; (c) the reactive power exchange between HV_ and MV_link-grid, QHV MV,t, at the STR primary , (d) the active power losses of the distribution grid, Ploss t , including losses of transformers, cables and overhead lines; (d) the active power losses of the distribution grid, Ploss t , including losses of transformers, cables and overhead lines; (e) the STR loading, LoadingSTR t ; e) the STR loading, LoadingSTR t ; (f) the mean loading of all DTRs, LoadingDTRs t , which is calculated as in (f) the mean loading of all DTRs, LoadingDTRs t , which is calculated as in g gt LoadingDTRs t = P32 k=1 LoadingDTR k,t 32 (11) LoadingDTRs t = P32 k=1 LoadingDTR k,t 32 (11) (11) where LoadingDTR k,t is the loading of the DTR k at time-point t, and 32 is the number of DTRs; (g) the voltage limit violation index, VIt, which is calculated as in where LoadingDTR k,t is the loading of the DTR k at time-point t, and 32 is the number of DTRs; (g) the voltage limit violation index, VIt, which is calculated as in g) the voltage limit violation index, VIt, which is calculated as in (g) the voltage limit violation index, VIt, which is calculated as in VIt = Pmt j=1  Uupper u,t −Uupper lim  ULV nom + Pnt j=1  Ulower lim −Ulower v,t  ULV nom (12) (12) where mt is the number of LV_link-grid nodes that violate the upper voltage limit at time-point t; nt is the number of LV_link-grid nodes that violate the lower voltage limit at time-point t; Uupper u,t is the voltage of the LV_link-grid node u with upper voltage limit violation at time-point t; Ulower v,t is the voltage of the LV_link-grid node v with lower voltage limit violation at time-point t; Uupper lim = 1.1·ULV nom= 0.44 kV is the upper voltage limit; and Ulower lim = 0.9·ULV nom= 0.36 kV is the lower voltage limit. 3.1. Behaviour of Distribution Grids Only LV_link-grid nodes are considered because the simulations show that no voltage limit violations appear in the MV_link-grid. where mt is the number of LV_link-grid nodes that violate the upper voltage limit at time-point t; h b f l k d d h l h l l l upper where mt is the number of LV_link-grid nodes that violate the upper voltage limit at time-point t; nt is the number of LV_link-grid nodes that violate the lower voltage limit at time-point t; Uupper u,t is the voltage of the LV_link-grid node u with upper voltage limit violation at time-point t; Ulower v,t is the voltage of the LV_link-grid node v with lower voltage limit violation at time-point t; Uupper lim = 1.1·ULV nom= 0.44 kV is the upper voltage limit; and Ulower lim = 0.9·ULV nom= 0.36 kV is the lower voltage limit. Only LV_link-grid nodes are considered because the simulations show that no voltage limit violations appear in the MV_link-grid. Table 2. Behaviour of the distribution grid with cable conductors in MV level at tcrit for different control setups. Control Setup QL(U) tot,tcrit (Mvar) QCD tot,tcrit (Mvar) QHV MV,tcrit (Mvar) Ploss tcrit (MW) LoadingSTR tcrit (%) LoadingDTRs tcrit (%) No control 0.00 0.00 −0.70 0.94 52.44 64.40 No CDs 2.07 0.00 −2.65 1.17 53.28 67.47 CDSTR MV 2.63 −3.17 0.00 1.23 50.95 67.74 CDDTR MV 3.03 −3.67 0.11 1.25 50.81 68.11 CDDTR LV 3.60 −4.24 0.11 1.33 50.35 60.74 Table 2. Behaviour of the distribution grid with cable conductors in MV level at tcrit for different control setups. 11 of 23 Energies 2019, 12, 3865 Table 3. Behaviour of the distribution grid with overhead conductors in MV level at tcrit for different control setups. Table 3. Behaviour of the distribution grid with overhead conductors in MV level at tcrit for different control setups. Control Setup QL(U) tot,tcrit (Mvar) QCD tot,tcrit (Mvar) QHV MV,tcrit (Mvar) Ploss tcrit (MW) LoadingSTR tcrit (%) LoadingDTRs tcrit (%) No control 0.00 0.00 −1.97 1.22 51.85 63.53 No CDs 2.11 0.00 −4.04 1.52 54.03 67.61 CDSTR MV 2.79 −4.66 0.00 1.60 48.93 67.90 CDDTR MV 3.80 −4.52 −1.08 1.63 49.04 69.42 CDDTR LV 4.40 −5.11 −1.06 1.72 48.50 58.49 Simulations show that the currents through the transformers, cables and overhead lines never exceed their thermal ratings. The behaviour of distribution grids strongly depends on the PV injections and the placement of CDs. 3.1.1. 3.1. Behaviour of Distribution Grids Distribution Grid with Cable Conductors in MV Level 3.1.1. Distribution Grid with Cable Conductors in MV Level Figure 10 shows the voltage profiles of the MV_link-grid with cable conductors and the backmost LV_link-grid for the critical time-point tcrit. Without any var control, the upper voltage limit is violated with a voltage limit violation index of 35.51. In the case of “no CDs”, “CDSTR MV ” and “CDDTR MV ”, no voltage limit violations appear, but if CDs are installed at the DTRs’ LV-busbars, the upper voltage limit is violated with a voltage limit violation index of 0.04. Energies 2019, 12, x FOR PEER REVIEW 11 of 23 Figure 10. Voltage profile of the MV_link-grid with cable conductors and the backmost LV_link-grid at t = tcrit . Figure 10. Voltage profile of the MV_link-grid with cable conductors and the backmost LV_link-grid at t = tcrit. Figure 10. Voltage profile of the MV_link-grid with cable conductors and the backmost LV_link-grid at t = tcrit . Figure 10. Voltage profile of the MV_link-grid with cable conductors and the backmost LV_link-grid at t = tcrit. Figure 11 shows the behaviour of the distribution grid with cable conductors in MV level over the 24 hour time horizon for all control setups. Figure 11a shows the total reactive power consumption of all L(U)s connected at the distribution grid. When “no control” is applied, no L(U)s are installed and as a result, there is no Q-consumption. The maximum total Q-consumption of L(U)s is reached at tcrit for all cases. The lowest value of 2.07 Mvar is achieved when no CDs are applied, while the highest one of 3.60 Mvar is reached when CDs are installed at the secondary sides of DTRs. Figure 11 shows the behaviour of the distribution grid with cable conductors in MV level over the 24 h time horizon for all control setups. Figure 11a shows the total reactive power consumption of all L(U)s connected at the distribution grid. When “no control” is applied, no L(U)s are installed and as a result, there is no Q-consumption. The maximum total Q-consumption of L(U)s is reached at tcrit for all cases. The lowest value of 2.07 Mvar is achieved when no CDs are applied, while the highest one of 3.60 Mvar is reached when CDs are installed at the secondary sides of DTRs. 3.1.1. Distribution Grid with Cable Conductors in MV Level Energies 2019, 12, 3865 is reached at tcrit fo while the highest o 12 of 23 applied, f DTRs 12 of 23 applied, f DTRs (a) (b) (c) (d) Energies 2019, 12, x FOR PEER REVIEW 12 of 23 Figure 11. Cont. (e) (f) Figure 11. Behaviour of the distribution grid with cable conductors in MV level for a 24 hour time horizon and different control strategies: (a) Q-consumption of L(U)s; (b) Q-contribution of CDs; (c) Q- exchange between HV_ and MV_link-grid; (d) active power losses; (e) STR loading; (f) mean DTR Figure 11. Behaviour of the distribution grid with cable conductors in MV level for a 24 h time horizon and different control strategies: (a) Q-consumption of L(U)s; (b) Q-contribution of CDs; (c) Q-exchange between HV_ and MV_link-grid; (d) active power losses; (e) STR loading; (f) mean DTR loading. (b) (a) (b) (a) Energies 2019, 12, x FOR PEER REVIEW (d) 12 of 23 11. Cont. (c) (d) (c) Figure 1 (e) (f) (f) (e) Figure 11. Behaviour of the distribution grid with cable conductors in MV level for a 24 hour time horizon and different control strategies: (a) Q-consumption of L(U)s; (b) Q-contribution of CDs; (c) Q- exchange between HV_ and MV_link-grid; (d) active power losses; (e) STR loading; (f) mean DTR Figure 11. Behaviour of the distribution grid with cable conductors in MV level for a 24 h time horizon and different control strategies: (a) Q-consumption of L(U)s; (b) Q-contribution of CDs; (c) Q-exchange between HV_ and MV_link-grid; (d) active power losses; (e) STR loading; (f) mean DTR loading. loading. Figure 11b shows the reactive power contribution of the CDs. In the cases of “no control” and “no CDs”, no CDs are installed, thus no Q-contribution is expected. In the other cases, the maximum Q-contribution of CDs appears at tcrit . The CDMV STR behaves inductive in time periods 0:00 to 9:20 a.m. and 03:00 to 12:00 p.m. to compensate the capacitive power produced by the cable. As the PV injection increases from 9:00 a.m., the L(U)s included in LV_link-grid begin to consume inductive power to eliminate the upper voltage limit violations. To compensate the additionally required inductive power, the reactive power production of CDMV STR changes from inductive to capacitive, reaching the maximum Q-injection of 3.17 Mvar. 3.1.1. Distribution Grid with Cable Conductors in MV Level When CDs are installed in distribution substation, they behave purely capacitive with a maximum Q-injection of 3.67 and 4.24 Mvar for the cases Figure 11b shows the reactive power contribution of the CDs. In the cases of “no control” and “no CDs”, no CDs are installed, thus no Q-contribution is expected. In the other cases, the maximum Q-contribution of CDs appears at tcrit. The CDSTR MV behaves inductive in time periods 0:00 to 9:20 a.m. and 03:00 to 12:00 p.m. to compensate the capacitive power produced by the cable. As the PV injection increases from 9:00 a.m., the L(U)s included in LV_link-grid begin to consume inductive power to eliminate the upper voltage limit violations. To compensate the additionally required inductive power, the reactive power production of CDSTR MV changes from inductive to capacitive, reaching the maximum Q-injection of 3.17 Mvar. When CDs are installed in distribution substation, they behave purely capacitive with a maximum Q-injection of 3.67 and 4.24 Mvar for the cases “CDDTR MV ” and “CDDTR LV ”, respectively. loading. Figure 11b shows the reactive power contribution of the CDs. In the cases of “no control” and “no CDs”, no CDs are installed, thus no Q-contribution is expected. In the other cases, the maximum Q-contribution of CDs appears at tcrit . The CDMV STR behaves inductive in time periods 0:00 to 9:20 a.m. and 03:00 to 12:00 p.m. to compensate the capacitive power produced by the cable. As the PV injection increases from 9:00 a.m., the L(U)s included in LV_link-grid begin to consume inductive power to eliminate the upper voltage limit violations. To compensate the additionally required inductive power, the reactive power production of CDMV STR changes from inductive to capacitive, reaching the maximum Q-injection of 3.17 Mvar. When CDs are installed in distribution substation, they behave purely capacitive with a maximum Q-injection of 3.67 and 4.24 Mvar for the cases Figure 11b shows the reactive power contribution of the CDs. In the cases of “no control” and “no CDs”, no CDs are installed, thus no Q-contribution is expected. In the other cases, the maximum Q-contribution of CDs appears at tcrit. The CDSTR MV behaves inductive in time periods 0:00 to 9:20 a.m. and 03:00 to 12:00 p.m. to compensate the capacitive power produced by the cable. 3.1.1. Distribution Grid with Cable Conductors in MV Level As the PV injection increases from 9:00 a.m., the L(U)s included in LV_link-grid begin to consume inductive power to eliminate the upper voltage limit violations. To compensate the additionally required inductive power, the reactive power production of CDSTR MV changes from inductive to capacitive, reaching the maximum Q-injection of 3.17 Mvar. When CDs are installed in distribution substation, they behave purely capacitive with a maximum Q-injection of 3.67 and 4.24 Mvar for the cases “CDDTR MV ” and “CDDTR LV ”, respectively. Energies 2019, 12, 3865 13 of 23 Figure 11c shows the reactive power exchange between HV_ and MV_link-grid. In the control setups “no control” and “no CDs”, the MV_link-grid draws reactive power from the HV_link-grid between approx. 9:30 a.m. and 3:00 p.m. In the remaining time horizon, the MV_link-grid injects reactive power into the HV_link-grid. In the case “no control”, two peaks of QHV MV,t are identified: one at tcrit, where the MV_link-grid draws 0.70 Mvar from the HV_link-grid; and the other at 10 p.m., where the MV_link-grid injects 0.80 Mvar into the HV_link-grid. This behaviour is caused by the capacitive nature of the load in the evening and the cable structure of the MV_link-grid. In the case of “no CDs”, the MV_link-grid draws the maximum reactive power of 2.65 Mvar from the HV_link-grid at tcrit. If the CD is installed at the STR MV-busbar, no reactive power is exchanged between HV_ and MV_link-grid over the all-time horizon. In the control setups “CDDTR MV ” and “CDDTR LV ”, the MV_link-grid injects reactive power into HV_link-grid over the all-time horizon, showing their minimum Q-injection of 0.11 Mvar at tcrit. Energies 2019, 12, x FOR PEER REVIEW 13 of 23 d h h l f h d b d I ll l Figure 11d shows the active power losses of the distribution grid. In all cases, losses increase drastically between approx. 8:00 a.m. and 4:30 p.m. due to the PV injection. The maximum values appear at tcrit, where the control setup “CDDTR LV ” provokes the highest grid losses of 1.33 MW. Figure 11d shows the active power losses of the distribution grid. In all cases, losses increase drastically between approx. 8:00 a.m. and 4:30 p.m. due to the PV injection. The maximum values appear at tcrit , where the control setup “CDLV DTR ” provokes the highest grid losses of 1.33 MW. 3.1.1. Distribution Grid with Cable Conductors in MV Level Fi 11 f h h STR d DTR l di i l I ll STR d DTR Figure 11e,f show the STR and mean DTR loading, respectively. In all cases, STR and mean DTR loading increase drastically between approx. 8:00 a.m. and 4:30 p.m. due to PV injections, reaching their maximum values at tcrit. For the STR loading, the highest value of 53.28% is reached for the control setup “no CDs”; while the lowest one of 50.35% is reached for the “CDDTR LV ” case. For the mean DTR loading, the highest value of 68.11% is reached for the control setup “CDDTR MV ”; while the lowest one of 60.74% is reached for the “CDDTR LV ” case. Figure 11e,f show the STR and mean DTR loading, respectively. In all cases, STR and mean DTR loading increase drastically between approx. 8:00 a.m. and 4:30 p.m. due to PV injections, reaching their maximum values at tcrit . For the STR loading, the highest value of 53.28% is reached for the control setup “no CDs”; while the lowest one of 50.35% is reached for the “CDLV DTR ” case. For the mean DTR loading, the highest value of 68.11% is reached for the control setup “CDMV DTR ”; while the lowest one of 60.74% is reached for the “CDLV DTR ” case. 3.1.2. Distribution Grid with Overhead Conductors in MV Level 3.1.2. Distribution Grid with Overhead Conductors in MV Level Figure 12 shows the voltage profiles of the MV_link-grid with overhead line conductors and the backmost LV_link-grid for the critical time-point tcrit. Figure 12 shows the voltage profiles of the MV_link-grid with overhead line conductors and the backmost LV_link-grid for the critical time-point tcrit . Figure 12 shows the voltage profiles of the MV_link-grid with overhead line conductors and the backmost LV_link-grid for the critical time-point tcrit. Figure 12 shows the voltage profiles of the MV_link-grid with overhead line conductors and the backmost LV_link-grid for the critical time-point tcrit . Figure 12. Voltage profile of the MV_link-grid with overhead line conductors and the backmost LV_link-grid at t = tcrit . Figure 12. Voltage profile of the MV_link-grid with overhead line conductors and the backmost LV_link-grid at t = tcrit. Figure 12. Voltage profile of the MV_link-grid with overhead line conductors and the backmost LV_link-grid at t = tcrit . Figure 12. Voltage profile of the MV_link-grid with overhead line conductors and the backmost LV_link-grid at t = tcrit. Without any var control, the upper voltage limit is violated with a voltage limit violation index of 58.38. In the case of “no CDs” and “CDMV STR ”, no voltage limit violations appear, but if CDs are installed at distribution substation, the upper voltage limit is violated with a voltage limit violation index of 1.79 and 7.73, respectively, for the cases “CDMV DTR ” and “CDLV DTR ”. Without any var control, the upper voltage limit is violated with a voltage limit violation index of 58.38. In the case of “no CDs” and “CDSTR MV ”, no voltage limit violations appear, but if CDs are installed at distribution substation, the upper voltage limit is violated with a voltage limit violation index of 1.79 and 7.73, respectively, for the cases “CDDTR MV ” and “CDDTR LV ”. 14 of 23 Energies 2019, 12, 3865 Figure 13 shows the behaviour of the distribution grid with overhead line conductors in MV level over the 24 h time horizon for all control setups. Figure 13a shows the total reactive power consumption of all L(U)s connected at the distribution grid. When “no control” is applied, no L(U)s are installed and as a result, there is no Q-consumption. The maximum total Q-consumption of L(U)s is reached at tcrit for all cases. 3.1.2. Distribution Grid with Overhead Conductors in MV Level 3.1.2. Distribution Grid with Overhead Conductors in MV Level The lowest value of 2.11 Mvar is achieved when no CDs are applied, while the highest one of 4.40 Mvar is reached when CDs are installed at the secondary sides of DTRs. LV_link-grid at t = tcrit . Without any var control, the upper voltage limit is violated with a voltage limit violation index of 58.38. In the case of “no CDs” and “CDMV STR ”, no voltage limit violations appear, but if CDs are installed at distribution substation, the upper voltage limit is violated with a voltage limit violation index of 1.79 and 7.73, respectively, for the cases “CDMV DTR ” and “CDLV DTR ”. while the highest one of 4.40 Mvar is reached when CDs are installed at the secondary sides of DTRs. (a) (b) Figure 13. Cont. Energies 2019, 12, x FOR PEER REVIEW 14 of 23 (c) (d) (e) (f) Figure 13. Behaviour of the distribution grid with overhead line conductors in MV level for a 24 hour time horizon and different control strategies: (a) Q-consumption of L(U)s; (b) Q-contribution of CDs; (c) Q-exchange between HV_ and MV_link-grid; (d) active power losses; (e) STR loading; (f) mean DTR loading. Figure 13 shows the behaviour of the distribution grid with overhead line conductors in MV level over the 24 hour time horizon for all control setups. Figure 13a shows the total reactive power Figure 13. Behaviour of the distribution grid with overhead line conductors in MV level for a 24 h time horizon and different control strategies: (a) Q-consumption of L(U)s; (b) Q-contribution of CDs; (c) Q-exchange between HV_ and MV_link-grid; (d) active power losses; (e) STR loading; (f) mean DTR loading. Figure 13b shows the reactive power contribution of the CDs. In the cases of “no control” and “no CDs”, no CDs are installed, thus no Q-contribution is expected. If CDs are installed, they behave l iti hi th i i Q t ib ti t t Th l t l f 4 52 M i (a) Energies 2019, 12, x FOR PEER REVIEW (b) 14 of 23 (b) (a) R REV 13. Cont. Figure (c) (d) (c) (f) (e) (f) (e) Figure 13. 3.1.2. Distribution Grid with Overhead Conductors in MV Level 3.1.2. Distribution Grid with Overhead Conductors in MV Level Behaviour of the distribution grid with overhead line conductors in MV level for a 24 hour time horizon and different control strategies: (a) Q-consumption of L(U)s; (b) Q-contribution of CDs; (c) Q-exchange between HV_ and MV_link-grid; (d) active power losses; (e) STR loading; (f) mean DTR loading. Figure 13. Behaviour of the distribution grid with overhead line conductors in MV level for a 24 h time horizon and different control strategies: (a) Q-consumption of L(U)s; (b) Q-contribution of CDs; (c) Q-exchange between HV_ and MV_link-grid; (d) active power losses; (e) STR loading; (f) mean DTR loading. Figure 13 shows the behaviour of the distribution grid with overhead line conductors in MV level over the 24 hour time horizon for all control setups. Figure 13a shows the total reactive power consumption of all L(U)s connected at the distribution grid. When “no control” is applied, no L(U)s Figure 13b shows the reactive power contribution of the CDs. In the cases of “no control” and “no CDs”, no CDs are installed, thus no Q-contribution is expected. If CDs are installed, they behave purely capacitive, reaching their maximum Q-contribution at tcrit. The lowest value of 4.52 Mvar is Energies 2019, 12, 3865 15 of 23 achieved when CDs are installed at the DTRs’ primary sides, while the highest one of 5.11 Mvar is reached when they are installed at the DTRs’ secondary sides. Figure 13c shows the reactive power exchange between HV_ and MV_link-grid. In the control setup “no control”, the MV_link-grid injects reactive power into the HV_link-grid between approx. 9:00 p.m. and 11:15 p.m. In the remaining time horizon, the MV_link-grid draws reactive power from the HV_link-grid; two peaks of QHV MV,t are identified: one at tcrit, where the MV_link-grid draws 1.97 Mvar from the HV_link-grid; and the other at 10 p.m., where the MV_link-grid injects 66 kvar into the HV_link-grid. This behaviour is caused by the capacitive nature of the load in the evening. If the CD is installed at the STR MV-busbar, no reactive power is exchanged between HV_ and MV_link-grid over the all-time horizon. In all other cases, the MV_link-grid draws reactive power from the HV_link-grid over the all-time horizon, with the maximum Q-exchange appearing at tcrit. In the case of “no CDs”, the MV_link-grid draws the maximum reactive power of 4.04 Mvar from the HV_link-grid. Figure 13d shows the active power losses of the distribution grid. 3.1.2. Distribution Grid with Overhead Conductors in MV Level 3.1.2. Distribution Grid with Overhead Conductors in MV Level In all cases, losses increase drastically between approx. 8:00 a.m. and 4:30 p.m. due to the PV injection. The maximum values appear at tcrit, where the control setup “CDDTR LV ” provokes the highest grid losses of 1.72 MW. Figure 13e,f show the STR and mean DTR loading, respectively. In all cases, STR and mean DTR loading increase drastically between approx. 8:00 a.m. and 4:30 p.m. due to PV injections, reaching their maximum values at tcrit. For the STR loading, the highest value of 54.03% is reached for the control setup “no CDs”; while the lowest one of 48.50% is reached for the “CDDTR LV ” case. For the mean DTR loading, the highest value of 69.42% is reached for the control setup “CDDTR MV ”; while the lowest one of 58.49% is reached for the “CDDTR LV ” case. 3.1.3. Effect of CD Placement of CDs (-) Cable CDSTR MV 0.0000 6.5051 34.4286 17.9049 23.9071 1 CDDTR MV 0.0000 6.6000 34.0946 18.5570 23.9116 32 CDDTR LV 0.0016 7.2863 33.8003 19.0885 23.5122 32 Overhead CDSTR MV 0.0000 8.3320 32.5502 17.4605 23.9019 1 CDDTR MV 0.1484 8.4739 32.3078 17.4682 24.1743 32 CDDTR LV 0.8307 8.8536 31.9763 17.3888 22.3732 32 The CD placement at the STR MV-bus bar supports the elimination of voltage limit violations in all cases, while the contrary is noticed when the CDs are placed on the DTR level. The placement of CDs on the MV side of DTRs provokes violations of the upper voltage limit in the case of overhead conductor type, VIavg = 0.1484. Meanwhile, when CDs are installed at the LV side of DTRs, limit violations appear in both cases, cable and overhead, with a VIavg of 0.0016 and 0.8307, respectively. The CD placement at the STR MV-bus bar supports the elimination of voltage limit violations in all cases, while the contrary is noticed when the CDs are placed on the DTR level. The placement of CDs on the MV side of DTRs provokes violations of the upper voltage limit in the case of overhead conductor type, VIavg = 0.1484. Meanwhile, when CDs are installed at the LV side of DTRs, limit violations appear in both cases, cable and overhead, with a VIavg of 0.0016 and 0.8307, respectively. The CD placement at the STR MV-bus bar supports the elimination of voltage limit violations in all cases, while the contrary is noticed when the CDs are placed on the DTR level. The placement of CDs on the MV side of DTRs provokes violations of the upper voltage limit in the case of overhead conductor type, VIavg = 0.1484. Meanwhile, when CDs are installed at the LV side of DTRs, limit violations appear in both cases, cable and overhead, with a VIavg of 0.0016 and 0.8307, respectively. 3.1.3. Effect of CD Placement Table 4 shows the relevant criteria used for the evaluation of different CD locations within the distribution grid with cable or overhead conductors in MV level. The energy loss, the average STR and DTR loadings, the average voltage limit violation index, and the energy exchange between MV_ and HV_link-grid are calculated according to Equations (A1)–(A5), Appendix B. Furthermore, one of the criteria applies to the number of compensation devices to be installed in each case. Table 4. Criteria used for the evaluation of different CD locations within the distribution grid with cable or overhead conductors in MV level. Table 4. Criteria used for the evaluation of different CD locations within the distribution grid with cable or overhead conductors in MV level. Conductor Type in MV Level Control Setup VIavg (-) Eloss (MWh) EHV MV (MWh) LoadingSTR avg (%) LoadingDTRs avg (%) No. of CDs (-) Cable CDSTR MV 0.0000 6.5051 34.4286 17.9049 23.9071 1 CDDTR MV 0.0000 6.6000 34.0946 18.5570 23.9116 32 CDDTR LV 0.0016 7.2863 33.8003 19.0885 23.5122 32 Overhead CDSTR MV 0.0000 8.3320 32.5502 17.4605 23.9019 1 CDDTR MV 0.1484 8.4739 32.3078 17.4682 24.1743 32 CDDTR LV 0.8307 8.8536 31.9763 17.3888 22.3732 32 The CD placement at the STR MV-bus bar supports the elimination of voltage limit violations in all cases, while the contrary is noticed when the CDs are placed on the DTR level. The placement of CDs on the MV side of DTRs provokes violations of the upper voltage limit in the case of overhead conductor type, VIavg = 0.1484. Meanwhile, when CDs are installed at the LV side of DTRs, limit violations appear in both cases, cable and overhead, with a VIavg of 0.0016 and 0.8307, respectively. Regarding the active power loss over the all-time horizon, i.e., the active energy loss, a clear trend is observed for both conductor types in MV level: while the CD placement at the STR MV-bus bar causes the lowest energy losses of 6.5051 and 8.3320 MWh for cable and overhead conductors in Table 4. Criteria used for the evaluation of different CD locations within the distribution grid with cable or overhead conductors in MV level. Conductor Type in MV Level Control Setup VIavg (-) Eloss (MWh) EHV MV (MWh) LoadingSTR avg (%) LoadingDTRs avg (%) No. 3.1.3. Effect of CD Placement g Regarding the active power loss over the all-time horizon, i.e., the active energy loss, a clear trend is observed for both conductor types in MV level: while the CD placement at the STR MV-bus bar causes the lowest energy losses of 6.5051 and 8.3320 MWh for cable and overhead conductors in Regarding the active power loss over the all-time horizon, i.e., the active energy loss, a clear trend is observed for both conductor types in MV level: while the CD placement at the STR MV-bus bar causes the lowest energy losses of 6.5051 and 8.3320 MWh for cable and overhead conductors in Regarding the active power loss over the all-time horizon, i.e., the active energy loss, a clear trend is observed for both conductor types in MV level: while the CD placement at the STR MV-bus bar causes the lowest energy losses of 6.5051 and 8.3320 MWh for cable and overhead conductors in Energies 2019, 12, 3865 16 of 23 MV level, respectively, the CD placement at DTRs’ LV-bus bars causes the highest ones of 7.2863 and 8.8536 MWh. level, respectively, the CD placement at DTRs’ LV-bus bars causes the highest ones of 7.2863 and 36 MWh. These results show that the compensation of the reactive power in distribution substation significantly deteriorates the effectiveness of L(U)-control, leading to very high losses and voltage limit violations in LV level. The amount of active power flowing from MV_ to HV_link-grid over the all-time horizon, i.e., active energy exchange, depends on the placement of the CDs, Figure 14. Their placement in the supplying substation supports the maximum active energy exchange in all cases. Energies 2019, 12, x FOR PEER REVIEW 16 of 23 (a) (b) Figure 14. Active energy exchange between MV_and HV_link-grid for different conductor types in MV level: (a) cable; (b) overhead line. Figure 14. Active energy exchange between MV_and HV_link-grid for different conductor types in MV level: (a) cable; (b) overhead line. (b) (a) (b) (a) Figure 14. Active energy exchange between MV_and HV_link-grid for different conductor types in MV level: (a) cable; (b) overhead line. Figure 14. Active energy exchange between MV_and HV_link-grid for different conductor types in MV level: (a) cable; (b) overhead line. Anyhow, due to the reduced active energy exchange, Figure 14, the lowest STR loading value results for this control setup. 3.1.3. Effect of CD Placement The highest average STR loading of 19.0885% for the grid with cable conductors in MV level appears when CDs are installed at the DTRs’ LV-bus bars. In the case of overhead conductor type, the highest value of 17.4682% is reached when CDs are installed at MV- bus bars of the DTRs. The number of CDs to install is very different. When placed at the STR MV-side, one CD per bus bar is required (or two CDs in double bus bar configurations), while the placement in the distribution The STR and DTR loading depends on the active and reactive power flows. In our simulations the CD set on the MV-bus bar of the supplying substation completely compensates the reactive power exchange at all times. Thus, the average STR loading is exclusively provoked by the active power flow, achieving the minimum value of 17.9049% for the grid with cable conductors in MV level. In the case of overhead conductor type, the lowest value of 17.3888% is reached when the CDs are placed at the LV side of the DTRs. In this case, active and reactive energy flow through the STR, because the CDs compensate the reactive power in DTR level. q ( g ) p substations normally requires as many CD devices as there are DTRs in place; which are in our case 32 CDs. Table 4. Criteria used for the evaluation of different CD locations within the distribution grid with cable or overhead conductors in MV level. Anyhow, due to the reduced active energy exchange, Figure 14, the lowest STR loading value results for this control setup. The highest average STR loading of 19.0885% for the grid with cable conductors in MV level appears when CDs are installed at the DTRs’ LV-bus bars. In the case of overhead conductor type, the highest value of 17.4682% is reached when CDs are installed at MV-bus bars of the DTRs. Conductor Type in MV Level Control Setup VIavg (-) Eloss (MWh) EMV HV (MWh) Loadingavg STR (%) Loadingavg DTRs (%) No. of CDs (-) CDMV STR 0.0000 6.5051 34.4286 17.9049 23.9071 1 The number of CDs to install is very different. 3.1.3. Effect of CD Placement When placed at the STR MV-side, one CD per bus bar is required (or two CDs in double bus bar configurations), while the placement in the distribution substations normally requires as many CD devices as there are DTRs in place; which are in our case 32 CDs. Cable MV CDMV DTR 0.0000 6.6000 34.0946 18.5570 23.9 DTR Figure 15 depicts a qualitative representation of the results given in Table 4. Cable CDLV DTR 0.0016 7.2863 33.8003 19.0885 23.5122 32 Overhead CDMV STR 0.0000 8.3320 32.5502 17.4605 23.9019 1 CDMV DTR 0.1484 8.4739 32.3078 17.4682 24.1743 32 CDDTR 0 8307 8 8536 31 9763 17 3888 22 3732 32 The effectiveness of the solution depends on the size of the surface of the pentagon. The smaller the surface of the latter, the more effective is the solution. Results show that the distribution grid performs best when CDs are placed at the MV-bus bar of the STR. 17 of 23 Energies 2019, 12, 3865 Figure 15 dep on a distribution (a) (b) Figure 15. Qualitative representation of the criteria used for the evaluation of various CD placements on a distribution grid with different conductor types in MV level: (a) cable; (b) overhead line. The effectiveness of the solution depends on the size of the surface of the pentagon. The smaller e surface of the latter, the more effective is the solution. Results show that the distribution grid rforms best when CDs are placed at the MV-bus bar of the STR. 2. Discussion Results have shown that the VVC chain strategy supports the integration of rooftop PVs on a ge scale. Figure 16 shows the most suitable setup of the VVC chain for a distribution grid with the ghest PV share operated by one DSO. The VVC chain is designed with a minimum number of condary and primary control units to reduce the associated investments and operating costs. erived from Equation (1), the VVC chain in the Y-axis is presented by: Two grid-link types, i.e., MV_ and CP_grid-link, are designed in this case. 3.1.3. Effect of CD Placement For the LV level, no id-link is designed for four reasons: MV_ and LV_link-grids have the same operator and as a result they do not have external interfaces between each other [16]; No reactive power is exchanged between LV_link-grids and CPs because of the Q-autarky of the latter; VCY-axis={VVSCMV൫voltPCOLTC MV , varPCRD MV, varPCDG MV, VVSCngb MV, varCnsMV HV൯, varLCL(U) LV , VSCCP൫varPCinv CP, varCnsCP LV = 0൯}. (13) (a) The effectiveness of the solution depends on t he surface of the latter, the more effective is the performs best when CDs are placed at the MV-bus .2. Discussion Results have shown that the VVC chain strat arge scale. Figure 16 shows the most suitable setu highest PV share operated by one DSO. The VVC econdary and primary control units to reduce Derived from Equation (1), the VVC chain in the Y Two grid-link types, i.e., MV_ and CP_grid-li grid-link is designed for four reasons: a) MV_ and LV_link-grids have the same ope VVCY-axis={VVSCMV൫voltPCOLTC MV , varPCRD MV, varPC VVSCCP൫varPCinv CP, varCnsCP LV = 0൯}. (b) he size of the surface of the pentagon. The smaller solution. Results show that the distribution grid bar of the STR. egy supports the integration of rooftop PVs on a p of the VVC chain for a distribution grid with the C chain is designed with a minimum number of the associated investments and operating costs. -axis is presented by: nk, are designed in this case. For the LV level, no rator and as a result they do not have external DG MV, VVSCngb MV, varCnsMV HV൯, varLCL(U) LV , (13) e latter, the mor hen CDs are plac gy supp of the V h i i (b) hey d (a) -grid Figure 15. Qualitative representation of the criteria used for the evaluation of various CD placements on a distribution grid with different conductor types in MV level: (a) cable; (b) overhead line. g p y interfaces between each other [16]; No reactive power is exchanged between LV_link-grids and CPs because of the Q-autarky of th l 3.2. Discussion latter; (c) No distribu For the LV level, no grid-link is designed for four reasons: 18 of 23 Energies 2019, 12, 3865 (a) MV_ and LV_link-grids have the same operator and as a result they do not have external interfaces between each other [16]; (a) MV_ and LV_link-grids have the same operator and as a result they do not have external interfaces between each other [16]; (b) No reactive power is exchanged between LV_link-grids and CPs because of the Q-autarky of the latter; (b) No reactive power is exchanged between LV_link-grids and CPs because of the Q-autarky of the latter; No distributed energy resources are foreseen to deliver reactive power to the LV_link-grids; (c) No distributed energy resources are foreseen to deliver reactive power to the LV_link-grids; (d) At each LV feeder with voltage limit violation potential is installed one locally con At each LV feeder with voltage limit violation potential is installed one locally controlled L(U). The MV_grid-link includes a VVSCMV that coordinates the Q-contribution of DGs, RDs and the neighbour grid-links with the voltage at the secondary side or OLTC position of the STR while respecting the reactive power constraints, varCnsHV MV, on the HV-MV intersection points and optimizing the network performance at the same time. The HV-MV intersection points correspond in many cases with TSO-DSO intersection points. varCnsHV MV is dynamic and therefore needs to be discussed and defined through real-time TSO and DSO cooperation in order to achieve an optimal solution, in both, transmission and distribution grids. Simulation results have shown that the uncontrolled reactive power flow provoked by the locally controlled units, varLCLV L(U), included in LV_link-grid is best compensated by the CD installed at the MV-bus bar of the STR. varLCLV L(U) s, installed at the end of each LV lateral with voltage limit violation potential, keep the voltage below the upper limit during the PV production period. The CP_grid-links have a VVSCCP varPCCP inv, varCnsLV CP= 0  that takes care to fully compensate the reactive power of the customer plant at all times. The interaction between the LV_link-grid and CPs in terms of reactive power is not existing and therefore no exchange of information between the DSO and the customers is required. Thus, the ICT challenge for the volt/var control is resolved at the LV level. The CPs inject or obtain exclusively active power into or from the LV_link-grid. 3.2. Discussion latter; (c) No distribu The VVSCMV is practically realized in real time in the frame of the industrial project Central Volt/var control in Presence of Distributed Generation (ZUQDE, Salzburg, Austria) [29,30]. The distribution state estimator was realized in a MV grid of European type with a symmetrical balanced behaviour. The VVSCMV voltPCMV OLTC, varPCMV DG, cosϕCnsHV MV  was successfully realized in closed loop. This project has indicated that the implementation of the proposed VVC chain strategy has great potential to be realized on an industrial scale. 3.2. Discussion latter; (c) No distribu Results have shown that the VVC chain strategy supports the integration of rooftop PVs on a large scale. Figure 16 shows the most suitable setup of the VVC chain for a distribution grid with the highest PV share operated by one DSO. The VVC chain is designed with a minimum number of secondary and primary control units to reduce the associated investments and operating costs. Derived from Equation (1), the VVC chain in the Y-axis is presented by: (d) At each LV feeder with voltage limit violation potential is installed one locally controlled L(U). The MV_grid-link includes a VVSCMV that coordinates the Q-contribution of DGs, RDs and the neighbour grid-links with the voltage at the secondary side or OLTC position of the STR while respecting the reactive power constraints, varCnsMV HV , on the HV-MV intersection points and optimizing the network performance at the same time The HV-MV intersection points correspond VVCY−axis= {VVSC MV voltPCMV OLTC, varPCMV RD , varPCMV DG, VVSCMV ngb, varCnsHV MV  , varLCLV L(U), VVSCCP varPCCP inv, varCnsLV CP= 0 o . (13) many cases with TSO-DSO intersection points. varCnsMV HV is dynamic and therefore needs to be ussed and defined through real-time TSO and DSO cooperation in order to achieve an optimal tion, in both, transmission and distribution grids. (13) o be mal Figure 16. The most suitable setup of the VVC chain for a distribution grid with the highest PV share operated by one DSO. Figure 16. The most suitable setup of the VVC chain for a distribution grid with the highest PV share operated by one DSO. Figure 16. The most suitable setup of the VVC chain for a distribution grid with the highest PV share operated by one DSO Figure 16. The most suitable setup of the VVC chain for a distribution grid with the highest PV share operated by one DSO. p y Two grid-link types, i.e., MV_ and CP_grid-link, are designed in this case. For the LV level, no grid-link is designed for four reasons: p y Two grid-link types, i.e., MV_ and CP_grid-link, are designed in this case. Appendix A Figure A1 shows the simplified one-line diagrams of the used MV_link-grid models. The YNyn6, 110 kV/20 kV, 18.5 MVA STR has a short-circuit voltage of 10.12% with a resistive part of 0.45%, core losses of 28.2 kW and an open-circuit current of 0.19%. Both MV_link-grids connect two PV-systems, each with a module-rating of PMV PV,r = 1 MW, and an inverter-rating of SMV inv,r = 1 MVA. Energies 2019, 12, x FOR PEER REVIEW 19 of 23 (a) (b) Figure A1. Detailed one-line diagrams of the MV_link-grid models with different conductor types: (a) cable; (b) overhead line. Figure A1. Detailed one-line diagrams of the MV_link-grid models with different conductor types: (a) cable; (b) overhead line. (a) (a) (b) Figure A1. Detailed one-line diagrams of the MV_link-grid models with different conductor types: (a) cable; (b) overhead line. Figure A1. Detailed one-line diagrams of the MV_link-grid models with different conductor types: (a) cable; (b) overhead line. Figure A1a shows the cable MV_link-grid. The cable segments have a resistance of 0.206 Ω/km, a reactance of 0.1222 Ω/km, a capacitance of 254 nF/km, and a limiting current of 419 A. Figure A1b shows the overhead line MV_link-grid. The line segments have a resistance of 0.358 Ω/km, a reactance of 0.376 Ω/km, a capacitance of 9.6 nF/km, and a limiting current of 350 A. Figure A1a shows the cable MV_link-grid. The cable segments have a resistance of 0.206 Ω/km, a reactance of 0.1222 Ω/km, a capacitance of 254 nF/km, and a limiting current of 419 A. Figure A1b shows the overhead line MV_link-grid. The line segments have a resistance of 0.358 Ω/km, a reactance of 0.376 Ω/km, a capacitance of 9.6 nF/km, and a limiting current of 350 A. 4. Conclusions Due to the current trends in distribution grids, i.e., implementation of distributed generation with local volt/var control, the local voltage increases, the process of reactive power management throughout the power grid becomes very difficult and the information and communications technology (ICT) related challenge follows up. Therefore, solving the problem of voltage control and reactive power management is of utmost importance to utilities, as they may favor the large scale integration of distributed generation. Results of this investigation have shown that the VVC chain strategy, which roots on LINK-based holistic architecture, supports the integration of rooftop PVs on a large scale. The inclusion of the L(U)+CP_Q-autarky control ensemble in the control chain eliminates the violation of the upper voltage limit at low voltage level, as well as the ICT challenges and social problems. The VVC chain is designed with a minimum number of secondary and primary control units to reduce the associated investments and operating costs. It consists of two volt/var secondary controls; one at medium voltage level (which also controls the TSO-DSO reactive power exchange), the other at the customer plant level. MV and LV grids have the best performance in terms of losses, loading of distribution and supplying transformers, number of installed compensation devices and active power production, when the compensation device is placed at the MV bus bar of supplying transformer. p pp y g One part of the VVC chain, VVSCMV, is industrially realized in real time in another project. Nevertheless, the industrial implementation of the entire VVC chain is the next step to prove the practical relevance of the results of this study. 19 of 23 Energies 2019, 12, 3865 Author Contributions: methodology, D.-L.S.; conceptualization A.I.; writing—original draft preparation, D.-L.S.; writing—review and editing, A.I. Funding: This research received no external funding. Acknowledgments: The authors acknowledge the TU Wien University Library for financial support throug Open Access Funding Programme. Acknowledgments: The authors acknowledge the TU Wien University Library for financial support through its Open Access Funding Programme. Conflicts of Interest: The authors declare no conflict of interest. flicts of Interest: The authors declare no conflict of interest. Appendix B Appendix B Different criteria are used to evaluate the simulated control setups in both distribution grid Different criteria are used to evaluate the simulated control setups in both distribution grid models. l Different criteria are used to evaluate the simulated control setups in both distribution grid models. A i l Th l Ploss d f h l d fl Different criteria are used to evaluate the simulated control setups in both distribution grid models. Active power loss—The active power loss Ploss t at time-point t is direct output of the load-flow i l ti d i l d th ti l f th STR d ll h d li bl d DTR Different criteria are used to evaluate the simulated control setups in both distribution grid models. Active power loss—The active power loss Pt loss at time-point t is direct output of the load-flow simulations and includes the active power losses of the STR and all overhead lines, cables and DTRs. Different criteria are used to evaluate the simulated control setups in both distribution grid models. Active power loss—The active power loss Ploss t at time-point t is direct output of the load-flow simulations and includes the active power losses of the STR and all overhead lines, cables and DTRs. The active energy loss Eloss over the all-time horizon is: l-time horizon is: Eloss = ∆t · ∑ Pt loss ∀t . (B1) Eloss = ∆t· X ∀t Ploss t . (A1) (B1) (A1) ∆t = 15 min is the time-step used for the simulations. ∆t = 15 min is the time-step used for the simulations. where ∆t 15 min is the time step used for the simulations. Average STR loading—The STR loading Loadingt STR at time-point t is a direct output of the load- flow simulations The average STR loading LoadingSTR over the all time horizon is: Average STR loading—The STR loading LoadingSTR t at time-point t is a direct output of the load-flow simulations. The average STR loading LoadingSTR avg over the all-time horizon is: avg Loadingavg STR = ∑ Loadingt STR ∀t N , (B2) LoadingSTR avg = P ∀t LoadingSTR t N , (A2) (B2) (A2) E i 2019 12 3865 20 f 23 Energies 2019, 12, 3865 20 of 23 where N = 96 is the number of conducted load-flow simulations per control setup and distribution grid model. Appendix B Appendix B Average DTR loading—The mean loading of all DTRs LoadingDTRs t at time-point t is calculated according to Equation (11). The average DTRs’ loading LoadingDTRs avg over the all-time horizon is: LoadingDTRs avg = P ∀t LoadingDTRs t N (A3) (A3) Average voltage limit violation index—The voltage limit violation index VIt at time-point t is calculated according to Equation (12). The average voltage limit violation index over the all-time horizon is: P VI Average voltage limit violation index—The voltage limit violation index VIt at time-point t is calculated according to Equation (12). The average voltage limit violation index over the all-time horizon is: VIavg = P ∀t VIt N (A4) (A4) Active energy exchange—The active power flow from MV_ to HV_link-grid PHV MV,t at the STR primary side at time-point t is direct output of the load-flow simulations. The total active energy exchange EHV MV between MV_ and HV_link-grid over the all-time horizon is: EHV MV = ∆t· X ∀t PHV MV,t (A5) (A5) Table A2 lists the nomenclature of all variables used for calculations. Table A2 lists the nomenclature of all variables used for calculations. Table A2. Nomenclature of all variables used for calculations. CP,Z t , CP,I t , CP,P t Active power ZIP coefficients for time-point t. CQ,Z t , CQ,I t , CQ,P t Reactive power ZIP coefficients for time-point t. EHV MV Active energy exchange between MV_ and HV_link-grid over the all-time horizon. Eloss Active energy loss over the all-time horizon. f P,load t Active power load profile factor at time-point t. f Q,load t Reactive power load profile factor at time-point t. f P,PV t Active power production profile factor at time-point t. LoadingDTR k,t Loading of the DTR k at time-point t. LoadingDTRs t Mean loading of all DTRs at time-point t. LoadingDTRs avg The average DTRs’ loading over the all-time horizon. LoadingSTR t The STR loading at time-point t. LoadingSTR avg The average STR loading over the all-time horizon. mt Number of LV_link-grid nodes that violate the upper voltage limit at time-point t. nt Number of LV_link-grid nodes that violate the lower voltage limit at time-point t. N Number of conducted load-flow simulations per control setup and distribution grid model. PCP inv,i,t Active power production of the PV-system of the CP i at time-point t. PCP load,i,t Active power consumption of the loads of the CP i at time-point t. PCP PV,r Module-rating of the PV-system of each CP. PLV CP,i,t Active power flow from the CP i to LV_link-grid at time-point t. Pload nom,t Active power consumption of each CP’s load for nominal grid voltage at time-point t. Pload peak Peak active power demand of each CP’s load. PMV inv,t Active power production of each PV-system connected to the MV_link-grid at time-point t. PMV PV,r Module-rating of each PV-system connected to the MV_link-grid. Ploss t Active power losses of the distribution grid at time-point t. PHV MV,t Active power flow from the MV_ to HV_link-grid at time-point t. QCP inv,i,t Reactive power production of the PV-system of the CP i at time-point t. QCP load,i,t Reactive power consumption of the loads of the CP i at time-point t. QLV CP,i,t Reactive power flow from the CP i to LV_link-grid at time-point t. Qload nom,t Reactive power consumption of each CP’s load for nominal grid voltage at time-point t. QL(U) tot,t Total reactive power consumption of all L(U)s included in the LV_link-grids at time-point t. Appendix C Table A1 lists all the abbreviations and the corresponding full forms used in the paper. Table A1. Abbreviations and corresponding full forms. BLiN Boundary link node MV Medium voltage BPN Boundary producer node OLTC On load tap changer BSN Boundary storage node OpEx Operational expenditures CapEx Capital expenditures PC Primary control CD Compensation device PV Photovoltaic CP Customer plant RD Reactive device DG Distributed generation RPM Reactive power margin DSO Distribution system operator SC Secondary control DSt Distributed storage STR Supplying transformer DTR Distribution transformer TSO Transmission system operator HV High voltage VCRD Voltage control reactive device ICT Information and communications technology VVC Volt/var control LC Local control VVSC Volt/var secondary control LV Low voltage cos ϕCnsHV MV cos ϕ constraint at the border to the HV_link-grid varPCLV DG/DSt Primary controls of DGs and DSts connected to the LV_link-grids varCnsHV MV Var constraint at the border to the HV_link-grid varPCLV RD Primary controls of RDs included in the LV_link-grids varCnsMV LV Var constraint at the border to the MV_link-grid varPCCP inv Primary controls of PV-inverters connected to CP_link-grids varCnsLV CP Var constraint at the border to the LV_link-grid voltPCMV OLTC Primary controls of the STR or other transformers with OLTC included in the MV_link-grid varLCLV L(U) Local controls of L(U)s included in the LV_link-grids VVSCMV VVSC of MV_grid-link varPCMV CD Primary controls of CDs included in the MV_link-grid VVSCLV VVSC of LV_grid-link varPCMV DG/DSt Primary controls of DGs and DSts connected to the MV_link-grid VVSCCP VVSC of CP_grid-link varPCMV RD Primary controls of RDs included in the MV_link-grid VVSCMV,LV ngb VVSC of neighbour MV_ or LV_grid-links varPCLV CD Primary controls of CDs included in the LV_link-grids VVSCLV,CP ngb VVSC of neighbour LV_ or CP_grid-links Table A1. Abbreviations and corresponding full forms. Table A1. Abbreviations and corresponding full forms. 21 of 23 Energies 2019, 12, 3865 Table A2 lists the nomenclature of all variables used for calculations. QCD tot,t Total reactive power contribution of all CDs included in MV_ or LV_link-grids at time-point t. QHV MV,t Reactive power flow from the MV_ to HV_link-grid at time-point t. SCP inv,r Inverter-rating of the PV-system of each CP. SMV inv,r Inverter-rating of each PV-system connected to the MV_link-grid. Ui,t Actual voltage at the BLiN of the CP i at time-point t. ULV nom Nominal voltage of LV_link-grids. Uupper u,t Voltage of the LV_link-grid node u with upper voltage limit violation at time-point t. Ulower v,t Voltage of the LV_link-grid node v with lower voltage limit violation at time-point t. Uupper lim Upper voltage limit. Ulower lim Lower voltage limit. VIt Voltage limit violation index at time-point t. 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Reference genes for normalising gene expression data in collagenase-induced rat intracerebral haemorrhage
BMC molecular biology
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PUBLISHED VERSION Cook, Naomi Louise; Kleinig, Timothy John; Van Den Heuvel, Corinna; Vink, Robert Reference genes for normalising gene expression data in collagenase-induced rat intracerebral haemorrhage BMC Molecular Biology, 2010; 11:7 Cook, Naomi Louise; Kleinig, Timothy John; Van Den Heuvel, Corinna; Vink, Robert Reference genes for normalising gene expression data in collagenase-induced rat intracerebral haemorrhage BMC Molecular Biology, 2010; 11:7 © 2010 Cook 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 electronic version of this article is the complete one and can be found online at: http://www.biomedcentral.com/1471-2199/11/7 PERMISSIONS http://www.biomedcentral.com/about/license Anyone is free:  to copy, distribute, and display the work;  to make derivative works;  to make commercial use of the work; Under the following conditions: Attribution  the original author must be given credit;  for any reuse or distribution, it must be made clear to others what the license terms of this work are;  any of these conditions can be waived if the authors gives permission. Tuesday, 29 October 2013 PERMISSIONS http://www.biomedcentral.com/about/license Anyone is free:  to copy, distribute, and display the work;  to make derivative works;  to make commercial use of the work; Under the following conditions: Attribution  the original author must be given credit;  for any reuse or distribution, it must be made clear to others what the license terms of this work are;  any of these conditions can be waived if the authors gives permission. Tuesday, 29 October 2013 PERMISSIONS http://www.biomedcentral.com/about/license Anyone is free:  to copy, distribute, and display the work;  to make derivative works;  to make commercial use of the work; Under the following conditions: Attribution  the original author must be given credit;  for any reuse or distribution, it must be made clear to others what the license terms of this work are;  any of these conditions can be waived if the authors gives permission. Tuesday, 29 October 2013 RESEARCH ARTICLE Open Access Open Access © 2010 Cook 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. Reference genes for normalising gene expression data in collagenase-induced rat intracerebral haemorrhage Naomi L Cook, Timothy J Kleinig, Corinna van den Heuvel, Robert Vink* Naomi L Cook, Timothy J Kleinig, Corinna van den Heuvel, Robert Vink* Abstract Background: The mechanisms of brain injury following intracerebral haemorrhage (ICH) are incompletely understood. Gene expression studies using quantitative real-time RT-PCR following ICH have increased our understanding of these mechanisms, however the inconsistent results observed may be related to inappropriate reference gene selection. Reference genes should be stably expressed across different experimental conditions, however, transcript levels of common reference genes have been shown to vary considerably. Reference gene panels have therefore been proposed to overcome this potential confounder. Results: The present study evaluated the stability of seven candidate reference genes in the striatum and overlying cortex of collagenase-induced ICH in rodents at survival times of 5 and 24 hours. Transcript levels of the candidate reference genes were quantified and ranked in order of stability using geNorm. When our gene of interest, transient receptor potential melastatin 2 (TRPM2), was normalised against each reference gene individually, TRPM2 mRNA levels were highly variable. When normalised to the four most stable reference genes selected for accurate normalisation of data, we found no significant difference between ICH and vehicle rats. Conclusion: The panel of reference genes identified in the present study will enable more accurate normalisation of gene expression data in the acute phase of experimental ICH. due to its wider dynamic range of quantification, higher sensitivity and precision, and a decreased risk of con- tamination compared to gel-based PCR [4-6]. Data obtained from real-time RT-PCR assays require a reli- able method of normalisation to correct for such factors as differences in quantity and quality of RNA samples and for efficiency of the reverse transcription reaction [7,8]. Several normalisation strategies exist, the most common being the use of one or more endogenously expressed reference genes [9]. A reference gene should be expressed at a stable level in different tissue types and be unaffected by the experimental condition under investigation [10]. However, several of the most com- monly used reference genes, such as glyceraldehyde-3- phosphate dehydrogenase (GAPDH) and b-actin, have been shown to have variable expression patterns and thus to be unsuitable for normalising real-time RT-PCR data in certain conditions [11-14]. The use of an inap- propriate reference gene for normalisation may lead to incorrect data interpretation [15]. In contrast, * Correspondence: robert.vink@adelaide.edu.au Discipline of Anatomy and Pathology, School of Medical Sciences, The University of Adelaide, Adelaide SA 5005, Australia Anyone is free: http://hdl.handle.net/2440/59375 http://hdl.handle.net/2440/59375 Cook et al. BMC Molecular Biology 2010, 11:7 http://www.biomedcentral.com/1471-2199/11/7 Background Intracerebral haemorrhage (ICH) accounts for around 10 - 15% of all strokes worldwide, with a higher propor- tion in Asian countries. Compared with ischaemic stroke, it causes disproportionate mortality and morbid- ity [1] and research efforts to understand its pathophy- siology are accordingly of high importance [2]. Brain injury following ICH is a dynamic process, due both to rebleeding and to downstream injury pathways activated by the blood clot [3]. However, no therapies addressing these secondary injury pathways have been proven beneficial. One approach to understanding these injury pathways is to study gene expression in animal models using quantitative real-time reverse transcription polymerase chain reaction (real-time RT-PCR). Real-time RT-PCR is the method of choice for quantifying mRNA transcripts Cook et al. BMC Molecular Biology 2010, 11:7 http://www.biomedcentral.com/1471-2199/11/7 Page 2 of 11 nm and 280 nm using a UV spectrophotometer. All samples had A260:A280 ratios of between 2.1 and 2.2, and A260:A230 ratios of between 2.2 and 2.6. RNA integrity was assessed using automated micro-capillary electrophoresis in the Agilent Bioanalyzer. An RNA Integrity Number (RIN) (reviewed in [36]) was assigned to each sample by the Agilent Bioanalyzer Expert 2100 software. All RIN values obtained were in the range of 8.6 - 9.4, representing high quality RNA with minimal degradation. nm and 280 nm using a UV spectrophotometer. All samples had A260:A280 ratios of between 2.1 and 2.2, and A260:A230 ratios of between 2.2 and 2.6. RNA integrity was assessed using automated micro-capillary electrophoresis in the Agilent Bioanalyzer. An RNA Integrity Number (RIN) (reviewed in [36]) was assigned to each sample by the Agilent Bioanalyzer Expert 2100 software. All RIN values obtained were in the range of 8.6 - 9.4, representing high quality RNA with minimal degradation. normalisation to the geometric mean of the expression of multiple reference genes [16] is considered to be a reliable and conservative approach [17]. Previous studies employing quantitative real-time RT- PCR in rat ICH models have utilised a single reference gene for normalisation, usually without including a reference gene validation protocol [18-22]. Therefore, as an example, we demonstrate the impact of different reference gene selection methods on determining the transcript levels of transient receptor potential melasta- tin 2 (TRPM2) channels following ICH. TRPM2 consists of a calcium-permeable channel fused to a protein kinase domain [23], and is highly expressed in the brain and immune cells [24,25]. Genes The relative standard curve method [30] was used to calculate TRPM2 mRNA level in the perihematomal region of ICH and vehicle rats with survival times of 5 h and 24 h, relative to each of the seven reference genes individually. Figure 1a shows TRPM2 mRNA level at 5 h post-ICH. Large variations were observed depending on which reference gene was used for normalisation. At the 24 hour time point, when TRPM2 data were nor- malised to GUSB only, a significant (p < 0.01) 1.85-fold increase in mean TRPM2 mRNA level was observed in the collagenase ICH rats compared to saline vehicles (Figure 1b). A significant (p < 0.05) 1.4-fold increase was found in TRPM2 mRNA level when data were nor- malised to HPRT only. When each of the other refer- ence genes was used individually for normalisation, there were no significant differences. Given the discre- pancy in these results and the large variation between samples, we proceeded with a reference gene evaluation study to determine the most stable reference genes in the collagenase model of ICH. Background It has been implicated in cell death pathways relating to oxidative stress [26,27] and ischaemia [28] and its mRNA level has been shown to increase in a time-dependent manner following transient middle cerebral artery occlusion in rats [29]. No studies to date have quantified the mRNA level of TRPM2 fol- lowing ICH. Reference Gene Stability The geNorm v3.5 application was used to determine the most stable reference genes out of the seven candidate genes tested. We were interested to see whether the sta- bility of the different reference genes was influenced by the different survival times of the collagenase ICH rats Results RNA Concentration and Integrity TRPM2 mRNA Level Normalised to Individual Reference Genes TRPM2 mRNA Level Normalised to Individual Reference Genes Real-time RT-PCR Primer pairs and reaction conditions were optimised using the standard cDNA pool prior to amplifying unknown cDNA samples. Melting curve analysis was consistent with a single reaction product for each gene, and product size was confirmed by 2% agarose gel elec- trophoresis stained with ethidium bromide and visua- lised with UV light (not shown). After validation of primer specificity, real-time PCR was carried out for unknown collagenase ICH and saline vehicle cDNA samples and serial dilutions from the cDNA pool. Reac- tion efficiencies were calculated automatically by the Corbett Rotor-Gene 6 software. The efficiency of all runs was between 95% and 105%. Minimum R2 values of 0.985 were accepted for each run, however, most runs had R2 values over 0.99. g We aimed to identify the most appropriate reference genes to normalise real-time RT-PCR data in the col- lagenase model of ICH in rats. Accordingly, real-time RT-PCR was used to measure transcript levels of seven reference genes: GAPDH, b-2-microglobulin (B2MG), RNA Polymerase II (POL2R), TATA Box Binding Pro- tein (TBP), hypoxanthine guanine phosphoribosyltrans- ferase (HPRT), succinate dehydrogenase complex, subunit A (SDHA) and b-glucuronidase (GUSB), plus our gene of interest, TRPM2. We initially normalised TRPM2 data to individual reference genes using the relative standard curve method [30]. Next, a reference gene validation study was conducted using the geNorm application [16] to rank reference genes in order of sta- bility. Other software programs are also available to assess the stability of candidate reference genes, includ- ing Normfinder [31] and BestKeeper [32]. Although dif- ferent algorithms are utilised in each application, studies have reported fairly consistent reference gene rankings between all three programs, particularly with regard to identifying the least stable genes [33-35]. However, we chose to use geNorm in the present study given that it also determines the number of reference genes required for accurate normalisation. The panel of stable reference genes identified by geNorm was then applied to normal- ise data regarding our gene of interest, TRPM2. The results presented herein will help to provide a guideline for selecting stable reference genes for normalising real- time RT-PCR data in future studies. RNA Concentration and Integrity The concentration of extracted total RNA was quanti- fied by measurement of the absorbance at 230 nm, 260 Cook et al. BMC Molecular Biology 2010, 11:7 http://www.biomedcentral.com/1471-2199/11/7 Page 3 of 11 Figure 1 Relative mRNA level of TRPM2 normalised to individual reference genes. TRPM2 mRNA level at (A) 5 hours and (B) 24 hours in the perihematomal brain region (RBG) of collagenase-induced ICH rats compared to saline vehicles. Bars represent mean of triplicate measurements from 5 animals per group ± SEM. Single asterisk denotes statistical significance (p < 0.05) between 24 h ICH and vehicle rats; double asterisk denotes statistical significance (p < 0.01) between 24 h ICH and vehicle rats, as assessed by t-tests. Figure 1 Relative mRNA level of TRPM2 normalised to individual reference genes. TRPM2 mRNA level at (A) 5 hours and (B) 24 hours in the perihematomal brain region (RBG) of collagenase-induced ICH rats compared to saline vehicles. Bars represent mean of triplicate measurements from 5 animals per group ± SEM. Single asterisk denotes statistical significance (p < 0.05) between 24 h ICH and vehicle rats; double asterisk denotes statistical significance (p < 0.01) between 24 h ICH and vehicle rats, as assessed by t-tests. In the combined group of all samples, B2MG and GUSB were found to be the most stable reference genes, while TBP was the least stable. Indeed, B2MG and GUSB were the two most stable reference genes in 3 out of the 5 groups tested, while TBP was ranked as the least stable gene in all 5 groups. Overall, HPRT dis- played variable stability; it was ranked among the least stable two genes in 3 out of 5 groups, but conversely was among the top two stable genes in the other 2 groups. POL2R was never one of the two most stable genes in any group, however, it was consistently ranked as the third most stable reference gene in 4 out of 5 groups. SDHA and GAPDH exhibited reasonable stabi- lity across the different groups. (5 hours and 24 hours), and whether the injury itself could produce localised fluctuations in reference genes between the perihematomal (RBG) and matching con- tralateral (LBG) regions of the brain. RNA Concentration and Integrity way ANOVA revealed statistically significant differences in relative mRNA level within the SDHA group. TRPM2 mRNA Level Normalised to Multiple Reference Genes way ANOVA revealed statistically significant differences in relative mRNA level within the SDHA group. The qBasePlus program was used to calculate the nor- malised mRNA level of TRPM2 in the perihematomal brain region at 5 h and 24 h post-ICH, relative to the most stable reference genes for each time point as deter- mined by geNorm (5 h: GAPDH, HPRT, POL2R and SDHA; 24 h: B2MG, GUSB and POL2R). qBasePlus uti- lises a modified version of the 2-ΔΔCt method of relative expression analysis [37] that takes into account multiple reference genes and gene-specific amplification efficien- cies [38]. There was no significant difference in mean TRPM2 transcript levels between collagenase ICH and saline vehicle animals (Figure 5). RNA Concentration and Integrity es in order of stability as determined by geNorm Ranking of candidate reference genes in order of stability as determined by geNorm. The most stable reference genes are listed at at the bottom. * indicates number of reference genes required for accurate normalisation of real-time RT-PCR data, revealed by pair and LBG refer to the perihematomal and the contralateral region, respectively, of collagenase ICH and vehicle rats. ce genes in order of stability as determined by geNorm. The most stable reference genes are listed at the top and the least stable mber of reference genes required for accurate normalisation of real-time RT-PCR data, revealed by pairwise variation analysis. RBG atomal and the contralateral region, respectively, of collagenase ICH and vehicle rats. Figure 2 shows geNorm output charts from the com- bined group of all collagenase-induced ICH and saline vehicle rats. All of the candidate reference genes had expression stability (M) values under the recommended cut-off value of 1.5 [16]. The highest M value obtained from all sample subsets tested was 0.867 (not shown). Figure 2a shows M values for each reference gene and ranks genes in order of stability from left to right. geN- orm does not discriminate between the two most stable genes since it relies on pairwise correlations. In Figure 2b, pairwise variation (V) analysis determined that four reference genes were optimal for accurate normalisation in the collective group, indicated at the V3/4 step of the chart. The ideal value of V (y-axis) is recommended to be under 0.15 [16] and was achieved in all sample subsets. Given that all of our candidate reference genes had M values well below 1.5 and that the pairwise variation threshold was achieved even when all seven genes were used for normalisation (V6/7 step in Figure 2b), we deter- mined whether even the least stable genes would serve as appropriate normalisation factors (i.e. meet the geNorm M and V thresholds) when the three most stable genes were removed from analysis. Accordingly, the values for B2MG, GUSB and POL2R were removed from the geN- orm input file and the stability of GAPDH, HPRT, SDHA and TBP were re-assessed. When these four reference genes were evaluated in the absence of the most stable genes, the M values were under 1.5 (not shown), how- ever, the threshold of 0.15 in the pairwise variation analy- sis was not achieved (far right bar in Figure 2b). RNA Concentration and Integrity Therefore, we used geNorm to ascertain the most stable reference genes in the following five groups: a collective group of all col- lagenase ICH and saline vehicle samples; 5 hour collage- nase ICH and saline vehicle samples; 24 collagenase ICH and all saline vehicle samples; RBG only from all collagenase ICH and saline vehicle samples; LBG only from all collagenase ICH and saline vehicle samples. The most stable reference genes for each group plus recommended number of reference genes for accurate normalisation are summarised in Table 1. (5 hours and 24 hours), and whether the injury itself could produce localised fluctuations in reference genes between the perihematomal (RBG) and matching con- tralateral (LBG) regions of the brain. Therefore, we used geNorm to ascertain the most stable reference genes in the following five groups: a collective group of all col- lagenase ICH and saline vehicle samples; 5 hour collage- nase ICH and saline vehicle samples; 24 collagenase ICH and all saline vehicle samples; RBG only from all collagenase ICH and saline vehicle samples; LBG only from all collagenase ICH and saline vehicle samples. The most stable reference genes for each group plus recommended number of reference genes for accurate normalisation are summarised in Table 1. Cook et al. BMC Molecular Biology 2010, 11:7 http://www.biomedcentral.com/1471-2199/11/7 Page 4 of 11 Table 1 Ranking of candidate reference genes in order of stability as determined by geNorm. All ICH and saline vehicle rats 5 hour ICH and saline vehicle rats 24 hour ICH and saline vehicle rats RBG of all ICH and saline vehicle rats LBG of all ICH and saline vehicle rats B2MG*/GUSB* GAPDH*/HPRT* B2MG*/GUSB* HPRT*/SDHA* B2MG*/GUSB* POL2R* POL2R* POL2R* GAPDH* POL2R* GAPDH* SDHA* GAPDH B2MG* SDHA* SDHA B2MG SDHA GUSB* GAPDH HPRT GUSB HPRT POL2R HPRT TBP TBP TBP TBP TBP Ranking of candidate reference genes in order of stability as determined by geNorm. The most stable reference genes are listed at the top and the least stable at the bottom. * indicates number of reference genes required for accurate normalisation of real-time RT-PCR data, revealed by pairwise variation analysis. RBG and LBG refer to the perihematomal and the contralateral region, respectively, of collagenase ICH and vehicle rats. Table 1 Ranking of candidate reference genes in order of stability as determined by geNorm. ndidate reference genes in order of stability as determined by geNorm. Discussion In this study we have evaluated reference genes for use as real-time RT-PCR normalising factors in collagenase- induced ICH. Based on our results, we conclude that use of a single normalisation reference gene is poten- tially hazardous, and suggest a panel of reference genes for more accurate transcript quantification. Real-time RT-PCR is a robust and sensitive technique for quantifying mRNA transcripts, and constitutes a powerful tool for increasing our understanding of the genomic response to ICH. It requires an appropriate normalisation strategy to control for error, the most common being the use of one or more endogenous reference genes [39]. A reference gene should be expressed at a stable level regardless of the experimental context, however, the expression of commonly used reference genes has been shown to vary considerably. Normalisation of real-time RT-PCR data using a single, non-validated reference gene may lead to inaccurate bio- logical conclusions, and previous studies have high- lighted the need to validate reference genes for each new experimental condition [7,9,15]. Figure 3 shows the average raw (i.e. not yet normal- ised) Ct values of the collagenase ICH and saline vehicle rats for each candidate reference gene. The raw Ct values of GAPDH were fairly consistent between groups, while HPRT exhibited more variation. The collagenase ICH samples generally had more raw Ct variation than the saline vehicle samples. In Figure 4, the raw Ct values were normalised to the geometric mean of the four most stable reference genes determined by geNorm (B2MG, GUSB, POL2R and GAPDH), in order to obtain the relative mRNA level of each reference gene. One- Our previous study [40] identified suitable reference genes for normalising real-time RT-PCR data in the rat Cook et al. BMC Molecular Biology 2010, 11:7 http://www.biomedcentral.com/1471-2199/11/7 Page 5 of 11 Figure 2 Ranking of candidate reference genes in order of stability by geNorm. (A) Expression stability values (M) of candidate reference genes in 5 h and 24 h collagenase-induced ICH and saline vehicle rats. geNorm ranks reference genes in order of least stable to most stable (left to right) by stepwise exclusion of the least stable gene. (B) Pairwise variation (V) analysis determines the optimal number of reference genes that should be used for accurate normalisation, with a threshold value of 0.15 [16]. In this case, the V3/4 step achieves the cut-off value, and therefore, the appropriate number of reference genes for accurate normalisation is four. Discussion The far right bar represents pairwise variation when the three most stable genes are removed from analysis and the stability of the remaining genes (GAPDH, HPRT, SDHA and TBP) are re-assessed. In this case the V threshold is not met. Figure 2 Ranking of candidate reference genes in order of stability by geNorm. (A) Expression stability values (M) of candidate reference genes in 5 h and 24 h collagenase-induced ICH and saline vehicle rats. geNorm ranks reference genes in order of least stable to most stable (left to right) by stepwise exclusion of the least stable gene. (B) Pairwise variation (V) analysis determines the optimal number of reference genes that should be used for accurate normalisation, with a threshold value of 0.15 [16]. In this case, the V3/4 step achieves the cut-off value, and therefore, the appropriate number of reference genes for accurate normalisation is four. The far right bar represents pairwise variation when the three most stable genes are removed from analysis and the stability of the remaining genes (GAPDH, HPRT, SDHA and TBP) are re-assessed. In this case the V threshold is not met. cerebral cortex and hippocampus following traumatic brain injury (TBI). The present study focuses on a dif- ferent neurological disorder with a different animal model, brain regions and survival times to our previous study. Our results demonstrate that the panels of stable reference genes in ICH are not the same as those identi- fied in our previous TBI reference gene study [40], con- sistent with the recommendation that reference gene validation be carried out whenever a new experimental variable is introduced [9]. Reference gene validation was thus a crucial prerequisite to gene expression analysis in ICH, and the current study has identified the appropri- ate panel of reference genes for further studies. Alternative normalisation strategies include the use of exogenous standards and normalisation to total RNA. Several groups have reported reliable normalisation with exogenous standards [41-43]. A known amount of exo- genous standard can be incorporated into the RNA extraction process, which will then be affected by the same experimental error as the RNA of interest [44]. Cook et al. BMC Molecular Biology 2010, 11:7 http://www.biomedcentral.com/1471-2199/11/7 Page 6 of 11 Figure 3 Average real-time RT-PCR cycle threshold (Ct) values o yet normalised) values from individual groups of collagenase ICH and represents the 25th and 75th percentile. Discussion Intriguingly, B2MG and GUSB were among the least stable genes in the 5 hour group, while GAPDH and HPRT were ranked 4th and 6th, respectively, at 24 hours post-ICH. These results clearly demonstrate that stability of a can- didate reference gene at one time point does not neces- sarily confer stability at another. Therefore, it is likely that the use of a single reference gene would be inade- quate for normalising real-time RT-PCR data from dif- ferent survival times of ICH. This is relevant because, as discussed, previous studies utilising real-time RT-PCR in rodent models of ICH generally have used one reference gene for normalisation without including a validation protocol. For example, HPRT was one of the most vari- ably expressed genes in our study, but has been used as a single normalising factor in other studies quantifying mRNAs of interest over a time course of ICH [21,22]. It is possible that studies using a single, non-validated reference gene for normalisation could fail to detect small changes in the mRNA species of interest [14], or erroneously conclude that changes have occurred [15]. Indeed, another study from our laboratory [40] demon- strated that the mRNA level of our gene of interest var- ied significantly when normalised to individual reference genes, but was far more reliable when a panel of vali- dated reference genes were used for normalisation. However, exogenous standards can be difficult to pro- duce and are subject to degradation, and do not control for differences in quality of RNA template [17]. Another approach is to normalise to total RNA mass, which con- sists predominantly of ribosomal RNA (rRNA) mole- cules. However, it has been shown that rRNA content is not always an accurate predictor of the mRNA fraction [16]. Furthermore, normalisation to total RNA does not control for errors introduced during reverse transcrip- tion or PCR [44]. To our knowledge, this is the only study which has validated reference genes for use as normalising factors in the collagenase model of ICH in rats. We quantified the transcript level of seven candidate reference genes (B2MG, GUSB, GAPDH, HPRT, POL2R, SDHA and TBP) and our gene of interest, TRPM2, in the perihe- matomal and matching contralateral regions in rats 5 and 24 hours following collagenase-induced ICH, as well as in saline vehicle controls. Discussion Bars represent mean of triplicate measurements from 5 animals, ± SEM. importance of validating the stability of reference genes to be used as normalising factors in real-time RT-PCR studies. When analysing all ICH and saline vehicle samples together, the most stable reference genes were B2MG and GUSB, with four reference genes recommended as the optimal number for accurate normalisation. The inclusion of additional reference genes further decreases the value of V, even when all seven genes are used for normalisation (V6/7). However, once the threshold value of 0.15 has been reached, additional reference genes do not significantly increase the reliability of normalisation [16]. Although the observed differences in reference gene stability were subtle, this does not imply that any reference gene combination would be appropriate. To illustrate this point, removing the 3 most stable refer- ence genes (B2MG, GUSB and POL2R) from our geN- orm input file showed that GAPDH, HPRT, SDHA and TBP assessed together do not meet the proposed cut-off value of V (0.15). Therefore, the most stable four genes, as determined by geNorm, should be used. Figure 5 Normalised mRNA level of TRPM2 relative to multiple reference genes. TRPM2 mRNA level in the perihematomal brain region (RBG) of collagenase-induced ICH animals was compared to saline vehicle controls, at 5 h and 24 h survival times. The most stable reference genes determined by geNorm were used for normalisation in each group (5 h: GAPDH, HPRT, POL2R, SDHA; 24 h: B2MG, GUSB, POL2R). Bars represent mean of triplicate measurements from 5 animals, ± SEM. Figure 5 Normalised mRNA level of TRPM2 relative to multiple reference genes. TRPM2 mRNA level in the perihematomal brain region (RBG) of collagenase-induced ICH animals was compared to saline vehicle controls, at 5 h and 24 h survival times. The most stable reference genes determined by geNorm were used for normalisation in each group (5 h: GAPDH, HPRT, POL2R, SDHA; 24 h: B2MG, GUSB, POL2R). Bars represent mean of triplicate measurements from 5 animals, ± SEM. y g We also compared the stability of the candidate refer- ence genes at individual time points following collage- nase-induced ICH (5 and 24 hours). The most stable genes at 5 hours (GAPDH and HPRT) were different to those at 24 hours (B2MG and GUSB). Discussion Whiskers represent the maxim Figure 3 Average real-time RT-PCR cycle threshold (Ct) values of candidate reference genes. Data are displayed as average raw Ct (not yet normalised) values from individual groups of collagenase ICH and saline vehicle rats. The line in the box indicates the median, while the box represents the 25th and 75th percentile. Whiskers represent the maximum and minimum values. Figure 3 Average real-time RT-PCR cycle threshold (Ct) values of candidate reference genes. D Figure 3 Average real-time RT-PCR cycle threshold (Ct) values of candidate reference genes. Data are displayed as average raw Ct (not yet normalised) values from individual groups of collagenase ICH and saline vehicle rats. The line in the box indicates the median, while the box represents the 25th and 75th percentile. Whiskers represent the maximum and minimum values. Cook et al. BMC Molecular Biology 2010, 11:7 http://www.biomedcentral.com/1471-2199/11/7 Page 7 of 11 Figure 4 Normalised mRNA levels of candidate reference genes. The mRNA level of each group (5 hour and 24 hour collagenase ICH and saline vehicle rats) was normalised to the geometric mean of the expression of the four most stable reference genes determined by geNorm (B2MG, GUSB, POL2R and GAPDH). Each bar represents the normalised mean of triplicate measurements from 5 animals, ± SEM. Asterisk denotes statistical significance (p < 0.05) between ICH 24 hour LBG and vehicle RBG samples by one-way ANOVA. Figure 4 Normalised mRNA levels of candidate reference genes. The mRNA level of each group (5 hour and 24 hour collagenase ICH and saline vehicle rats) was normalised to the geometric mean of the expression of the four most stable reference genes determined by geNorm (B2MG, GUSB, POL2R and GAPDH). Each bar represents the normalised mean of triplicate measurements from 5 animals, ± SEM. Asterisk denotes statistical significance (p < 0.05) between ICH 24 hour LBG and vehicle RBG samples by one-way ANOVA. Cook et al. BMC Molecular Biology 2010, 11:7 http://www.biomedcentral.com/1471-2199/11/7 Page 8 of 11 Figure 5 Normalised mRNA level of TRPM2 relative to multiple reference genes. TRPM2 mRNA level in the perihematomal brain region (RBG) of collagenase-induced ICH animals was compared to saline vehicle controls, at 5 h and 24 h survival times. The most stable reference genes determined by geNorm were used for normalisation in each group (5 h: GAPDH, HPRT, POL2R, SDHA; 24 h: B2MG, GUSB, POL2R). RNA Extraction Total RNA was extracted from the left and right stria- tum with overlying cortex (’LBG’ (basal ganglia) and RBG, respectively) of collagenase ICH rats (n = 5 from each time point) and saline vehicle rats (n = 5) using the RNeasy Lipid Tissue kit (Qiagen, Doncaster, Austra- lia) according to the manufacturer’s instructions. Fifty mg tissue was used in each RNA extraction, which included an on-column DNase treatment step (Qiagen). RNA was quantified by UV spectrometry using the Nanophotometer (Implen, Australia) to measure absor- bance at 230, 260 and 280 nm. RNA integrity was evalu- ated using the Agilent Bioanalyzer RNA 6000 Nano Chip (Series II) kit. Discussion Initially, data regarding TRPM2 transcript level in the perihematomal region were normalised to individual reference genes using the relative standard curve method of analysis [30]. At the 24 h survival time point, when TRPM2 data were nor- malised to either GUSB or HPRT, significant differences were found. However, no differences were found when normalised to other commonly-used reference genes. There was considerable variation between samples when data were analysed in this way (Figure 1). However, with normalisation to a reference gene panel far more consis- tent results were obtained (Figure 5). Had we used HPRT or GUSB as a single reference gene for normali- sation, it is likely that we would have drawn erroneous conclusions about our data. These results highlight the The candidate reference genes in the present study also showed differences in stability between the perihe- matomal brain and the uninjured contralateral region. Page 9 of 11 Cook et al. BMC Molecular Biology 2010, 11:7 http://www.biomedcentral.com/1471-2199/11/7 Cook et al. BMC Molecular Biology 2010, 11:7 http://www.biomedcentral.com/1471-2199/11/7 with bone wax and the scalp wound closed with wound clips after irrigation with bupivacaine. As might be expected, differences in gene stability were most marked in the perihaematomal region. A cascade of pathological processes including excitotoxicity, oedema and inflammation are initiated by ICH [18], all of which may impact reference gene expression. Animals recovered in their home cage in a climate- and light-controlled environment with free access to food and water. Animals were killed by decapitation under deep isoflurane anesthesia either at 5 or 24 hours post-surgery. The brain was quickly extracted and a 4 mm thick slice taken of cortex and basal ganglia, incor- porating the injection site at the centre and the bulk of the perihematomal brain. This was bisected into right and left hemispheres and immediately snap-frozen for RNA extraction. Conclusions We have evaluated the expression stability of seven can- didate reference genes following collagenase-induced ICH in rats. We have identified B2MG, GUSB, POL2R and GAPDH as an appropriate panel of reference genes to be used in the acute phase (5 and 24 hours) of experimental ICH. When analysed in subsets of these samples (according to survival time and brain region), variations in the stability of the reference genes were revealed. Our gene of interest, TRPM2, was inconsis- tently elevated following injury when normalised to indi- vidual reference genes. However, when normalised to a panel of the most stable genes, no significant differences were seen. These results emphasise the importance of identifying and validating suitable reference genes to prevent erroneous conclusions. The results of the pre- sent study will enable more accurate normalisation of real-time RT-PCR data at 5 and 24 hour time points fol- lowing collagenase-induced ICH. Collagenase-induced ICH Complementary DNA was synthesised using the Super- Script III Reverse Transcription kit (Invitrogen, Mt Waverley, Australia). Two μg total RNA was added to 250 ng random hexamers (Geneworks, Adelaide, Austra- lia), 1 mM each dNTP (Invitrogen) and nuclease-free water to 13 μL. Reactions were heated to 65°C for 5 minutes then immediately placed on ice for 1 minute. To each tube, 4.75 μL 5× First Strand Buffer, 1 μL RNase OUT (Invitrogen), 0.02 M dithiothreitol and 200 units SuperScript III reverse transcriptase were added. Reactions containing nuclease-free water in place of enzyme served as negative controls. Reactions were incubated at 25°C for 5 minutes, 55°C for 60 minutes and 70°C for 15 minutes. cDNA was diluted to 10 ng/ μL with nuclease-free water and stored at -20°C. Real-time PCR All animal protocols were approved by the Institute of Medical and Veterinary Science and the University of Adelaide Animal Ethics committees and were con- formed to guidelines issued by the Australian National Health and Medical Research Council. Adult male Sprague-Dawley rats (n = 15) weighing 300-340 g were used in the study, and obtained four days prior to surgery to ameliorate any acute stress- induced changes in gene expression. The study used col- lagenase to induce intracerebral hemorrhage [45]. Briefly, animals were anesthetized using isoflurane (1.5 - 2.0%) in a 30:70 mix of oxygen and nitrogen via a nose cone and placed in a Kopf stereotaxic frame. A rectal thermometer was inserted and temperature maintained between 36.5 - 37.5°C with a thermostatically-controlled heat pad. The scalp was shaved and cleaned, and bupi- vacaine instilled. A midline scalp incision was made, the skull exposed and a burrhole drilled 0.7 mm anterior and 3.0 mm lateral to bregma. Using a syringe driver (Harvard Instruments), 0.2 U type VII bacterial collage- nase (Sigma C0773) in 2 μL normal saline, or saline alone (’vehicle’) was infused over 4 minutes, via a 30 G needle, into the centre of the striatum (6.0 mm anterior to bregma). The needle was left in place for five min- utes, and then withdrawn slowly. The hole was sealed Acknowledgements NLC and TJK are grateful recipients of postgraduate scholarships from the National Health and Medical Research Council of Australia. TJK’s scholarship was co-awarded by the National Heart Foundation (Grant ID 465450). This work was also supported, in part, by the Neurosurgical Research Foundation, and by a Pfizer neurosciences research grant for TJK. μ Real-time PCR Amplification was carried out in a Rotor-Gene 3000 (Corbett Research, Mortlake, Australia) with an initial UDG incubation of 50°C for 2 minutes, initial denatura- tion of 95°C for 2 minutes, followed by 40 cycles of: 95° C for 15 sec denaturation, primer-specific annealing temperature for 15 sec (see [40]), and 72°C for 15 sec extension. TRPM2 annealing temperature was 60°C. Fluorescence data were collected during the extension step of each cycle. Specificity of amplicons were verified by melting curve analysis after 40 cycles (72°C to 95°C) and 2% agarose gel electrophoresis stained with ethi- dium bromide and visualised under UV light. All cDNA samples were run in triplicate. Negative controls con- taining water instead of cDNA were present in all runs. No-reverse transcriptase controls were included for each gene to test for genomic DNA amplification. had been determined. Statistical analyses were carried out using unpaired Student’s t-tests and one-way analy- sis of variance (ANOVA) with Newman-Keuls post-hoc tests. Data Analysis 4. Bustin SA: Absolute quantification of mRNA using real-time reverse transcription polymerase chain reaction. Journal of Molecular Endocrinology 2000, 25:169-193. 4. Bustin SA: Absolute quantification of mRNA using real-time reverse transcription polymerase chain reaction. Journal of Molecular Endocrinology 2000, 25:169-193. Standard curves made from serial dilutions of pooled cDNA were used to calculate PCR efficiency (E) using the formula: E (%) = (10[-1/slope] - 1) × 100. Raw values of input RNA were determined for each sample from the standard curve using the Corbett Rotor-Gene 6 soft- ware. The relative standard curve method [30] was applied to these raw quantities to calculate TRPM2 mRNA level relative to each of the seven reference genes individually. The cycle threshold (Ct) of an indivi- dual sample reflects the cycle at which a detectable number of PCR products have accumulated above back- ground fluorescence [47]. Ct values were calculated from the standard curve, entered into the qBasePlus software [38] and used to generate an input file for geN- orm v3.5 [16]. geNorm determined the most stable reference genes out of the panel of candidate genes using expression stability analysis by pairwise correla- tions. An expression stability measure, M, was assigned to each gene, which was used to rank candidate refer- ence genes in order of stability. The most stable refer- ence genes were determined in the following groups: all collagenase ICH and saline vehicle samples; 5 hour col- lagenase ICH and saline vehicle samples; 24 collagenase ICH and all saline vehicle samples; RBG only from all collagenase ICH and saline vehicle samples; LBG only from all collagenase ICH and saline vehicle samples. Normalised mRNA levels of each gene were calculated using qBasePlus once the most stable reference genes 5. Klein D: Quantification using real-time PCR technology: applications and limitations. Trends in Molecular Medicine 2002, 8(6):257-260. 5. Klein D: Quantification using real-time PCR technology: applications and limitations. Trends in Molecular Medicine 2002, 8(6):257-260. 6. Bustin SA, Benes V, Garson JA, Hellemans J, Huggett J, Kubista M, Mueller R, Nolan T, Pfaffl MW, Shipley GL, et al: The MIQE guidelines: minimum information for publication of quantitative real-time PCR experiments. Clinical Chemistry 2009, 55(4):611-622. 7. Radonić A, Thulke S, Mackay IM, Landt O, Siegert W, Nitsche A: Guideline to reference gene selection for quantitative real-time PCR. Biochemical and Biophysical Research Communications 2004, 313:856-862. 7. Authors’ contributions NLC carried out the reference gene study, performed the statistical analyses and drafted the manuscript. TJK carried out the animal surgery, and assisted with statistical analyses and drafting the manuscript. CVDH supervised the study, participated in study design and provided useful discussion. RV participated in study design and coordination and provided useful discussion. All authors read and approved the final manuscript. Received: 24 July 2009 Accepted: 20 January 2010 Published: 20 January 2010 Received: 24 July 2009 Accepted: 20 January 2010 Published: 20 January 2010 Received: 24 July 2009 μ Real-time PCR The candidate reference genes used in this study were: GAPDH, B2MG, POL2R, TBP, HPRT, SDHA and GUSB. Primer sequences for reference genes have been described elsewhere [40]. TRPM2 primer sequences were (5’ - 3’): forward, GAAGGAAAGAGGGGGTGTG and reverse, CATTGGTGATGGCGTTGTAG [46], with a product size of 101 base pairs. Real-time RT-PCR amplifications for reference genes and TRPM2 were car- ried out using 10 μL 2× Invitrogen Platinum SYBR Green SuperMix-UDG, 300 nM forward and reverse Page 10 of 11 Cook et al. BMC Molecular Biology 2010, 11:7 http://www.biomedcentral.com/1471-2199/11/7 primers (400 nM for POL2R and GAPDH), 1 μL cDNA and nuclease-free water in a total volume of 20 μL. One hundred mM MgCl2 (Invitrogen) was added to POL2R reactions. A set of standards was included in each run, comprising five-fold serial dilutions made from aliquots of pooled cDNA, derived from an RNA pool of all sam- ples. Serial dilutions contained the following amounts of cDNA: 50 ng, 10 ng, 2 ng and 0.4 ng. The standard ser- ies encompassed the unknown cDNA concentrations. Amplification was carried out in a Rotor-Gene 3000 (Corbett Research, Mortlake, Australia) with an initial UDG incubation of 50°C for 2 minutes, initial denatura- tion of 95°C for 2 minutes, followed by 40 cycles of: 95° C for 15 sec denaturation, primer-specific annealing temperature for 15 sec (see [40]), and 72°C for 15 sec extension. TRPM2 annealing temperature was 60°C. Fluorescence data were collected during the extension step of each cycle. Specificity of amplicons were verified by melting curve analysis after 40 cycles (72°C to 95°C) and 2% agarose gel electrophoresis stained with ethi- dium bromide and visualised under UV light. All cDNA samples were run in triplicate. Negative controls con- taining water instead of cDNA were present in all runs. No-reverse transcriptase controls were included for each gene to test for genomic DNA amplification. primers (400 nM for POL2R and GAPDH), 1 μL cDNA and nuclease-free water in a total volume of 20 μL. One hundred mM MgCl2 (Invitrogen) was added to POL2R reactions. A set of standards was included in each run, comprising five-fold serial dilutions made from aliquots of pooled cDNA, derived from an RNA pool of all sam- ples. Serial dilutions contained the following amounts of cDNA: 50 ng, 10 ng, 2 ng and 0.4 ng. The standard ser- ies encompassed the unknown cDNA concentrations. 1. Ferro JM: Update on intracerebral haemorrhage. Journal of Neurology 2006, 253(8):985-999. 1. Ferro JM: Update on intracerebral haemorrhage. Journal of Neurology 2006, 253(8):985-999. 1. Ferro JM: Update on intracerebral haemorrhage. Journal of Neurology 2006, 253(8):985-999. 2. Morgenstern LB, Woodbury-Harris K, Marler JR: Priorities for clinical research in intracerebral haemorrhage: report from a National Institute of Neurological Disorders and Stroke workshop. Stroke 2005, 36(3):e23-41 g p 3. Xi G, Keep RF, Hoff JT: Mechanisms of brain injury after intracerebral haemorrhage. 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BMC Molecular Biology 2010 11:7. doi:10.1186/1471-2199-11-7 Cite this article as: Cook et al.: Reference genes for normalising gene expression data in collagenase-induced rat intracerebral haemorrhage. BMC Molecular Biology 2010 11:7. 28. Aarts M, Iihara K, Wei W-L, Xiong Z-G, Arundine M, Cerwinski W, MacDonald JF, Tymianski M: A key role for TRPM7 channels in anoxic neuronal death. Cell 2003, 115:863-877. 29. Fonfria E, Mattei C, Hill K, Brown JT, Randall A, Benham CD, Skaper SD, Campbell CA, Crook B, Murdock PR, et al: TRPM2 is elevated in the tMCAO stroke model, transcriptionally regulated, and functionally expressed in C13 microglia. Journal of Receptors and Signal Transduction 2006, 26:179-198. 30. Pfaffl MW: A new mathematical model for relative quantification in real- time RT-PCR. Nucleic Acids Research 2001, 29(9):e45. 31. Data Analysis Radonić A, Thulke S, Mackay IM, Landt O, Siegert W, Nitsche A: Guideline to reference gene selection for quantitative real-time PCR. Biochemical and Biophysical Research Communications 2004, 313:856-862. 8. Bustin SA: Real-time, fluorescence-based quantitative PCR: a snapshot of current procedures and preferences. Expert Review of Molecular Diagnosis 2005, 5(4):493-498. 9. Dheda K, Huggett JF, Bustin SA, Johnson MA, Rook G, Zumla A: Validation of housekeeping genes for normalizing RNA expression in real-time PCR. BioTechniques 2004, 37:112-119. 10. Stürzenbaum SR, Kille P: Control genes in quantitative molecular biological techniques: the variability of invariance. Comparative Biochemistry and Physiology Part B 2001, 130:281-289. 11. Bas A, Forsberg G, Hammarström S, Hammarström M-L: Utility of the housekeeping genes 18S rRNA, b-actin, glyceraldehyde-3-phosphate- dehydrogenase for normalization in real-time quantitative reverse transcriptase-polymerase chain reaction analysis of gene expression in human T lymphocytes. Scandinavian Journal of Immunology 2004, 59:566-573. 12. Glare EM, Divjak M, Bailey MJ, Walters EH: b-actin and GAPDH housekeeping gene expression in asthmatic airways is variable and not suitable for normalising mRNA levels. Thorax 2002, 57:765-770. 13. Zhong H, Simons JW: Direct comparison of GAPDH, b-actin, cyclophilin, and 28S rRNA as internal standards for quantifying RNA levels under hypoxia. Biochemical and Biophysical Research Communications 1999, 259:523-526. 14. Olsvik PA, Lie KK, Jordal A-EO, Nilsen TO, Hordvik I: Evaluation of potential reference genes in real-time RT-PCR studies of Atlantic salmon. BMC Molecular Biology 2005, 6(21). Page 11 of 11 Cook et al. BMC Molecular Biology 2010, 11:7 http://www.biomedcentral.com/1471-2199/11/7 15. Dheda K, Huggett JF, Chang JS, Kim LU, Bustin SA, Johnson MA, Rook GAW, Zumla A: The implications of using an inappropriate reference gene for real-time reverse transcription PCR data normalization. Analytical Biochemistry 2005, 344:141-143. 35. Pérez R, Tupac-Yupanqui I, Dunner S: Evaluation of suitable reference genes for gene expression studies in bovine muscular tissue. BMC Molecular Biology 2008, 9(79). 36. Schroeder A, Mueller O, Stocker S, Salowsky R, Leiber M, Gassmann M, Lightfoot S, Menzel W, Granzow M, Ragg T: The RIN: an RNA integrity number for assigning integrity values to RNA measurements. BMC Molecular Biology 2006, 7(3). 16. Vandesompele J, De Preter K, Pattyn F, Poppe B, Van Roy N, De Paepe A, Speleman F: Accurate normalization of real-time quantitative RT-PCR data by geometric averaging of multiple internal control genes. Genome Biology 2002, 3(7):research0034.0031-0034.0011. 37. Livak KJ, Schmittgen TD: Analysis of relative gene expression data using real-time quantitative PCR and the 2-ΔΔCT method. Data Analysis Methods 2001, 25:402-408. 17. Wong ML, Medrano JF: Real-time PCR for mRNA quantitation. BioTechniques 2005, 39:75-85. 18. 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Cook N, Heuvel Van Den C, Donkin J, Vink R: Validation of reference genes for normalization of real-time quantitative RT-PCR data in traumatic brain injury. Journal of Neuroscience Research 2009, 87:34-41. 21. Wasserman JK, Schlichter LC: Minocycline protects the blood-brain barrier and reduces oedema formation following intracerebral haemorrhage in the rat. Experimental Neurology 2007, 207:227-237. 41. Ke LD, Chen Z, Yung WKA: A reliability test of standard-based quantitative PCR: exogenous vs. endogenous standards. Molecular and Cellular Probes 2000, 14:127-135. 22. Wasserman JK, Zhu X, Schlichter LC: Evolution of the inflammatory response in the brain following intracerebral haemorrhage and effects of delayed minocycline treatment. Brain Research 2007, 1180:140-154. 42. Moriya Y, Nakamura T, Okamura N, Sakaeda T, Horinouchi M, Tamura T, Aoyama N, Kasuga M, Okumura K: Comparison of synthetic DNA templates with authentic cDNA templates in terms of quantification by real-time quantitative reverse transcription polymerase chain reaction. Biological and Pharmaceutical Bulletin 2006, 29(3):535-539. 23. Perraud A-L, Fleig A, Dunn CA, Bagley LA, Launay P, Schmitz C, Stokes AJ, Zhu Q, Bessman MJ, Penner R, et al: ADP-ribose gating of the calcium- permeable LTRPC2 channel revealed by Nudix motif homology. Nature 2001, 411:595-599. 43. Smith RD, Brown B, Ikonomi P, Schechter AN: Exogenous reference RNA for normalisation of real-time quantitative PCR. BioTechniques 2003, 34:88-91. 24. Nagamine K, Kudoh J, Minoshima S, Kawasaki K, Asakawa S, Ito F, Shimizu N: Molecular cloning of a novel putative Ca2+ channel protein (TRPC7) highly expressed in brain. Genomics 1998, 54:124-131. 44. 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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 "BioMed Central will be the most significant development for disseminating the results of biomedical research in our lifetime." Sir Paul Nurse, Cancer Research UK Your research papers will be: available free of charge to the entire biomedical community peer reviewed and published immediately upon acceptance cited in PubMed and archived on PubMed Central yours — you keep the copyright Submit your manuscript here: http://www.biomedcentral.com/info/publishing_adv.asp BioMedcentral Publish with BioMed Central and every scientist can read your work free of charge Publish with BioMed Central and every scientist can read your work free of charge 32. Pfaffl MW, Tichopad A, Prgomet C, Neuvians TP: Determination of stable housekeeping genes, differentially regulated target genes and sample integrity: BestKeeper - Excel-based tool using pair-wise correlations. Biotechnology Letters 2004, 26:509-515. 33. Cappelli K, Felicetti M, Capomaccio S, Spinsanti G, Silvestrelli M, Supplizi AV: Exercise induced stress in horses: selection of the most stable reference genes for quantitative RT-PCR normalization. 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Avelumab (anti–PD-L1) as first-line switch-maintenance or second-line therapy in patients with advanced gastric or gastroesophageal junction cancer: phase 1b results from the JAVELIN Solid Tumor trial
Journal for immunotherapy of cancer
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RESEARCH ARTICLE Open Access Chung et al. Journal for ImmunoTherapy of Cancer (2019) 7:30 https://doi.org/10.1186/s40425-019-0508-1 Chung et al. Journal for ImmunoTherapy of Cancer (2019) 7:30 https://doi.org/10.1186/s40425-019-0508-1 (2019) 7:30 Chung et al. Journal for ImmunoTherapy of Cancer https://doi.org/10.1186/s40425-019-0508-1 Avelumab (anti–PD-L1) as first-line switch-maintenance or second-line therapy in patients with advanced gastric or gastroesophageal junction cancer: phase 1b results from the JAVELIN Solid Tumor trial ung1*, Hendrik-Tobias Arkenau2, Jeeyun Lee3, Sun Young Rha1, Do-Youn Oh4, Lucjan Wyrwicz5 Hyun Cheol Chung1*, Hendrik-Tobias Arkenau2, Jeeyun Lee3, Sun Young Rha1, Do-Youn Oh4, Lucjan Wyrwicz5, Yoon-Koo Kang6, Keun-Wook Lee7, Jeffrey R. Infante8, Sung Sook Lee9, Margaret Kemeny10, Ulrich Keilholz11, Bohuslav Melichar12, Alain Mita13, Ruth Plummer14, Denis Smith15, Arnold B. Gelb16, Huiling Xiong16, Janet Hong16, Vikram Chand16,18 and Howard Safran17 © 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. * Correspondence: unchung8@yuhs.ac p g y Prior presentation Interim analyses were presented at the American Society of Clinical Oncology (ASCO) Gastrointestinal Cancers Symposium, San Francisco, CA, 21-23 January 2016 (abstract 167) and the ASCO Annual Meeting, Chicago, IL, 3-7 June 2016 (abstract 4009). Analyses reported in the current manuscript were presented at the American Association for Cancer Research Annual Meeting, Chicago, IL, 14-18 April 2018. 1Yonsei Cancer Center, Yonsei University College of Medicine, Yonsei University Health System, Seoul 03722, South Korea Full list of author information is available at the end of the article Abstract Background: We evaluated the antitumor activity and safety of avelumab, a human anti–PD-L1 IgG1 antibody, as first-line switch-maintenance (1 L-mn) or second-line (2 L) treatment in patients with advanced gastric/gastroesophageal cancer (GC/GEJC) previously treated with chemotherapy. Methods: In a phase 1b expansion cohort, patients without (1 L-mn) or with (2 L) disease progression following first-line chemotherapy for advanced GC/GEJC received avelumab 10 mg/kg intravenously every 2 weeks. Endpoints included best overall response, progression-free survival (PFS), overall survival (OS), and safety. (Continued on next page) (Continued on next page) * Correspondence: unchung8@yuhs.ac Background patients [9]. PD-L1 is a key therapeutic target for reactivat- ing antitumor immune responses [10]. Additionally, PD-L1 is expressed in ≈30 to 60% of GC/GEJC specimens, with a higher frequency seen in certain pathological and genomic subtypes [11]. Immunotherapy with anti–PD-1 antibodies has been associated with durable antitumor responses in early-phase studies of patients with GC/GEJC [10, 12, 13]. Background Gastric cancer (GC) is an aggressive disease that repre- sents the third leading cause of cancer-related death worldwide [1]. Gastroesophageal junction cancer (GEJC) has similar biology, prognosis, and treatment guidelines as GC [2, 3]. First-line (1 L) standard of care for ad- vanced inoperable GC/GEJC is based on combination fluoropyrimidine and platinum treatment, with trastuzu- mab added for HER2+ tumors. Second-line (2 L) options include regimens based on irinotecan, taxanes, and/or ramucirumab [2, 3]. However, cytotoxic regimens are associated with cumulative toxicity that may restrict long-term treatment, resulting in limited duration of re- sponse and overall survival (OS). Maintenance therapy, ie, continued treatment with an agent administered in the 1 L induction regimen or sequential treatment with a different agent until progression (switch maintenance), has the potential to extend durations of response and OS, particularly when an agent with a different mechan- ism of action is employed, while avoiding potential addi- tive toxicity associated with further chemotherapy or combination treatment. As such, maintenance therapy has become an established strategy for several advanced tumors [4, 5]. Although the role of maintenance therapy in treating GC/GEJC is less well defined, observational and retrospective studies of maintenance fluoropyrimidine treatment in advanced GC/GEJC have shown that this approach is feasible and may improve progression-free survival (PFS) compared with observation alone [6–8]. Avelumab is a human anti–PD-L1 monoclonal antibody that has been approved in various countries for the treat- ment of metastatic Merkel cell carcinoma and in the United States and Canada for the treatment of advanced urothelial carcinoma progressing after platinum-containing chemo- therapy. In phase 1 and 2 studies across various advanced cancers, avelumab has demonstrated a tolerable safety pro- file and durable antitumor activity [14–16]. In preclinical studies, avelumab activated both adaptive and innate im- mune effector cells [17, 18], suggesting an additional mech- anism of action compared with other approved anti–PD-1/ PD-L1 antibodies. To investigate the efficacy and safety of avelumab in the treatment of advanced GC/GEJC, we enrolled a cohort of patients in the phase 1 JAVELIN Solid Tumor trial. Background Patients were enrolled following 1 L chemotherapy; those without disease progression received avelumab as 1 L switch maintenance (1 L-mn subgroup), and those with disease progression received avelumab as 2 L treat- ment (2 L subgroup). To our knowledge, this is the first study of an anti–PD-L1 agent administered as switch- maintenance therapy in this disease. In recent years, much attention has been focused on anticancer therapies that activate the immune response. In a randomized phase 2 study of patients with advanced GC/GEJC, switch-maintenance ipilimumab (anti–CTLA-4) after 1 L chemotherapy did not improve immune-related PFS or OS compared with best supportive care, which in- cluded continued fluoropyrimidine chemotherapy in most © The Author(s). 2019 Open Access This article is distributed under the terms of the Creative Commons Attribution 4.0 International License (http://creativecommons.org/licenses/by/4.0/), which permits unrestricted use, distribution, and reproduction in any medium, provided you give appropriate credit to the original author(s) and the source, provide a link to the Creative Commons license, and indicate if changes were made. The Creative Commons Public Domain Dedication waiver (http://creativecommons.org/publicdomain/zero/1.0/) applies to the data made available in this article, unless otherwise stated. Page 2 of 10 Chung et al. Journal for ImmunoTherapy of Cancer (2019) 7:30 (2019) 7:30 (Continued from previous page) Results: Overall, 150 patients were enrolled (1 L-mn, n = 90; 2 L, n = 60) and median follow-up in the 1 L-mn and 2 L subgroups was 36.0 and 33.7 months, respectively. The confirmed objective response rate was 6.7% in both subgroups (95% CI, 2.5–13.9% and 1.8–16.2%, respectively), including complete responses in 2.2% of the 1 L-mn subgroup (n = 2). In the 1 L-mn and 2 L subgroups, median duration of response was 21.4 months (95% CI, 4.0–not estimable) and 3.5 months (95% CI, 2.8–8.3) and disease control rates were 56.7 and 28.3%, respectively. Median PFS in the 1 L-mn and 2 L subgroups was 2.8 months (95% CI, 2.3–4.1) and 1.4 months (95% CI, 1.3–1.5), with 6-month PFS rates of 23.0% (95% CI, 14.7–32.4%) and 7.9% (95% CI, 2.6–17.2%), and median OS was 11.1 months (95% CI, 8.9–13.7) and 6.6 months (95% CI, 5.4–9.4), respectively. In the 1 L-mn subgroup, median OS measured from start of 1 L chemotherapy was 18.7 months (95% CI, 15.4–20.6). Across both subgroups, 20.7% had an infusion-related reaction of any grade. Other common treatment-related adverse events (TRAEs) of any grade included fatigue (10.0%) and nausea (6.7%). Treatment-related serious adverse events occurred in 4.0% of patients. Overall, 8.7% had a grade ≥3 TRAE, including 1 treatment-related death. Conclusion: Avelumab showed clinical activity and an acceptable safety profile in patients with GC/GEJC. Trial registration: ClinicalTrials.gov NCT01772004; registered 21 January 2013. Keywords: Avelumab, Metastatic, Gastric, Esophagogastric junction, Adenocarcinoma, Maintenance Statistical methods Enrollment of 150 patients was planned for this cohort based on the anticipated sample size required to estimate and provide 95% Clopper-Pearson confidence intervals (CIs) for potential objective response rates (ORR; propor- tion of patients with a confirmed CR or PR; eg, 10% [5.7– 16.0%] for 15 responders or 20% [13.9–27.3%] for 30 responders). Safety and antitumor activity were analyzed in all patients who received ≥1 dose of avelumab. Time-to-event endpoints (PFS, OS, duration of response, and duration of follow-up) were estimated using the Kaplan–Meier method and CIs for the median were calculated using the Brookmeyer–Crowley method. Study design and patients JAVELIN Solid Tumor (NCT01772004) is an inter- national, open-label, phase 1 trial. In the phase 1b, non- randomized expansion cohort reported here, eligible Chung et al. Journal for ImmunoTherapy of Cancer (2019) 7:30 Chung et al. Journal for ImmunoTherapy of Cancer Page 3 of 10 patients had histologically confirmed, unresectable, locally advanced or metastatic GC/GEJC, and previous treatment with 1 L combination chemotherapy; patients with prior neoadjuvant platinum-based doublet or triplet chemotherapy who were not candidates for surgery were also eligible. Patients should not have received >1 line of prior treatment for metastatic disease, and patients with prior checkpoint inhibitor or trastuzumab treatment were ineligible (Additional file 1: Table S1). Patients in the 2 L subgroup were not permitted to have received anticancer treatment within 28 days before the start of study treatment, whereas in the 1 L-mn subgroup, patients were permitted to be enrolled within 28 days if all toxicity from prior therapy had resolved to grade ≤1. A fresh or archival tumor specimen was required, but patients were not preselected based on PD-L1 status (ie, all-comer design). Patients were enrolled in accordance with an approved protocol, international standards of good clinical practice, and institutional safety monitoring, and written informed consent was obtained. The study protocol was approved by the institutional review board or independent ethics committee at each center. cutoffs of ≥1% of tumor cells positive for partial or complete membrane PD-L1 staining of any intensity. HER2 and microsatellite status were recorded retrospectively from medical records when available. Procedures and assessments Patients received avelumab 10 mg/kg intravenously every 2 weeks until confirmed disease progression, unaccept- able toxicity, or protocol-based criteria for withdrawal [15]. Premedication with diphenhydramine and acetaminophen was required 30 to 60 min before all avelumab infusions. Safety was assessed at each biweekly trial visit and in- cluded assessment of adverse events (AEs), physical examination, clinical laboratory tests (hematology, hep- atic panels, and serum chemistry), and documentation of concurrent medications. AEs and laboratory abnormal- ities were classified and graded according to National Cancer Institute Common Terminology Criteria for Adverse Events version 4.0. A serious AE (SAE) was de- fined as any untoward event that was life-threatening, required hospitalization, resulted in disability, was a con- genital anomaly, or resulted in death. Immune-related AEs were identified using a prespecified list of Medical Dictionary for Regulatory Activities terms plus medical review. Clinical activity was assessed by investigators using Response Evaluation Criteria in Solid Tumors version 1.1. Radiographic tumor assessments were performed at baseline and every 6 weeks. In patients achieving a partial response (PR) or complete response (CR), a confirmatory CT or MRI scan was done ≥28 days later (preferably at the scheduled 6-week interval). PD-L1 expression was assessed in tumor cells using a proprietary immunohistochemistry assay (PD-L1 IHC 73-10; Dako, Carpinteria, CA), as described previously [15, 19]; in this report, PD-L1 status was defined using Outcomes Primary endpoints for the entire JAVELIN Solid Tumor trial were dose-limiting toxicities during the first 3 weeks of treatment in the phase 1a dose-escalation part (reported previously [20]) and confirmed best overall response adjudicated by independent review in specified efficacy expansion cohorts (not including the GC/GEJC cohort reported here). Secondary endpoints assessed in the current cohort included investigator-assessed best overall response, duration of response, PFS, OS, safety, and evaluation of PD-L1 expression [15]. Results Patients Five patients remained on avelumab treatment at data cutoff, all in the 1 L-mn subgroup (5.6%). Results Patients Between 13 February 2014 and 11 August 2015, 150 patients with histologically confirmed GC/GEJC were enrolled, including 90 without disease progression after 1 L chemotherapy (1 L-mn subgroup) and 60 with progressive disease (2 L subgroup), per investigator assessment (Table 1). In the 1 L-mn subgroup, 27.8% had achieved a PR with prior chemotherapy; in the 2 L subgroup, prior responses were PR in 13.3% and CR in 1.7%. The median interval between end of prior chemo- therapy and start of avelumab was 45 days (1 L-mn subgroup) and 77 days (2 L subgroup). Across both subgroups, 30.7% had PD-L1+ tumors. At data cutoff (30 September 2017), patients in the 1 L-mn and 2 L subgroups had received a median (range) of 7 (1–79) and 4.5 (1–44) avelumab doses, and median duration of treatment was 3.2 months (interquartile range [IQR], 1.4–6.1) and 2.2 months (IQR, 1.4–5.2), respectively. Median duration of follow-up was 36.0 months (IQR, 33.7–37.7) in the 1 L-mn subgroup and 33.7 months (IQR, 27.9–34.9) in the 2 L subgroup. In both subgroups, the most common reason for treatment Chung et al. Results Patients Journal for ImmunoTherapy of Cancer (2019) 7:30 Page 4 of 10 Table 1 Baseline characteristics in the first-line switch-maintenan and second-line subgroups Characteristics 1 L-mn subgroup (n = 90) 2 L subgroup (n = 60) Median age (IQR), years 59 (52.0–67.0) 62.5 (51.5–66 Sex, n (%) Male 68 (75.6) 46 (76.7) Female 22 (24.4) 14 (23.3) ECOG PS, n (%) 0 37 (41.1) 23 (38.3) 1 53 (58.9) 37 (61.7) Geographic region, n (%) North America 31 (34.4) 32 (53.3) Asia 34 (37.8) 10 (16.7) Europe 25 (27.8) 18 (30.0) Race, n (%) White 44 (48.9) 36 (60.0) Asian 35 (38.9) 13 (21.7) Black 4 (4.4) 4 (6.7) Other 7 (7.8) 7 (11.7) Histology, n (%) Tubular 18 (20.0) 3 (5.0) Signet ring 17 (18.9) 13 (21.7) Mucinous 4 (4.4) 4 (6.7) Papillary 1 (1.1) 0 Other/not specified 1 (1.1) 0 Unknown 37 (41.1) 39 (65.0) PD-L1 expression status based on ≥1% cutoff on tumor cells, n (%) PD-L1+ 26 (28.9) 20 (33.3) PD-L1− 51 (56.7) 25 (41.7) Not evaluable 13 (14.4) 15 (25.0) HER2 status, n (%) HER2− 62 (68.9) 29 (48.3) HER2+ 4 (4.4) 5 (8.3) Unknown 24 (26.7) 26 (43.3) Microsatellite status, n (%) Low 1 (1.1) 0 Stable 21 (23.3) 17 (28.3) High 2 (2.2) 2 (3.3) Unknown 66 (73.3) 41 (68.3) Prior gastrectomy, n (%) 24 (26.7) 14 (23.3) Metastatic disease status at study entry, n (%) M0 5 (5.6) 2 (3.3) M1 85 (94.4) 58 (96.7) Tumor size at baselinea Median (IQR), mm 33 (19–52) 44 (25–69.5) Unknown, n (%) 1 (1.1) 1 (1.7) Table 1 Baseline characteristics in the first-line switch-maintenance and second-line subgroups Table 1 Baseline characteristics in the first-line switch-maintenance and second-line subgroups (Continued) Characteristics 1 L-mn subgroup (n = 90) 2 L subgroup (n = 60) Best response to prior anticancer therapy, n (%) Complete response 0 1 (1.7) Partial response 25 (27.8) 8 (13.3) Stable disease 59 (65.6) 23 (38.3) Progressive disease 0 22 (36.7) Not evaluable or unknown 6 (6.7) 5 (8.3) Prior anticancer therapy (any setting), n (%) 90 (100) 60 (100) Number of prior lines of anticancer therapy for metastatic or locally advanced disease, n (%) 0 1 (1.1) 5 (8.3) 1 87 (96.7) 53 (88.3) 2 2 (2.2) 1 (1.7) Unknown 0 1 (1.7) Median prior lines (range) 1.0 (0–2) 1.0 (0–2) Interval from end of prior chemotherapy to start of avelumab therapy Median (IQR), days 45 (35–64) 77 (49–135) Data missing, n (%) 8 (8.9) 15 (25.0) aSum of the longest diameters of target lesions Abbreviations: 1 L-mn first-line switch-maintenance, 2 L second line, ECOG PS Eastern Cooperative Oncology Group performance status, IQR interquartile range Table 1 Baseline characteristics in the first-line switch-maintenance and second-line subgroups (Continued) Interval from end of prior chemotherapy to start of avelumab therapy discontinuation was disease progression (1 L-mn, 75.6%; discontinuation was disease progression (1 L-mn, 75.6%; 2 L, 71.7%); other reasons were AE (13.3%, 10.0%), death (3.3%, 8.3%), withdrawal of consent (1.1%, 6.7%), loss to follow-up (0%, 1.7%), protocol noncompliance (1.1%, 0%), and physician decision (0%, 1.7%). Antitumor activity: 1 L-mn subgroup y g p The confirmed ORR (additional effect after the end of chemotherapy) was 6.7% (n = 6; 95% CI, 2.5–13.9%) (Additional file 1: Table S2). Notably, 2 patients (2.2%) had a CR; both patients were Asian and had stable dis- ease (SD) as best response to prior chemotherapy, and PD-L1 status was positive in 1 patient and not evaluable in the other. Four patients (4.4%) had a PR, which repre- sented additional tumor shrinkage following prior chemotherapy; best response to prior chemotherapy in these patients was PR (n = 2) and SD (n = 2) (Additional file 1: Table S3). Forty-five patients (50.0%) had SD of any duration as best response (disease control rate, 56.7%). Early and durable responses were observed (Fig. 1a and Additional file 1: Figure S1A), with a median time to response of 1.4 months (IQR, 1.3–4.1), a median duration of response of 21.4 months (95% CI, 4.0–not estimable), and an estimated 66.7% (95% CI, 19.5– 90.4%) of responses lasting ≥6 months. Responses were Chung et al. Journal for ImmunoTherapy of Cancer (2019) 7:30 Page 5 of 10 Chung et al. Journal for ImmunoTherapy of Cancer 200 180 160 140 120 100 80 60 40 20 -20 -40 -60 -80 -100 0 0 2 4 6 8 10 12 14 16 Time since treatment initiation (months) Change in target lesions from baseline (%) 6 3 8 3 4 3 2 3 0 3 8 2 6 2 4 2 2 2 0 2 8 1 First occurence of new lesion Patient off treatment Complete response Partial response Stable disease Progressive disease Not evaluable A 200 180 160 140 120 100 80 60 40 20 -20 -40 -60 -80 -100 0 0 2 4 6 8 10 12 14 16 Time since treatment initiation (months) Change in target lesions from baseline (%) 6 3 8 3 4 3 2 3 0 3 8 2 6 2 4 2 2 2 0 2 8 1 First occurence of new lesion Patient off treatment Partial response Stable disease Progressive disease Not evaluable B Fig. 1 Change in sum of target lesion diameters over time with avelumab in evaluable patients. a First-line switch-maintenance subgroup (n = 81). b Second-line subgroup (n = 52). Color coding is based on best overall response per Response Evaluation Criteria in Solid Tumors version 1.1. Antitumor activity: 1 L-mn subgroup Dotted lines indicate the 3-month timepoint and changes in target lesion size of −30, 0, and +20% 200 180 160 140 120 100 80 60 40 20 -20 -40 -60 -80 -100 0 0 2 4 6 8 10 12 14 16 Time since treatment initiation (months) Change in target lesions from baseline (%) 6 3 8 3 4 3 2 3 0 3 8 2 6 2 4 2 2 2 0 2 8 1 First occurence of new lesion Patient off treatment Complete response Partial response Stable disease Progressive disease Not evaluable A Time since treatment initiation (months) 200 180 160 140 120 100 80 60 40 20 -20 -40 -60 -80 -100 0 0 2 4 6 8 10 12 14 16 Time since treatment initiation (months) Change in target lesions from baseline (%) 6 3 8 3 4 3 2 3 0 3 8 2 6 2 4 2 2 2 0 2 8 1 First occurence of new lesion Patient off treatment Partial response Stable disease Progressive disease Not evaluable B B Fig. 1 Change in sum of target lesion diameters over time with avelumab in evaluable patients. a First-line switch-maintenance subgroup (n = 81). b Second-line subgroup (n = 52). Color coding is based on best overall response per Response Evaluation Criteria in Solid Tumors version 1.1. Dotted lines indicate the 3-month timepoint and changes in target lesion size of −30, 0, and +20% Fig. 1 Change in sum of target lesion diameters over time with avelumab in evaluable patients. a First-line switch-maintenance subgroup (n = 81). b Second-line subgroup (n = 52). Color coding is based on best overall response per Response Evaluation Criteria in Solid Tumors version 1.1. Dotted lines indicate the 3-month timepoint and changes in target lesion size of −30, 0, and +20% Duration of avelumab treatment in these 2 patients was 36.2 months (ongoing) and 23.5 months, respectively. Another patient had SD before disease progression at week 13 (due to a new lesion), followed by a 100% reduction in target lesions sustained for >6 months. ongoing at data cutoff in 2 patients, including 1 patient with a CR. In evaluable patients with PD-L1+ or PD-L1− tumors, confirmed ORR was 7.7% (2/26; 95% CI, 0.9– 25.1%) vs 3.9% (2/51; 95% CI, 0.5–13.5%). Antitumor activity: 1 L-mn subgroup Of 81 patients evaluable for change in size of target lesions, 13 (16.0%) had shrinkage of ≥30% and 37 (45.7%) had shrinkage of any level (Additional file 1: Figure S2A). No correlation was seen between response to prior chemotherapy and tumor shrinkage on avelumab. g Median PFS and OS measured from start of avelumab therapy (ie, not including prior chemotherapy) were 2.8 months (95% CI, 2.3–4.1) and 11.1 months (95% CI, 8.9–13.7), respectively. The 6-month and 12-month PFS rates were 23.0% (95% CI, 14.7–32.4%) and 13.0% (95% CI, 6.6–21.6%), respectively, and the 12-month OS rate was 46.2% (95% CI, 35.6–56.1%) (Fig. 2a and b). In patients from Asian and non-Asian countries, median OS was 12.4 months (95% CI, 9.7–20.0) and 9.4 months Two patients had prolonged shrinkage in target lesions with avelumab after documented progressive disease (due to >30% increase in target lesion size vs baseline at weeks 7–13 in 1 patient and a new lesion in the other patient), suggestive of pseudoprogression. Chung et al. Journal for ImmunoTherapy of Cancer (2019) 7:30 Chung et al. Antitumor activity: 1 L-mn subgroup Journal for ImmunoTherapy of Cancer Page 6 of 10 100 90 80 70 60 50 40 30 20 10 0 0 2 4 6 8 10 12 14 16 Time since treatment initiation (months) Progression-free survival (%) 18 20 22 24 26 28 30 32 34 36 90 Number at risk 55 36 17 13 8 8 5 5 5 5 5 5 4 4 4 4 2 0 Median PFS: 2.8 months (95% CI, 2.3–4.1) 6-month PFS rate: 23.0% (95% CI, 14.7–32.4%) 12-month PFS rate: 13.0% (95% CI, 6.6–21.6%) 100 90 80 70 60 50 40 30 20 10 0 0 2 4 6 8 10 12 14 16 Time since treatment initiation (months) Overall survival (%) 18 20 22 24 26 28 30 32 34 38 36 90 83 78 70 60 49 41 33 29 26 24 19 18 15 11 11 10 7 0 3 Number at risk Median OS: 11.1 months (95% CI, 8.9–13.7) 12-month OS rate: 46.2% (95% CI, 35.6–56.1%) 100 90 80 70 60 50 40 30 20 10 0 0 2 4 6 8 10 12 14 16 Time since prior treatment initiation (months) Overall survival (%) 18 20 22 24 26 28 30 32 34 36 38 40 42 44 46 48 85 Number at risk 85 85 83 75 72 63 56 49 44 36 30 26 22 22 18 16 15 14 12 9 6 4 2 0 Median OS: 18.7 months (95% CI, 15.4–20.6) 12-month OS rate: 74.1% (95% CI, 63.4–82.1%) 24-month OS rate: 31.7% (95% CI, 22.2–41.7%) 100 90 80 70 60 50 40 30 20 10 0 0 2 4 6 8 10 12 14 16 Time since treatment initiation (months) Progression-free survival (%) 18 Median PFS: 1.4 months (95% CI, 1.3–1.5) 6-month PFS rate: 7.9% (95% CI, 2.6–17.2%) 12-month PFS rate: 2.0% (95% CI, 0.2–9.1%) 20 22 24 26 28 30 32 34 36 60 Number at risk 19 12 4 3 3 1 1 1 0 0 0 0 0 0 0 0 0 0 100 90 80 70 60 50 40 30 20 10 0 0 2 4 6 8 10 12 14 16 Time since treatment initiation (months) Overall survival (%) 18 20 22 24 26 28 30 32 34 36 38 60 54 40 33 24 18 13 11 10 8 8 8 6 5 3 3 1 1 1 0 Number at risk Median OS: 6.6 months (95% CI, 5.4–9.4) 12-month OS rate: 25.6% (95% CI, 14.9–37.6%) A C B D E Fig. Antitumor activity: 1 L-mn subgroup e OS in the 2 L subgroup (n = 60) 100 90 80 70 60 50 40 30 20 10 0 0 2 4 6 8 10 12 14 16 Time since treatment initiation (months) Progression-free survival (%) 18 20 22 24 26 28 30 32 34 36 90 Number at risk 55 36 17 13 8 8 5 5 5 5 5 5 4 4 4 4 2 0 Median PFS: 2.8 months (95% CI, 2.3–4.1) 6-month PFS rate: 23.0% (95% CI, 14.7–32.4%) 12-month PFS rate: 13.0% (95% CI, 6.6–21.6%) A 100 90 80 70 60 50 40 30 20 10 0 0 2 4 6 8 10 12 14 16 Time since prior treatment initiation (months) Overall survival (%) 18 20 22 24 26 28 30 32 34 36 38 40 42 44 46 48 85 Number at risk 85 85 83 75 72 63 56 49 44 36 30 26 22 22 18 16 15 14 12 9 6 4 2 0 Median OS: 18.7 months (95% CI, 15.4–20.6) 12-month OS rate: 74.1% (95% CI, 63.4–82.1%) 24-month OS rate: 31.7% (95% CI, 22.2–41.7%) C C A 90 55 36 17 13 8 8 5 5 5 5 5 5 4 4 4 4 2 0 100 90 80 70 60 50 40 30 20 10 0 0 2 4 6 8 10 12 14 16 Time since treatment initiation (months) Overall survival (%) 18 20 22 24 26 28 30 32 34 38 36 90 83 78 70 60 49 41 33 29 26 24 19 18 15 11 11 10 7 0 3 Number at risk Median OS: 11.1 months (95% CI, 8.9–13.7) 12-month OS rate: 46.2% (95% CI, 35.6–56.1%) B 100 D 100 90 80 70 60 50 40 30 20 10 0 0 2 4 6 8 10 12 14 16 Time since treatment initiation (months) Progression-free survival (%) 18 Median PFS: 1.4 months (95% CI, 1.3–1.5) 6-month PFS rate: 7.9% (95% CI, 2.6–17.2%) 12-month PFS rate: 2.0% (95% CI, 0.2–9.1%) 20 22 24 26 28 30 32 34 36 60 Number at risk 19 12 4 3 3 1 1 1 0 0 0 0 0 0 0 0 0 0 D B Median PFS: 1.4 months (95% CI, 1.3–1.5) 6-month PFS rate: 7.9% (95% CI, 2.6–17.2%) 12-month PFS rate: 2.0% (95% CI, 0.2–9.1%) 90 83 78 70 60 49 41 33 29 26 24 19 18 15 11 11 10 7 0 3 60 19 12 4 3 3 1 1 1 0 0 0 0 0 0 0 0 0 0 100 90 80 70 60 50 40 30 20 10 0 0 2 4 6 8 10 12 14 16 Time since treatment initiation (months) Overall survival (%) 18 20 22 24 26 28 30 32 34 36 38 60 54 40 33 24 18 13 11 10 8 8 8 6 5 3 3 1 1 1 0 Number at risk Median OS: 6.6 months (95% CI, 5.4–9.4) 12-month OS rate: 25.6% (95% CI, 14.9–37.6%) E Fig. Antitumor activity: 1 L-mn subgroup 2 Kaplan–Meier estimates of progression-free survival (PFS) and overall survival (OS). a PFS from start of avelumab in the first-line switch- maintenance (1 L-mn) subgroup (n = 90). b OS from start of avelumab in the 1 L-mn subgroup (n = 90). c OS from start of 1 L chemotherapy in the 1 L-mn subgroup (n = 90). d PFS in the second-line (2 L) subgroup (n = 60). Antitumor activity: 1 L-mn subgroup 2 Kaplan–Meier estimates of progression-free survival (PFS) and overall survival (OS). a PFS from start of avelumab in the first-line switch- maintenance (1 L-mn) subgroup (n = 90). b OS from start of avelumab in the 1 L-mn subgroup (n = 90). c OS from start of 1 L chemotherapy in the 1 L-mn subgroup (n = 90). d PFS in the second-line (2 L) subgroup (n = 60). e OS in the 2 L subgroup (n = 60) 100 E Antitumor activity: 2 L subgroup (95% CI, 7.4–13.7), respectively. Median OS measured from start of 1 L chemotherapy was 18.7 months (95% CI, 15.4–20.6) overall (Fig. 2c), and 20.6 months (95% CI, 17.1–28.1) and 15.8 months (95% CI, 12.3–19.9) in patients from Asian and non-Asian countries, respect- ively. In PD-L1+ and PD-L1−subgroups, median PFS was 3.0 (95% CI, 1.4–4.1) and 2.7 (95% CI, 1.4–3.6) months (hazard ratio [HR], 0.844 [95% CI, 0.505–1.411]) and median OS was 15.9 (95% CI, 11.4–20.7) and 10.4 (95% CI, 8.3–12.4) months (HR, 0.588 [95% CI, 0.342–1.009]), respectively (Additional file 1: Figures S3A and S4A). The ORR was 6.7% (95% CI, 1.8–16.2%; PR in 4 patients), and the disease control rate was 28.3% (13 patients [21.7%] had SD as best response) (Additional file 1: Table S2). Me- dian time to and duration of response were 2.0 months (IQR, 1.3–2.7) and 3.5 months (95% CI, 2.8–8.3), respect- ively. An estimated 25.0% (95% CI, 0.9–66.5%) of responses lasted ≥6 months (Fig. 1b and Additional file 1: Figure S1B). Of 52 evaluable patients, 7 (13.5%) had target lesion shrink- age of ≥30% and 16 (30.8%) had shrinkage of any level (Additional file 1: Figure S2B). Median PFS was 1.4 months The ORR was 6.7% (95% CI, 1.8–16.2%; PR in 4 patients), and the disease control rate was 28.3% (13 patients [21.7%] had SD as best response) (Additional file 1: Table S2). Me- dian time to and duration of response were 2.0 months (IQR, 1.3–2.7) and 3.5 months (95% CI, 2.8–8.3), respect- ively. An estimated 25.0% (95% CI, 0.9–66.5%) of responses lasted ≥6 months (Fig. 1b and Additional file 1: Figure S1B). Of 52 evaluable patients, 7 (13.5%) had target lesion shrink- age of ≥30% and 16 (30.8%) had shrinkage of any level (Additional file 1: Figure S2B). Median PFS was 1.4 months Chung et al. Antitumor activity: 1 L-mn subgroup Journal for ImmunoTherapy of Cancer (2019) 7:30 Page 7 of 10 Table 2 Any-grade TRAEs occurring in ≥10% of patients or grade ≥3 in any patient and infusion-related reactions in the first-line switch-maintenance or second-line subgroup Patients, n (%) 1 L-mn subgroup (n = 90) 2 L subgroup (n = 60) Any grade Grade 3 Grade 4 Grade 5 Any grade Grade 3 Grade 4 Grade 5 Any TRAEa 57 (63.3) 7 (7.8) 0 1 (1.1) 28 (46.7) 3 (5.0) 2 (3.3) 0 Fatigue 10 (11.1) 2 (2.2) 0 0 5 (8.3) 0 0 0 Decreased appetite 3 (3.3) 0 0 0 2 (3.3) 1 (1.7) 0 0 Asthenia 1 (1.1) 0 0 0 3 (5.0) 2 (3.3) 0 0 Colitis 2 (2.2) 1 (1.1) 0 0 0 0 0 0 Elevated amylase 2 (2.2) 0 0 0 1 (1.7) 1 (1.7) 0 0 Elevated lipase 2 (2.2) 1 (1.1) 0 0 1 (1.7) 0 1 (1.7) 0 Elevated γ-glutamyltransferase 2 (2.2) 1 (1.1) 0 0 0 0 0 0 Anemia 1 (1.1) 1 (1.1) 0 0 1 (1.7) 1 (1.7) 0 0 Decreased platelet count 1 (1.1) 1 (1.1) 0 0 1 (1.7) 0 1 (1.7) 0 Abdominal pain 1 (1.1) 1 (1.1) 0 0 0 0 0 0 Adrenal insufficiency 1 (1.1) 0 0 0 1 (1.7) 1 (1.7) 0 0 Autoimmune hepatitisb 1 (1.1) 0 0 1 (1.1) 0 0 0 0 Decreased hemoglobin 1 (1.1) 1 (1.1) 0 0 0 0 0 0 Hepatic failureb 1 (1.1) 0 0 1 (1.1) 0 0 0 0 Hyperglycemia 1 (1.1) 1 (1.1) 0 0 0 0 0 0 Hypokalemia 1 (1.1) 1 (1.1) 0 0 0 0 0 0 Peripheral motor neuropathy 1 (1.1) 1 (1.1) 0 0 0 0 0 0 Infusion-related reactionc 20 (22.2) 1 (1.0) 0 0 11 (18.3) 0 0 0 aThe incidence of treatment-related infusion-related reaction based on the single MedDRA preferred term is not listed bOccurred in the same patient cIncludes adverse events categorized as infusion-related reaction, drug hypersensitivity, or hypersensitivity reaction that occurred on the day of infusion or day after infusion, in addition to signs and symptoms of infusion-related reaction that occurred on the same day of infusion and resolved within 2 days (including adverse events classified by investigators as related or unrelated to treatment) 1 L-mn first-line switch-maintenance, 2 L second line, TRAE treatment-related adverse event Table 2 Any-grade TRAEs occurring in ≥10% of patients or grade ≥3 in any patient and infusion-related reactions in the first-line switch-maintenance or second-line subgroup Table 2 Any-grade TRAEs occurring in ≥10% of patients or grade ≥3 in any patient and infusion-related reactions in the first-line i h i d li b Occurred in the same patient cIncludes adverse events categorized as infusion-related reaction, drug hypersensitivity, or hypersensitivity reaction that occurred on the day of infusion or day after infusion, in addition to signs and symptoms of infusion-related reaction that occurred on the same day of infusion and resolved within 2 days (including adverse events classified by investigators as related or unrelated to treatment) 1 L-mn first-line switch-maintenance 2 L second line TRAE treatment-related adverse event y g ) 1 L-mn first-line switch-maintenance, 2 L second line, TRAE treatment-related adverse event (6.0%), and pruritus (5.3%). Antitumor activity: 1 L-mn subgroup Thirteen patients (8.7%) had a grade ≥3 TRAE (1 L-mn subgroup, 8 [8.9%]; 2 L subgroup, 5 [8.3%]), most commonly fatigue, asthenia, anemia, and elevated lipase (n = 2 each; 1.3%). One patient (0.7%) had a grade 3 infusion-related reaction (1 L-mn subgroup). Two patients (both 2 L subgroup) had a grade 4 TRAE: elevated lipase (n = 1) and decreased platelet count (n = 1). Overall, 81 patients (54.0%) had a SAE, which was related to treatment in 6 patients (4.0%; 3 in each subgroup). One treatment-related death occurred (1 L-mn subgroup) in a patient with peritoneal metastases and ascites at study entry who developed grade 5 autoimmune hepatitis and hepatic failure. Twenty-three patients (15.3%) had an immune-related AE, including grade ≥3 in 3 patients (2.0%): colitis (grade 3, 1 L-mn), autoimmune hepatitis/hepatic failure (grade 5, 1 L-mn), and adrenal insufficiency (grade 3, 2 L). Avelumab was permanently discontinued following a TRAE in 8 patients (5.3% overall; 1 L-mn: 6 [6.7%]; 2 L: 2 [3.3%]), of whom 3 (2.0%) discontinued because of an infusion-related reaction. (95% CI, 1.3–1.5), and 6-month and 12-month PFS rates were 7.9% (95% CI, 2.6–17.2%) and 2.0% (95% CI, 0.2– 9.1%), respectively (Fig. 2d). Median OS was 6.6 months (95% CI, 5.4–9.4), and the 12-month OS rate was 25.6% (95% CI, 14.9–37.6%) (Fig. 2e). In patients from Asian and non-Asian countries, median OS was 8.3 months (95% CI, 2.0–10.4) and 6.3 months (95% CI, 4.3–9.4), respectively. No significant difference was seen in PFS and OS based on PD-L1 status (Additional file 1: Figures S3B and S4B). (95% CI, 1.3–1.5), and 6-month and 12-month PFS rates were 7.9% (95% CI, 2.6–17.2%) and 2.0% (95% CI, 0.2– 9.1%), respectively (Fig. 2d). Median OS was 6.6 months (95% CI, 5.4–9.4), and the 12-month OS rate was 25.6% (95% CI, 14.9–37.6%) (Fig. 2e). In patients from Asian and non-Asian countries, median OS was 8.3 months (95% CI, 2.0–10.4) and 6.3 months (95% CI, 4.3–9.4), respectively. No significant difference was seen in PFS and OS based on PD-L1 status (Additional file 1: Figures S3B and S4B). Occurred in the same patient cIncludes adverse events categorized as infusion-related reaction, drug hypersensitivity, or hypersensitivity reaction that occurred on the day of infusion or day after infusion, in addition to signs and symptoms of infusion-related reaction that occurred on the same day of infusion and resolved within 2 days (including adverse events classified by investigators as related or unrelated to treatment) 1 L mn first line switch maintenance 2 L second line TRAE treatment related adverse event aThe incidence of treatment-related infusion-related reaction based on the single MedDRA preferred term is not listed bOccurred in the same patient Discussion were initiated. In a randomized phase 3 trial of nivolumab vs placebo as third-line or later treatment in Asian patients with GC/GEJC (n = 493), the ORR was 11.2% vs 0% (P<.0001), median PFS was 1.6 vs 1.5 months (P<.0001), and median OS was 5.3 vs 4.1 months (P<.0001), respect- ively [13]. However, to date no improvement in OS has been shown in studies comparing single-agent checkpoint inhibitors with chemotherapy, such as trials of 2 L pem- brolizumab vs paclitaxel (KEYNOTE-061) [29] and third-line avelumab vs physician choice of chemotherapy (JAVELIN Gastric 300) [30]. Results from phase 3 trials assessing alternative anti–PD-1/PD-L1–based regimens in the 1 L setting, such as switch-maintenance (sequential) or combination (concurrent) approaches, are needed. In this single-arm phase 1b cohort of 150 patients with previously treated advanced GC/GEJC, avelumab showed evidence of durable antitumor activity as 1 L-mn and 2 L therapy. The ORR was 6.7% in both subgroups, although median durations of response were 21.4 months in the 1 L-mn subgroup and 3.5 months in the 2 L subgroup. Remarkably, 2 patients (2.2%) in the 1 L-mn subgroup had a CR after achieving only SD on prior chemotherapy. Avelumab showed a tolerable safety profile, including a low rate of grade ≥3 TRAEs (8.7%) and immune-related AEs (any grade, 15.3%; grade ≥3, 2.0%), similar to observations in other tumor types [21]. Detailed guidance for recognizing and managing immune-related AEs with this class of agents have been published by consensus groups [22, 23]. The incidence of TRAEs of any grade was higher in the 1 L-mn sub- group compared with the 2 L subgroup (63.3% vs 46.7%), which may be due to the longer treatment dur- ation and shorter interval from end of prior chemother- apy to start of avelumab in the 1 L-mn subgroup, although the incidence of grade ≥3 TRAEs was similar in both subgroups (8.9% vs 8.3%, respectively). pp Available data indicate that the benefits seen with anti–PD-1/PD-L1 antibodies in GC/GEJC may be lim- ited to a small proportion of patients. Thus, predictive biomarkers to identify subpopulations more likely to respond to immunotherapy are a focus of ongoing research [10]. In this study, clinical activity was seen both in PD-L1+ and PD-L1−tumors including similar ORR and PFS and a nonsignificant trend in the 1 L-mn subgroup for longer OS in PD-L1+ tumors. Discussion It should be noted that the PD-L1 assay used in this study differs from those used in studies of other approved anti–PD-1/ PD-L1 antibodies. Also, in the present study, PD-L1 status was based solely on tumor cell expression, whereas in studies of pembrolizumab in patients with GC/GEJC, in which antitumor activity was associated with PD-L1 expression, PD-L1 status was determined based on expression on tumor or immune cells (combined positive score). In addition, responses to pembrolizumab in patients with GC/GEJC have been associated with microsatellite instability–high/mismatch repair–deficient status and Epstein-Barr virus status [12, 31]. In the current trial, microsatellite status was available for only a small number of patients and findings were inconclusive. Assessment of novel biomarkers is planned for future avelumab studies in GC/GEJC. g p ( p y) Approximately 70% of patients achieve a response or SD with standard 1 L chemotherapy [24, 25]; however, duration of OS is usually short [2, 3]. In the 1 L-mn sub- group, median PFS was 2.8 months (6-month rate, 23.0%), median OS measured from the start of avelumab was 11.1 months (12-month rate, 46.2%), and median OS measured from the start of prior chemotherapy was 18.7 months. Thus, the OS seen in the 1 L-mn subgroup, which enrolled patients without disease progression fol- lowing chemotherapy, is encouraging for this subgroup of patients. Administering immunotherapy sequentially after completion of 1 L chemotherapy may enhance the immunostimulatory effects of chemotherapy while redu- cing the toxicity that may result when anti–PD-1 anti- bodies are administered in combination with other agents (eg, chemotherapy or ipilimumab) [26, 27]. To further assess this strategy, a randomized phase 3 trial is comparing avelumab switch-maintenance treatment with continuation of 1 L platinum-based chemotherapy in patients with advanced GC/GEJC (JAVELIN Gastric 100; NCT02625610). Conclusion The data in the present phase 1b study demonstrate that avelumab administered as maintenance therapy (after disease control with standard chemotherapy) has antitumor activity and acceptable safety in patients with advanced GC/GEJC, supporting further investigations of this treatment approach. Several early-phase studies assessed anti–PD-1 monother- apy in patients with chemotherapy-treated (later-line) GC/ GEJC outside of the maintenance setting [12, 13, 27, 28], and median PFS and OS reported in non–PD-L1–selected populations were 2.0 months and 5.5–6.2 months, respect- ively. Survival data for avelumab (anti–PD-L1) in the 2 L subgroup (median PFS and OS of 1.4 and 6.6 months, respectively) appear consistent with these studies. Subse- quently, phase 3 trials assessing later-line treatment with anti–PD-1/PD-L1 monotherapy in advanced GC/GEJC Safety Across both subgroups, 85 of 150 patients (56.7%) had a treatment-related AE (TRAE) of any grade, including 57 of 90 (63.3%) in the 1 L-mn subgroup and 28 of 60 (46.7%) in the 2 L subgroup. Patterns of TRAEs were similar in both subgroups (Table 2 and Additional file 1: Table S4). Overall, infusion-related reactions and related symptoms occurred in 20.7%. Time to onset of infusion-related reaction was first or second infusion in 29 of 31 cases (93.5%). Other common TRAEs (any grade in ≥5%) were fatigue (10.0%), nausea (6.7%), chills Chung et al. Journal for ImmunoTherapy of Cancer (2019) 7:30 Page 8 of 10 Page 8 of 10 Availability of data and materials For all new products or new indications approved in both the European Union and the United States after January 1, 2014, Merck KGaA, Darmstadt, Germany will share patient-level and study-level data after deidentification, as well as redacted study protocols and clinical study reports from clinical tri- als in patients. These data will be shared with qualified scientific and medical researchers, upon researcher’s request, as necessary for conducting legitimate research. Such requests must be submitted in writing to the company’s data sharing portal. More information can be found at https://www.merckgroup. com/en/research/our-approach-to-research-and-development/healthcare/ clinical-trials/commitment-responsible-data-sharing.html. Where Merck KGaA has a co-research, co-development or co-marketing/co-promotion agree- ment or where the product has been out-licensed, it is recognized that the responsibility for disclosure may be dependent on the agreement between parties. Under these circumstances, Merck KGaA will endeavour to gain agreement to share data in response to requests. AstraZeneca; stock and other ownership interests: Bristol-Myers Squibb. y HTA, JL, SYR, DYO, LW, JRI, SSL, MK, DS, and HS declare that they have no competing interests. HTA, JL, SYR, DYO, LW, JRI, SSL, MK, DS, and HS declare that they have no competing interests. Publisher’s Note Springer Nature remains neutral with regard to jurisdictional claims in published maps and institutional affiliations. Springer Nature remains neutral with regard to jurisdictional claims in published maps and institutional affiliations. Funding This trial was sponsored by Merck KGaA and is part of an alliance between Merck KGaA and Pfizer, Inc., New York, NY, USA. Medical writing support was provided by ClinicalThinking and was funded by Merck KGaA and Pfizer. HX: employment: EMD Serono Inc. (a business of Merck KGaA, Darmstadt, Germany). JH: employment: EMD Serono Inc. (a business of Merck KGaA, Darmstadt, Germany). VC: employment: EMD Serono Inc. (a business of Merck KGaA, Darmstadt, Germany) when the study was performed; current employment at VC: employment: EMD Serono Inc. (a business of Merck KGaA, Darmstadt, Germany) when the study was performed; current employment at AstraZeneca; stock and other ownership interests: Bristol-Myers Squibb. HTA, JL, SYR, DYO, LW, JRI, SSL, MK, DS, and HS declare that they have no competing interests. Abbreviations BM: honoraria: Bristol-Myers Squibb, Merck & Co, Merck KGaA, Novartis, Roche; consulting or advisory role: Bristol-Myers Squibb, Merck & Co, Merck KGaA, Novartis, Roche; travel, accommodations, expenses: Bristol-Myers Squibb, Novartis, Roche. 1 L-mn: First-line switch-maintenance; 2 L: Second-line; AE: Adverse event; CR: Complete response; ECOG PS: Eastern Cooperative Oncology Group performance status; GC: Gastric cancer; GEJC: Gastroesophageal junction cancer; HR: Hazard ratio; ORR: Objective response rate; OS: Overall survival; PFS: Progression-free survival; PR: Partial response; SAE: Serious adverse event; SD: Stable disease; TRAE: Treatment-related adverse event 1 L-mn: First-line switch-maintenance; 2 L: Second-line; AE: Adverse event; CR: Complete response; ECOG PS: Eastern Cooperative Oncology Group performance status; GC: Gastric cancer; GEJC: Gastroesophageal junction cancer; HR: Hazard ratio; ORR: Objective response rate; OS: Overall survival; PFS: Progression-free survival; PR: Partial response; SAE: Serious adverse event; SD: Stable disease; TRAE: Treatment-related adverse event p RP: honoraria: Bristol-Myers Squibb, Roche/Genentech; consulting or advisory role: AstraZeneca, BioMarin, Clovis Oncology, Karus Therapeutics, Mission Therapeutics, Merck & Co, Novartis, Roche/Genentech, Tesaro, Vertex; speakers bureau: Novartis; research funding: AstraZeneca/MedImmune, Clovis Oncology, Eisai, Etherapeutics, Ipsen, Vertex; patents, royalties, other intellectual property: named on patent for use of PARP inhibitor (rucaparib); travel, accommodations, expenses: Merck & Co, Roche/Genentech. ABG: employment: EMD Serono Inc. (a business of Merck KGaA, Darmstadt, Germany) when the study was performed; stock ownership: Halozyme Therapeutics. Author details 1 1Yonsei Cancer Center, Yonsei University College of Medicine, Yonsei University Health System, Seoul 03722, South Korea. 2Sarah Cannon Research Institute/ University College London, London, UK. 3Samsung Medical Center, Sungkyunkwan University School of Medicine, Seoul, South Korea. 4Seoul National University Hospital, Cancer Research Institute, Seoul National University College of Medicine, Seoul, South Korea. 5Centrum Onkologii-Instytut im. M. Sklodowskiej Curie, Warszawa, Poland. 6Asan Medical Center, University of Ulsan College of Medicine, Seoul, South Korea. 7Seoul National University Bundang Hospital, Seoul National University College of Medicine, Seongnam, South Korea. 8Sarah Cannon Research Institute/Tennessee Oncology, PLLC, Nashville, USA. 9Inje University College of Medicine, Busan, South Korea. 10Queens Cancer Center, Mt Sinai School of Medicine, New York, USA. 11Charité Comprehensive Cancer Center, Charitéplatz 1, Berlin, Germany. 12Palacky University Medical School and Teaching Hospital, I.P. Pavlova 6, Olomouc, Czech Republic. 13Samuel Oschin Comprehensive Cancer Institute, Cedars-Sinai Medical Center, Los Angeles, USA. 14Northern Centre for Cancer Care and Newcastle University, Freeman Hospital, Newcastle upon Tyne, UK. 15Medical Oncology, Bordeaux University Hospital, Bordeaux CEDEX, France. 16EMD Serono, Inc, Billerica, USA. 17Brown University, Providence, USA. 18Present address: AstraZeneca Pharmaceuticals LP, Gaithersburg, USA. Authors’ contributions P i i f d i Provision of study materials or patients: HCC, HTA, JL, SYR, DYO, LW, YKK, KWL, JRI, SSL, MK, UK, BM, AM, RP, DS, HS. Collection and assembly of data: all authors. Data analysis and interpretation: all authors. Manuscript writing: all authors. Final approval of manuscript: all authors. Accountable for all aspects of the work: all authors. Additional file Additional file 1: Table S1. Eligibility criteria. Table S2. Response to avelumab in the 1 L-mn and 2 L subgroups. Table S3. Best response to avelumab compared with best response to prior anticancer therapy in the 1 L-mn subgroup. Table S4. Overall summary of safety. Figure S1. Additional file 1: Table S1. Eligibility criteria. Table S2. Response to avelumab in the 1 L-mn and 2 L subgroups. Table S3. Best response to avelumab compared with best response to prior anticancer therapy in the 1 L-mn subgroup. Table S4. Overall summary of safety. Figure S1. Page 9 of 10 Page 9 of 10 Page 9 of 10 Chung et al. Journal for ImmunoTherapy of Cancer (2019) 7:30 Chung et al. Journal for ImmunoTherapy of Cancer KWL: research funding: AstraZeneca/MedImmune, Daiichi Sankyo, Five Prime Therapeutics, Green Cross Corp, Macrogenics, Merck KGaA, Merck & Co, Ono Pharmaceutical, Taiho Pharmaceutical. Time to and duration of response in responding patients. Figure S2. Best change in sum of target lesion diameters from baseline with avelumab in evaluable patients. Figure S3. Progression-free survival by PD-L1 expression status (≥1% tumor cell cutoff) in evaluable patients. Figure S4. Overall survival by PD-L1 expression status (≥1% tumor cell cutoff) in evaluable patients. (DOCX 794 kb) Time to and duration of response in responding patients. Figure S2. Best change in sum of target lesion diameters from baseline with avelumab in evaluable patients. Figure S3. Progression-free survival by PD-L1 expression status (≥1% tumor cell cutoff) in evaluable patients. Figure S4. Overall survival by PD-L1 expression status (≥1% tumor cell cutoff) in evaluable patients. (DOCX 794 kb) UK: honoraria: AstraZeneca, Bristol-Myers Squibb, Glycotope GmbH, Merck KGaA, Merck & Co, Novartis, Pfizer, Roche/Genentech; consulting or advisory role: AstraZeneca, Bristol-Myers Squibb, Merck Serono, Merck & Co, Pfizer; speakers bureau: AstraZeneca, Bristol-Myers Squibb, Glycotope GmbH, Merck Serono, MSD Oncology, Novartis; research funding: Pfizer; travel, accommodations, expenses: AstraZeneca, Merck KGaA, Merck & Co. Acknowledgements The authors thank the patients and their families, investigators, coinvestigators, and study teams at each of the participating centers and at Merck KGaA, Darmstadt, Germany, and EMD Serono Research & Development Institute, Inc., Billerica, MA, USA (a business of Merck KGaA, Darmstadt, Germany). ABG: employment: EMD Serono Inc. (a business of Merck KGaA, Darmstadt, Germany) when the study was performed; stock ownership: Halozyme Therapeutics. 1. Ferlay J, Soerjomataram I, Dikshit R, Eser S, Mathers C, Rebelo M, et al. Cancer incidence and mortality worldwide: sources, methods and major patterns in GLOBOCAN 2012. Int J Cancer. 2015;136:E359–86. 2. NCCN Clinical Practice Guidelines in Oncology. Gastric Cancer. V2.2018. https://www.nccn.org/professionals/physician_gls/pdf/gastric.pdf. Accessed 15 Oct 2018. Consent for publication Not applicable. Received: 24 October 2018 Accepted: 13 January 2019 Received: 24 October 2018 Accepted: 13 January 2019 Ethics approval and consent to participate The trial was conducted in accordance with the ethics principles of the Declaration of Helsinki and the International Council for Harmonisation Guidelines on Good Clinical Practice. The protocol was approved by the institutional review board or independent ethics committee of each center. All patients provided written informed consent before enrollment. References 10. Alsina M, Moehler M, Hierro C, Guardeño R, Tabernero J. Immunotherapy for gastric cancer: a focus on immune checkpoints. 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Bevacizumab in combination with chemotherapy as first-line therapy in advanced gastric cancer: a randomized, double-blind, placebo-controlled phase III study. J Clin Oncol. 2011;29:3968–76. y 7. Eren OO, Ozturk MA, Sonmez OU, Oyan B. Safety, feasibility, and efficacy of capecitabine maintenance in patients with advanced gastric cancer: a retrospective study. Am J Ther. 2016;23:e1493–7. 8. Li W, Zhao X, Wang H, Liu X, Zhao X, Huang M, et al. Maintenance treatment of uracil and tegafur (UFT) in responders following first-line fluorouracil-based chemotherapy in metastatic gastric cancer: a randomized phase 2 study. Oncotarget. 2017;8:37826–34. 26. Wainberg ZA, Jalal S, Muro K, Yoon HH, Garrido M, Golan T, et al. KEYNOTE-059 update: efficacy and safety of pembrolizumab alone or in combination with chemotherapy in patients with advanced gastric or gastroesophageal (G/GEJ) cancer. Ann Oncol. 2017;28 [abstract LBA28]. 9. Bang YJ, Cho JY, Kim YH, Kim JW, Di Bartolomeo M, Ajani JA, et al. Efficacy of sequential ipilimumab monotherapy vs best supportive care for unresectable locally advanced/metastatic gastric or gastroesophageal junction cancer. Clin Cancer Res. 2017;23:5671–8. 27. Janjigian YY, Ott PA, Calvo E, Kim JW, Ascierto PA, Sharma P, et al. Nivolumab ± ipilimumab in pts with advanced (adv)/metastatic chemotherapy-refractory (CTx-R) gastric (G), esophageal (E), or gastroesophageal junction (GEJ) cancer: CheckMate 032 study. J Clin Oncol. 2017;35(Suppl 15) [abstract 4014]. References HCC: consultant/advisory role: Bristol-Myers Squibb, Celltrion Healthcare, Eli Lilly, Merck & Co, Merck KGaA, Quintiles, Taiho Pharmaceutical; speakers bureau: Eli Lilly, Foundation Medicine, Merck KGaA; research grants: Bristol-Myers Squibb, Eli Lilly, GlaxoSmithKline, Merck & Co, Merck KGaA, Ono Pharmaceutical, Taiho Pharmaceutical. 1. Ferlay J, Soerjomataram I, Dikshit R, Eser S, Mathers C, Rebelo M, et al. 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Metformin-Induced Mitochondrial Complex I Inhibition: Facts, Uncertainties, and Consequences
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Metformin-Induced Mitochondrial Complex I Inhibition: Facts, Uncertainties, and Consequences INSERM, LBFA, Université Grenoble Alpes, Grenoble, France Metformin is the most widely prescribed drug to treat patients with type II diabetes, for whom retrospective studies suggest that metformin may have anticancer properties. However, in experiments performed with isolated cells, authors have reported both pro- and anti-apoptotic effects of metformin. The exact molecular mechanism of action of metformin remains partly unknown despite its use for over 60 years and more than 17,000 articles in PubMed. Among the various widely recognized or recently proposed targets, it has been reported consistently that metformin is capable of inhibiting mitochondrial respiratory chain Complex I. Since most of the effects of metformin have been replicated by other inhibitors of Complex I, it has been suggested that the mechanism of action of metformin involved the inhibition of Complex I. However, compared to conventional Complex I inhibitors, the metformin-induced inhibition of Complex I has unique characteristics. Among these, the most original one is that the concentrations of metformin required to inhibit Complex I are lower in intact cells than in isolated mitochondria. Experiments with isolated mitochondria or Complex I were generally performed using millimolar concentrations of metformin, while plasma levels remain in the micromolar range in both human and animal studies, highlighting that metformin concentration is an important issue. In order to explain the effects in animals based on observations in cells and mitochondria, some authors proposed a direct effect of the drug on Complex I involving an accumulation of metformin inside the mitochondria while others proposed an indirect effect (the drug no longer having to diffuse into the mitochondria). This brief review attempts to: gather arguments for and against each hypothesis concerning the mechanism by which metformin inhibits Complex I and to highlight remaining questions about the toxicity mechanism of metformin for certain cancer cells. Edited by: Frederic Bost, Centre National de la Recherche Scientifique (CNRS), France Reviewed by: Gayathri K. Natarajan, Medical College of Wisconsin, United States Adam Mickiewicz, University in Poznan, Poland *Correspondence: Eric Fontaine eric.fontaine@univ-grenoble-alpes.fr *Correspondence: Eric Fontaine eric.fontaine@univ-grenoble-alpes.fr Specialty section: This article was submitted to Cellular Endocrinology, a section of the journal Frontiers in Endocrinology Received: 27 April 2018 Accepted: 27 November 2018 Published: 17 December 2018 Citation: Fontaine E (2018) Metformin-Induced Mitochondrial Complex I Inhibition: Facts, Uncertainties, and Consequences. Front. Endocrinol. 9:753. doi: 10.3389/fendo.2018.00753 Specialty section: This article was submitted to Cellular Endocrinology, a section of the journal Frontiers in Endocrinology Keywords: metformin, mitochondria, Complex I, pharmacokinetic, cell death, cancer, permeability transition Keywords: metformin, mitochondria, Complex I, pharmacokinetic, cell death, cancer, permeability transition MINI REVIEW published: 17 December 2018 doi: 10.3389/fendo.2018.00753 MINI REVIEW published: 17 December 2018 doi: 10.3389/fendo.2018.00753 INTRODUCTION Received: 27 April 2018 Accepted: 27 November 2018 Published: 17 December 2018 Metformin is a drug with pleiotropic effects. It takes part in glucose homeostasis, mainly by inhibiting liver glucose production (1). It also modifies the production of reactive oxygen species and affects cell death processes (2, 3). Most of these effects have been traced to the inhibition of mitochondrial respiratory chain Complex I for two main reasons: First, over the past 20 years, different laboratories have reproducibly observed that metformin inhibits mitochondrial respiratory chain Complex I (4–20). Second, these pleiotropic effects have been reproduced by well identified Complex I inhibitors [gluconeogenesis (21, 22), cell death (18, 23–28)]. METFORMIN PHARMACOKINETICS Metformin is a hydrophilic compound charged positively at physiological pH. Its hydrophilicity limits its permeability through lipid membranes. Metformin enters and leaves cells by the presence of several transporters including Organic Cation Transporters (OCTs) and multidrug and toxin extrusion (MATE) transporters (29). This leads to a steady-state concentration of metformin inside cells, depending on both the amount and activity of such transporters as well as metformin plasma concentration. Despite this, many authors have hypothesized that metformin accumulates in mitochondria (5, 13, 15, 44). This scenario may reconcile the observation that millimolar concentrations of metformin are necessary to inhibit Complex I in isolated mitochondria (see below) while, when used at the therapeutic dose, the plasma metformin concentration remains in the micromolar range in both humans and animals (31, 36, 42). The pharmacological inhibition or the genetic ablation of OCTs reduce the distribution of metformin to the liver, small intestine and kidney (30–32) while the overexpression of OCT1 in HEK293 and CHO cells increases metformin uptake (30, 33). The pharmacological inhibition or the genetic ablation of MATE1 cause hepatic and kidney accumulation of metformin (32, 34). In humans, the genomic variations of metformin transporters can affect its pharmacokinetics (concentration, clearance, volume of distribution) (35, 36) suggesting that such genomic variations affect metformin concentration in tissues. From a theoretical point of view, this hypothesis is plausible. Indeed, because the mitochondrial respiratory chain transfers protons from the matrix to the intermembrane space, mitochondria build up and maintain an electrical mitochondrial membrane potential that drives the accumulation of positively charged molecules into mitochondria, provided the molecule crosses the membrane. In these conditions, Nernst equation indicates that for a physiological mitochondrial membrane potential of −180 mV the thermodynamic equilibrium is reached after a 1,000-fold accumulation of a positively charged molecule if the molecule has one charge. Since metformin is a positively charged molecule and assuming the presence of a still unknown carrier for metformin in the inner membrane, its mitochondrial concentration could reach the millimolar range despite a cytosolic concentration within the micromolar range (see Figure 1). In addition, assuming a plasma membrane potential of −36 mV and the absence of kinetic constraints on metformin transporters (OCT and MATE), the cytosolic concentration of metformin would be 4 times that of plasma. Citation: Fontaine E (2018) Metformin-Induced Mitochondrial Complex I Inhibition: Facts, Uncertainties, and Consequences. Front. Endocrinol. 9:753. doi: 10.3389/fendo.2018.00753 December 2018 | Volume 9 | Article 753 Frontiers in Endocrinology | www.frontiersin.org 1 Metformin and Mitochondrial Complex I Fontaine the studies (35, 40–42). With a volume of distribution of 1.12 ± 0.08 L/kg in healthy volunteers (40), metformin is not supposed to accumulate dramatically in tissues. The amount of metformin in the liver ranges from 2 to 5 times that of plasma -depending on the studies (32, 35, 42, 43)- and increases up to 10 times that of plasma in small intestinal walls (32). However, the mechanism by which metformin affects the activity of Complex I remains debated. In order to clarify whether the different conclusions found in the literature may be due to methodological differences, this review compares results obtained in vivo or with intact cells, to results obtained with isolated mitochondria or isolated Complex I. In this last case, authors tend to assume that metformin accumulates in mitochondria, here we will discuss evidence supporting or not this assumption. Finally, since pro- and anti-apoptotic effects of metformin are observed in intact cells, we will examine the role of metformin concentrations as a potential cause of these conflicting observations. Thus, the pharmacokinetic studies indicate that metformin enters but does not accumulate in large amounts in cells. Whether its metabolic activity depends on its diffusion inside the cells is supported by several but not all studies. Once in the cell, as metformin inhibits Complex I it is tempting to speculate that metformin penetrates the mitochondria. The composition of the mitochondrial matrix (the space delimited by the inner mitochondrial membrane) is different from that of the cytosol. In order to maintain such a different metabolite composition, the inner membrane is impermeable to almost all hydrophilic molecules which enter or leave the mitochondria through specific transporters. Among the numerous recognized mitochondrial carriers, no specific carrier for metformin has been identified yet. Frontiers in Endocrinology | www.frontiersin.org December 2018 | Volume 9 | Article 753 METFORMIN PHARMACOKINETICS Whether the activities of the metformin transporters (i.e., the metformin concentration in tissues) affect the metabolic effects of metformin is not systematically reported in the literature. On the one hand, metformin failed to reduce fasting plasma glucose concentration in OCT1-knockout mice submitted to a high- fat diet for 8 weeks and failed to suppress glucagon-stimulated glucose production in OCT1−/−hepatocytes (30). On the other hand, the effect of metformin on glucose tolerance tests was similar in animal controls and OCT1/2-knockout animals (31). A broad variation in clinical efficacy of metformin has long been recognized as well as a reduced function polymorphism of OCT1 in humans. However, if some authors reported a decreased effect of metformin in type-2 diabetes patients carrying reduced function polymorphism of OCT1 (30, 36), others did not observe such a correlation (37, 38). However, the hypothesis that metformin accumulates in mitochondria contradicts several observations. First of all, the accumulation of numerous positive charges in the matrix compensated by proton extrusion by the respiratory chain, should lead to a collapse of mitochondrial membrane potential associated with an increase in delta pH. However, note that metformin did not depolarize isolated mitochondria (8). To the best of my knowledge, no study correlating metformin concentration in tissue (or cells) and metformin-induced Complex I inhibition was ever published. Secondly, assuming that the total mitochondrial volume represents approximately 20% of hepatocytes, a 1,000-fold accumulation of metformin inside mitochondria would represent an approximately 200-fold accumulation of metformin in liver Drugs that are extensively sequestered in organelles have a very large apparent volume of distribution and a prolonged half- life in vivo (39). Metformin is not metabolized and is secreted by the kidneys with a half-life of 1.74–7.3 h in humans depending on December 2018 | Volume 9 | Article 753 2 Fontaine Metformin and Mitochondrial Complex I FIGURE 1 | Mechanism of action of metformin on complex I: the direct hypothesis and the indirect hypothesis. Metformin enters cells via Organic Cation Transporters (OCTs) and leaves cells via multidrug and toxin extrusion (MATE) transporters. Assuming a plasma membrane potential of 35 mV and a passive mechanism of OCTs and MATE, the cytosolic metformin concentration is expected to be 4 times that of the plasma concentration. METFORMIN PHARMACOKINETICS In the direct hypothesis (left), metformin is supposed to enter mitochondria via a hypothetical carrier reaching a matrix concentration 1,000 times that of the cytosol for mitochondrial membrane potential of 180 mV. In the indirect hypothesis (right), metformin does not enter mitochondria but stimulates a hypothetical signaling pathway that eventually modifies Complex I conformation, making it less active. FIGURE 1 | Mechanism of action of metformin on complex I: the direct hypothesis and the indirect hypothesis. Metformin enters cells via Organic Cation Transporters (OCTs) and leaves cells via multidrug and toxin extrusion (MATE) transporters. Assuming a plasma membrane potential of 35 mV and a passive mechanism of OCTs and MATE, the cytosolic metformin concentration is expected to be 4 times that of the plasma concentration. In the direct hypothesis (left), metformin is supposed to enter mitochondria via a hypothetical carrier reaching a matrix concentration 1,000 times that of the cytosol for mitochondrial membrane potential of 180 mV. In the indirect hypothesis (right), metformin does not enter mitochondria but stimulates a hypothetical signaling pathway that eventually modifies Complex I conformation, making it less active. compounds in the mitochondrial matrix at a concentration in the millimolar range. If metformin accumulated spontaneously in the mitochondria, the addition of molecules targeting the mitochondria would be unnecessary, which is clearly not the case. (without accounting for accumulation in the cytosol). Such an accumulation is 2 orders of magnitude higher than that measured by several groups (32, 35, 42, 43). Thirdly, a large mitochondrial accumulation is not compatible with the low volume of distribution of metformin and its short half-live (see above). Fourthly, using radioactive [14C] metformin, the radioactivity was not found to accumulate in liver mitochondria of rats treated orally with metformin (45) and no radioactivity was measured inside mitochondria when Xenopus laevis oocytes were exposed to concentrations of metformin that led to Complex I inhibition (6). Importantly, Complex I remained inhibited after mitochondrial isolation. Although this result does not definitively exclude a possible accumulation of metformin in mitochondria as a cause of Complex I inhibition (accumulated metformin may diffuse during the isolation procedure), it rules out the hypothesis that the inhibition of Complex I by metformin requires metformin inside mitochondria. Frontiers in Endocrinology | www.frontiersin.org CHARACTERISTICS OF COMPLEX I INHIBITION ACCORDING TO THE MODELS USED Although this may seem odd, it has been reported by several different laboratories that the concentrations required to inhibit Complex I are lower for intact cells than for isolated mitochondria (4–6, 13, 19). Note however that the characteristics of Complex I inhibition reveals some differences depending on whether metformin acts on intact cells (animal models, infused organs, isolated cells) or directly on isolated mitochondria or isolated Complex I (see Table 1). In summary, unlike the less hydrophilic biguanides (46), the accumulation of metformin inside the mitochondria is not supported by direct measurements, is not consistent with the pharmacokinetic data, and would require a transporter that has not yet been discovered. The incubation of isolated Complex I or submitochondrial particles in the presence of millimolar concentrations of metformin leads to an inhibition of Complex I that can be complete (13) with an IC50 ranging from 19 to 79 mM depending on laboratories (5, 9, 13). It should be noted that there is no membrane potential in these particular conditions of incubation, thus no possibility of metformin accumulation. In other words, the concentrations tested are the actual concentrations to which Complex I is exposed. Derivatives combining a molecule of metformin at different alkyl chain lengths containing a triphenylphosphonium cation (a liposoluble cation known to accumulate in mitochondria according to membrane potential) have been synthesized (47, 48) in order to increase the anti-cancer effect of metformin (see below). These different compounds accumulate in cells (47), depolarize mitochondria (48) and inhibit Complex I with an IC50 in the micromolar range (47, 48), which according to Nernst equation is consistent with the accumulation of The incubation of isolated mitochondria in the presence of millimolar concentrations of metformin leads to a rather fast (within a few minutes) inhibition of Complex I with an “apparent” IC50 also in the millimolar range (5). December 2018 | Volume 9 | Article 753 3 Metformin and Mitochondrial Complex I Fontaine TABLE 1 | Main differences in the characteristics of Complex I inhibition according to the model used. CHARACTERISTICS OF COMPLEX I INHIBITION ACCORDING TO THE MODELS USED Live animal, perfused organ, intact cells Isolated mitochondria, isolated Complex I References References Speed of inhibition Time dependent (4) (5) Immediate (minutes) (14) (13) Type of inhibition Partial (4) (5) (18) Total (13) Affinity Apparent IC50 approximately 1 mM after 30 min in isolated rat hepatocytes Apparent IC50 250 µM and 330 µM after 6 h incubation in 143B and HepG2 cells respectively (4) (13) Apparent IC50 15 mM in isolated mitochondria IC50 79 mM in sub-mitochondrial particles IC50 66 mM in immunocaptured Complex I IC50 19 mM in isolated Complex I (5) (5) (9) (13) Inhibition in State-3? Yes (5) (11) (12) (14) (15) Yes (5) (8) (9) (10) (12) (14) (15) Inhibition in State-4? Yes (4) (7) (14) No (4) (8) (10) (15) Inhibition after uncoupling Yes (4) (7) (11) (14) (19) No (4) (15) (19) Inhibition after the removal of metformin Yes (4) (14) (20) No for metformin concentration ≤2 mM (13) NADH/NAD+ Increases (4) (5) (18) Decreases (19) metformin concentration (hours are required for micromolar concentrations of metformin) (5, 50). Contrary to what is observed in isolated Complex I, the inhibition is not total and plateaus at approximately 40% of the Vmax (4). Consistent with a pure effect on Complex I, the inhibition leads to an increase in the NADH/NAD+ ratio (as assessed by the Lactate/pyruvate and 3-hydroxybutyrate/ acetoacetate ratios) (4, 5). Importantly, once cells are permeabilized (i.e., once mitochondria can be studied as if they were isolated) the inhibition is observed in State 3, but also in State 4 and after uncoupling (4, 11, 19). Finally, Complex I remains inhibited in mitochondria isolated from either rat exposed to metformin or liver perfused with metformin, even after uncoupling (4, 14) or when NADH:quinone oxidoreductase activity (i.e., Complex I activity) is studied directly using broken mitochondria (4). Note that the isolation procedure removes most of (if not all) the free metformin, while uncoupling (either chemical or after inner membrane rupture) would release the putative accumulated metformin. Although these results do not exclude a possible binding of metformin in mitochondrial membrane, they rule out the hypothesis that the inhibition of Complex I by metformin could depend on membrane potential. This observation is not easily reconcilable with the proposal that metformin accumulates in mitochondria. CHARACTERISTICS OF COMPLEX I INHIBITION ACCORDING TO THE MODELS USED Indeed, assuming that metformin did accumulate in mitochondria, Complex I inhibition would have been observed at micromolar concentrations of metformin (corresponding to millimolar concentrations inside mitochondria), which has not been reported. One could argue that at millimolar concentrations of metformin, the inhibition of Complex I would depolarize mitochondria, preventing metformin accumulation. However, it has been shown that millimolar concentrations of metformin did not depolarize isolated mitochondria (8). In these particular conditions of incubation (isolated mitochondria exposed to millimolar concentrations of metformin), it has to be noted that the inhibition of Complex I is observed almost exclusively during ATP synthesis (also called State 3) and disappears when mitochondria are depolarized (uncoupled State) or at rest (also called State 4) (4, 8, 15). Such behavior is not observed with rotenone (the reference inhibitor of Complex I) but is typical of biguanide-induced inhibition of Complex I in isolated mitochondria (49). It has been proposed that the reason why the inhibition is not observed after uncoupling might be due to the fact that the driving force for metformin accumulation within mitochondria disappears in these particular conditions. Although, as stated above, the accumulation of metformin in mitochondria is not supported by any evidence, this hypothesis does not explain the lack of inhibition in State 4, a situation in which the driving force (the membrane potential) is higher than in State 3. Frontiers in Endocrinology | www.frontiersin.org OTHER MITOCHONDRIAL EFFECTS OF METFORMIN In intact cells the inhibition of oxygen consumption is strictly located on Complex I. This conclusion comes from the observation that metformin has no effect on oxygen consumption when electrons feed the respiratory chain downstream Complex I (using succinate for example) regardless of the respiratory State (3, 4 and uncoupled) (4). Curiously, it has been reported that the inhibition of oxygen consumption in isolated mitochondria is accompanied by NADH oxidation (19). This observation is not expected in case of a simple Complex I inhibition, suggesting an uncoupling effect of metformin in this particular condition. On the contrary, using isolated mitochondria and millimolar concentrations of metformin, some authors reported inhibitory effects on complexes III and IV (16). High concentrations of metformin have been reported to inhibit ATP hydrolysis but not The incubation of intact cells in the presence of metformin leads to a slower inhibition of Complex I depending on December 2018 | Volume 9 | Article 753 Frontiers in Endocrinology | www.frontiersin.org 4 Metformin and Mitochondrial Complex I Fontaine ATP synthesis (13), suggesting an unconventional effect on the ATP synthase. ATP synthesis (13), suggesting an unconventional effect on the ATP synthase. ATP synthesis (13), suggesting an unconventional effect on the ATP synthase. The first one (in chronological order, but second in popularity) proposes that in vivo and in intact cells, metformin triggers a signaling pathway that in turn induces the inhibition of Complex I (4). Although such a signaling pathway is yet unknown, it has been reported that Complex I exists in two different functional conformations (active and inactive) (52), while reactive thiols of several Complex I subunits have been identified as targets for post-translational modifications (53, 54). However, whether metformin affects reactive thiols in Complex I has not been published yet. Some evidence suggests that Complex I can interact with ATP synthase (51). So we may infer that in this particular condition of incubation (isolated mitochondria exposed to millimolar concentrations of metformin), ATP synthesis possibly sensitizes Complex I to metformin. Although speculative, this personal suggestion could account for the observation that millimolar concentrations of metformin inhibit Complex I almost exclusively in State 3. In summary, on intact cells metformin acts slowly but the effect is visible at micromolar concentrations. The inhibition affects only Complex I in all the respiratory states and does not depend on mitochondrial membrane potential. WHERE DOES METFORMIN ACT ON COMPLEX I? Apparently contradictory effects are found in the literature regarding the effects of metformin on cell death. Some authors have put forward its protective effects against cell death (3) while others have reported its induction of cell death especially in cancer cells (2). Yet, all of them have concluded that the observed effects are due to the inhibition of Complex I (see below). The respiratory chain is a sequence of redox reactions which couple an electron flux with a vectorial transfer of protons. Mammalian respiratory chain complex I is a large protein complex with at least 45 subunits. It includes a hydrophobic part embedded in the inner membrane involved in proton transfer and a hydrophilic part protruding into the matrix in which electrons pass from NADH to ubiquinone via a succession of redox reactions. Complex I inhibitors rotenone and piericidin bind at, or close to, the ubiquinone binding site, inhibiting both electron flux and proton extrusion. Using artificial electron acceptors, a rotenone-insensitive NADH oxidation which is not coupled with proton pumping (i.e., a non-physiological pathway) can occur in Complex I. OTHER MITOCHONDRIAL EFFECTS OF METFORMIN On isolated mitochondria (or isolated Complex I), metformin acts rapidly but the effect requires millimolar concentrations. The inhibition does not only affect Complex I and Complex I inhibition is not observed in all the respiratory states. The second hypothesis necessarily involves an accumulation of metformin in the mitochondria that would be driven by mitochondrial membrane potential. Although proposed by several authors, this hypothesis is not yet supported by any evidence (see above). METFORMIN PREVENTS CELL DEATH WHEN IT IS DUE TO PTP OPENING The permeability transition pore (PTP) is a channel located in the inner membrane normally closed in order to maintain a high mitochondrial membrane potential required for ATP synthesis. Once permanently opened, the membrane potential collapses (55), leading to a drastic inhibition of ATP synthesis. Beyond this uncoupling effect, PTP opening has many other effects: It allows the thermodynamic equilibrium of the mitochondrial and cytosolic redox potentials, leading to an increase in cytosolic NAD(P)H concentration (56). It partly inhibits Complex I (57), reallocating the electron flux for the production of reactive oxygen species (58). Finally, it leads to the release of mitochondrial pro-apoptotic proteins both in isolated mitochondria (secondary to mitochondrial swelling leading to the rupture of the outer membrane) (59) and in intact cells (most probably by a distinct but still unknown mechanism) (56, 60–62). Using isolated Complex I and millimolar concentrations of metformin, it has been shown that metformin does not inhibit NADH oxidation due to artificial electron acceptors, behaves as a non-competitive inhibitor of the physiological electron pathway and preferentially binds Complex I when the enzyme is in its “deactive” conformation (13). However, the exact localization where metformin acts in this condition of incubation remains unknown. Moreover, the exact mechanism leading to the inhibition of Complex I in intact cells using micromolar concentrations of metformin and where exactly it inhibits the electron flux in Complex I has not been reported. As there are several signaling pathways involved in cell death, there are many factors activating these pathways. To discriminate whether a given condition leading to cell death involves PTP opening or not, experiments are performed in the presence or absence of a recognized PTP inhibitor (generally cyclosporine A, but not exclusively). Using this approach, it has been reproducibly observed that PTP opening occurs when cell death is triggered by calcium overload or oxidative stress (63). HYPOTHETICAL MECHANISMS OF ACTION To account for the fact that the concentration of metformin required to observe the inhibition of Complex I on whole cells is lower than the concentration required to observe the inhibition on mitochondria, two hypotheses have been proposed in the literature (see Figure 1). December 2018 | Volume 9 | Article 753 Frontiers in Endocrinology | www.frontiersin.org 5 Metformin and Mitochondrial Complex I Fontaine The molecular nature of the PTP has long been a subject of dispute but recent and compelling data from different laboratories suggest that the PTP might involve ATP synthase (51, 64). Surprisingly, the reference Complex I inhibitor rotenone has been shown to inhibit PTP opening in all the tested cells and tissues (either spontaneously or in the presence of cyclosporine A) (23, 65). Although rotenone induces an energetic stress, it also prevents cell death in the same models as cyclosporine A (23) and does inhibit Complex I and PTP opening with a similar concentration dependence (65). Piericidin, another well recognized Complex I inhibitor also inhibits PTP opening (23). Thus, the activity of Complex I can be said to be a regulator of PTP opening. Moreover, several ubiquinone analogs (known to bind with Complex I among others) have been proved to regulate PTP opening and cell death (57, 66–69). totally prevented by the overexpression of a metformin-resistant Saccharomyces cerevisiae NADH dehydrogenase NDI1 (15), very elegantly demonstrating that the toxicity of metformin is due to its effect on Complex I. The suggestion that metformin’s toxicity is related to an energetic stress raises several questions: Why is metformin less toxic in non-cancer cells that are yet more dependent on mitochondrial ATP production? How can metformin protect against PTP-induced cell death despite its effect on ATP production? In other words, what triggers that a same inhibition of Complex I either prevents or induces cell death? Again, part of the answer could be found in the comparison of metformin concentrations. While millimolar concentrations of metformin are generally used to induce cell death in vitro, micromolar concentrations are sufficient to prevent PTP- opening induced cell death. Although it has been shown that cellular energy status is inversely correlated with metformin concentrations (11, 79), a 24-h incubation with 100 µM metformin did not affect the AMP/ATP ratio in primary cultured hepatocytes (11). HYPOTHETICAL MECHANISMS OF ACTION This suggests that the metformin concentration used to prevent PTP opening (100 µM, overnight) was not sufficient to induce a lethal decrease in energy status. On the contrary, this confirms that the concentrations used to kill cells dramatically affect the energy status. Note however that some authors have reported an anti-apoptotic effect even at millimolar concentrations of metformin, suggesting that some cells are able to overcome energy stress (75, 80). Knowing that metformin partly inhibits Complex I, we tested whether it also inhibited PTP opening and related cell death. We found that, metformin was less potent than rotenone but also inhibited PTP opening (50). Suggesting a common mechanism of action with rotenone, the effect of metformin was not additive with that of rotenone, whereas it was additive with that of cyclosporine A (65). At present, metformin has been shown to prevent PTP opening in endothelial cells (50), KB cells (7), INS-1 insulinoma cells (61), HeLa cells (65), LNCaP cells (70), A375 cells (70), primary cortical neurons (71) and kidney mitochondria (72). Accordingly, metformin prevents cell death induced by oxidative stress in endothelial cells (50) and KB cells (7), etoposide in primary neurons (71), gentamicin in kidneys (72), hyperglycemia in endothelial (50) and INS-1 cells (61), hyperfructosemia in INS-1 cells (61) and ischemia reperfusion in INS-1 cells (73). Many other works have found a protective effect of metformin (particularly during oxidative stress or ischemia reperfusion injury) without having studied the role of the PTP (18, 74–77). However, if the mechanism by which metformin kills isolated cells can be traced to a collapse in energy status, the concentrations that prevent cancer growth in animal models are in the micromolar range. The practical assumption of metformin accumulation in mitochondria has obviously been retained, but one can wonder: why are normal cells preserved? Alternative or complementary explanations must exist. 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CONCLUSIONS AND PROPOSAL As explained several times in this manuscript, the concentration with which experiments were conducted is the main misleading point regarding the effect of metformin on Complex I. On the one hand, it is obvious that the assumption that metformin accumulates in mitochondria suits many authors. This hypothesis can bridge the gap between concentrations measured in vivo and those used in vitro. On the other hand, two different laboratories that attempted to measure such an accumulation put forward a total absence of metformin accumulation in mitochondria (6, 45) in which Complex I was nevertheless inhibited (6). Furthermore, although the pharmacokinetic data are indirect evidence, they are not compatible with an accumulation of metformin in mitochondria. Cancer cells are known to be generally highly glycolytic (Warburg effect) and are thus not supposed to be very sensitive to mitochondrial poison. But is it so simple? As soon as cells consume oxygen at the mitochondrial level, they are supposed to produce mitochondrial ATP. Thus, even if the proportion of mitochondrial ATP production is reduced in cancer cells, this mitochondrial ATP production exists and its reduction could be toxic. Supporting this proposal, it has been reported that metformin inhibits the proliferation of HCT116 p53−/−cancer cells in the presence of glucose, while it induces cell death in case of glucose deprivation (15). Moreover, the effect of metformin is December 2018 | Volume 9 | Article 753 6 Fontaine Metformin and Mitochondrial Complex I generally accepted hypothesis of metformin accumulation in mitochondria. Facing the facts, one must admit that there is either a technical mistake in the studies that did not find metformin accumulation in mitochondria or there is absolutely no experiment performed at millimolar concentrations of metformin that reflect what occurs in vivo. This includes a lot of articles both on its antidiabetic role and on its anticancer effect. There is an urgent need to solve this problem for good, and this could be performed easily by fast cell fractionation coupled to mass spectrometry (or other technics to detect metformin) in order to confirm if metformin is found in large amount in mitochondria of cells exposed to metformin. Currently the published evidence does not support the ACKNOWLEDGMENTS I thank Christophe Cottet and Emily Tubbs for the corrections of the English version of this paper. I thank Christophe Cottet and Emily Tubbs for the corrections of the English version of this paper. AUTHOR CONTRIBUTIONS The author confirms being the sole contributor of this work and has approved it for publication. REFERENCES Effect of transient and permanent permeability transition pore opening on NAD(P)H localization in intact cells. J Biol Chem. (2009) 284:15117–25. doi: 10.1074/jbc.M900926200 37. 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Frontiers in Endocrinology | www.frontiersin.org December 2018 | Volume 9 | Article 753 REFERENCES No use, distribution or reproduction is permitted which does not comply with these terms. 73. Lablanche S, Cottet-Rousselle C, Argaud L, Laporte C, Lamarche F, Richard MJ, et al. Respective effects of oxygen and energy substrate deprivation on beta cell viability. Biochim Biophys Acta (2015) 1847:629–39. doi: 10.1016/j.bbabio.2015.04.002 December 2018 | Volume 9 | Article 753 Frontiers in Endocrinology | www.frontiersin.org 9
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https://www.biorxiv.org/content/biorxiv/early/2019/07/20/708719.full.pdf
English
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Plasma-derived HIV Nef+ exosomes persist in ACTG384 study participants despite successful virological suppression
bioRxiv (Cold Spring Harbor Laboratory)
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cc-by
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. CC-BY 4.0 International license a certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made available under The copyright holder for this preprint (which was not this version posted July 20, 2019. ; https://doi.org/10.1101/708719 doi: bioRxiv preprint . CC-BY 4.0 International license a certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made available under The copyright holder for this preprint (which was not this version posted July 20, 2019. ; https://doi.org/10.1101/708719 doi: bioRxiv preprint . CC-BY 4.0 International license a certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made available under The copyright holder for this preprint (which was not this version posted July 20, 2019. ; https://doi.org/10.1101/708719 doi: bioRxiv preprint . CC-BY 4.0 International license a certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made available under The copyright holder for this preprint (which was not this version posted July 20, 2019. ; https://doi.org/10.1101/708719 doi: bioRxiv preprint 1 Plasma-derived HIV Nef+ exosomes persist in ACTG384 study participants despite successful 1 Plasma-derived HIV Nef+ exosomes persist in ACTG384 study participants despite successful 1 Plasma-derived HIV Nef+ exosomes persist in ACTG384 study participants despite successful 2 virological suppression. Andrea D. Raymond 1, Michelle J. Lang 1, Jane Chu 1, Tamika Campbell-Sims 1, 2 virological suppression. Andrea D. Raymond 1, Michelle J. Lang 1, Jane C 3 Mahfuz Khan1, Vincent C. Bond 1, Richard B. Pollard*, David M. Asmuth*, and Michael D. Powell 1. 4 Morehouse School of Medicine1, *University of California-Davis, and the ACTG 384 team 6 Short title: HIV Nef+ exosomes impact immune recovery 7 Keywords: Nef, exosomes, microvesicles, discordant CD count/viral load, CD4 recovery, 8 ACTG384 study 18 1 1 . CC-BY 4.0 International license a ertified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made available under The copyright holder for this preprint (which was not this version posted July 20, 2019. ; https://doi.org/10.1101/708719 doi: bioRxiv preprint . CC-BY 4.0 International license a certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made available under The copyright holder for this preprint (which was not this version posted July 20, 2019. ; https://doi.org/10.1101/708719 doi: bioRxiv preprint . CC-BY 4.0 International license a certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made available under The copyright holder for this preprint (which was not this version posted July 20, 2019. ; https://doi.org/10.1101/708719 doi: bioRxiv preprint 19 Abstract 19 Abstract 20 Human Immunodeficiency Virus (HIV) accessory protein Negative factor (Nef) is detected in the 21 plasma of HIV+ individuals associated with exosomes. The role of Nef+ exosomes (exNef) in HIV 22 pathogenesis is unknown. We perform a retrospective longitudinal analysis to determine 23 correlative clinical associations of exNef plasma levels in ARV-treated HIV+ patients with or 24 without immune recovery. exNef concentration in a subset of AIDS Clinical Trial Group (ACTG) 25 384 participants with successful virological suppression and with either high (∆ >100 CD4 cell 26 recovery/High Immunological Responders (High-IR) or low (∆ ≤100 CD4 cell recovery/ Low 27 Immunologic Responders (Low-IR) immunologic recovery was measured and compared for 28 study weeks 48, 96, and 144. CD4 recovery showed a negative correlation with exNef at study 29 week 144 (r = -0.3573, *p=.0366). Plasma exNef concentration in high IRs negatively correlated 30 with naïve CD4 count and recovery (r = -0.3249, *p = 0. 0348 (High-IR); r =0.2981, *p= #0.0513 (Low- 31 IR)). However, recovery of CD4 memory cells positively correlated with exNef (r =.4534, 32 *p=.0358) in Low-IRs but not in High-IRs. Regimen A (Didanosine, Stavudine, Efavirenz) lowered 33 exNef levels in IRs by 2-fold compared to other regimens. Nef+ exosomes persist in ART-treated 34 HIV+ individuals despite undetectable viral loads, negatively correlates with naive and memory 35 CD4 T cell restoration and may be associated with reduced immunological recovery. Taken 36 together, these data suggest that exNef may represent a novel mechanism utilized by HIV to 37 promote immune dysregulation 20 Human Immunodeficiency Virus (HIV) accessory protein Negative factor (Nef) is detected in the 38 39 2 2 . CC-BY 4.0 International license a ertified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made available under The copyright holder for this preprint (which was not this version posted July 20, 2019. ; https://doi.org/10.1101/708719 doi: bioRxiv preprint . CC-BY 4.0 International license a certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made available under The copyright holder for this preprint (which was not this version posted July 20, 2019. ; https://doi.org/10.1101/708719 doi: bioRxiv preprint 40 Introduction 41 The prognosis for patients infected with HIV has improved significantly with the advent of 42 combination antiretroviral therapy (cART) which can lead to suppression of plasma viremia 43 usually associated with marked improvements in CD4+ T cell counts [1, 2]. However, a subset of 44 patients do not experience a robust immune recovery despite viral suppression. While host 45 factors such as age and baseline CD4+ T-cells with a naïve phenotype have been observed to be 46 negatively associated with the magnitude of CD4+ T-cell count improvement [1, 3-6], virological 47 factors that contribute to these immunologic outcomes are less well defined. 48 Immune reconstitution can be defined as an increase in the number of peripheral CD4 T- 49 cells to greater than 350-500 cells/dL after 4 years of effective cART [3]. Discordant responses 50 characterized by a lack of immune recovery despite viral suppression occur in 7-39% of 51 participants receiving cART [4-7]. The reasons for this phenomenon are not understood. 52 The HIV-encoded Negative factor (Nef) has been implicated in pathogenesis based on 53 studies examining HIV-infected long term non-progressors (LTNP) and elite controllers (EC). 54 LTNPs remain asymptomatic for years with stable CD4 counts while ECs control plasma HIV 55 RNA in the absence of ART [12]. Although several hypotheses have been presented to account 56 for the protective factors leading to delayed disease progression, replication defective virions/nef- 57 defective virions have been associated with these populations in some cases [9-11]. Recent 58 reports have shown that the viruses within some LTNPs and ECs are actually replication 59 competent and control of HIV may be due to host and/or viral factors that maintain low levels of 60 viremia [11]. HIV-1 Nef may be integral to the control of viral replication and/or T-cell responses 61 in an HIV-infected individual. Early in vitro studies have shown that soluble Nef is cytotoxic to 48 Immune reconstitution can be defined as an increase in the number of peripheral CD4 T- 49 cells to greater than 350-500 cells/dL after 4 years of effective cART [3]. Discordant responses 50 characterized by a lack of immune recovery despite viral suppression occur in 7-39% of 51 participants receiving cART [4-7]. The reasons for this phenomenon are not understood. 3 52 The HIV-encoded Negative factor (Nef) has been implicated in pathogenesis based on 53 studies examining HIV-infected long term non-progressors (LTNP) and elite controllers (EC). 40 Introduction 54 LTNPs remain asymptomatic for years with stable CD4 counts while ECs control plasma HIV 55 RNA in the absence of ART [12]. Although several hypotheses have been presented to account 56 for the protective factors leading to delayed disease progression, replication defective virions/nef- 57 defective virions have been associated with these populations in some cases [9-11]. Recent 58 reports have shown that the viruses within some LTNPs and ECs are actually replication 59 competent and control of HIV may be due to host and/or viral factors that maintain low levels of 60 viremia [11]. HIV-1 Nef may be integral to the control of viral replication and/or T-cell responses 61 in an HIV-infected individual. Early in vitro studies have shown that soluble Nef is cytotoxic to 3 52 The HIV-encoded Negative factor (Nef) has been implicated in pathogenesis based on 53 studies examining HIV-infected long term non-progressors (LTNP) and elite controllers (EC). 54 LTNPs remain asymptomatic for years with stable CD4 counts while ECs control plasma HIV 55 RNA in the absence of ART [12]. Although several hypotheses have been presented to account 56 for the protective factors leading to delayed disease progression, replication defective virions/nef- 57 defective virions have been associated with these populations in some cases [9-11]. Recent 58 reports have shown that the viruses within some LTNPs and ECs are actually replication 59 competent and control of HIV may be due to host and/or viral factors that maintain low levels of 60 viremia [11]. HIV-1 Nef may be integral to the control of viral replication and/or T-cell responses 61 in an HIV-infected individual. Early in vitro studies have shown that soluble Nef is cytotoxic to 3 3 . CC-BY 4.0 International license a certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made available under The copyright holder for this preprint (which was not this version posted July 20, 2019. ; https://doi.org/10.1101/708719 doi: bioRxiv preprint . CC-BY 4.0 International license a certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made available under The copyright holder for this preprint (which was not this version posted July 20, 2019. 40 Introduction It is made available under The copyright holder for this preprint (which was not this version posted July 20, 2019. ; https://doi.org/10.1101/708719 doi: bioRxiv preprint . CC-BY 4.0 International license a ertified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made available under The copyright holder for this preprint (which was not this version posted July 20, 2019. ; https://doi.org/10.1101/708719 doi: bioRxiv preprint e Leukemia Virus) [28-32]. Given the immunomodulatory functions of exosomes, we to explore whether exNef may selectively impair CD4+ T-cells recovery during cART . CC-BY 4.0 International license a by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made available under The copyright holder for this preprint (which was not this version posted July 20, 2019. ; https://doi.org/10.1101/708719 doi: preprint . CC-BY 4.0 International license a certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made available under The copyright holder for this preprint (which was not this version posted July 20, 2019. ; https://doi.org/10.1101/708719 doi: bioRxiv preprint 83 Murine Leukemia Virus) [28-32]. Given the immunomodulatory functions of exosomes, we 84 sought to explore whether exNef may selectively impair CD4+ T-cells recovery during cART. 85 For this study, Nef concentration was determined in plasma-derived exosomes isolated 86 from a subset of the ACTG-384 cohort with and without immune recovery post-cART. We show 87 that Nef+ exosomes persist and can be detected in study participants with undetectable viral 88 loads even after 144 weeks of therapy. This suggests that exNef production is independent of 89 plasma viral load. Interestingly, we demonstrate that Low-IRs have significantly higher levels of 90 exNef compared to High- IRs at 144 weeks post-treatment. Recovery of naive CD4 T-cells 91 (CD45RA+, CD62L+) and total CD4 cells negatively correlated with exNef. Overall, we report that 92 Nef+ exosomes are detected in the plasma despite viral suppression and that exNef is negatively 93 associated with changes in naïve and total CD4 count. Taken together these results suggest that 94 exNef may ultimately affect CD4 T-cell recovery and be a biomarker of immune recovery. . CC-BY 4.0 International license a certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. 40 Introduction It is made available under 5 84 sought to explore whether exNef may selectively impair CD4+ T-cells recovery during cART. 85 For this study, Nef concentration was determined in plasma-derived exosomes isolated 86 from a subset of the ACTG-384 cohort with and without immune recovery post-cART. We show 87 that Nef+ exosomes persist and can be detected in study participants with undetectable viral 88 loads even after 144 weeks of therapy. This suggests that exNef production is independent of 89 plasma viral load. Interestingly, we demonstrate that Low-IRs have significantly higher levels of 90 exNef compared to High- IRs at 144 weeks post-treatment. Recovery of naive CD4 T-cells 91 (CD45RA+, CD62L+) and total CD4 cells negatively correlated with exNef. Overall, we report that 92 Nef+ exosomes are detected in the plasma despite viral suppression and that exNef is negatively 93 associated with changes in naïve and total CD4 count. Taken together these results suggest that 94 exNef may ultimately affect CD4 T-cell recovery and be a biomarker of immune recovery. 95 96 Material and Methods 97 Study Population. A subset of cryopreserved plasma samples (n = 240) taken from participants 98 in ACTG 384 were used in this study [1] (Robbins, GK 2003; Shafer 2003). As previously 99 described ACTG 384 was a factorial multi-center randomized controlled trial conducted in the 100 United States and Italy that compared sequential three-drug regimens for treatment of HIV 101 infection. Nine hundred eighty ART-naive HIV-1+ subjects were randomized and treated with 102 stavudine/didanosine or zidovudine/lamivudine with nelfinavir, efavirenz, or both nelfinavir 103 and efavirenz. If virological failure occurred, then participants were placed on another regimen 85 For this study, Nef concentration was determined in plasma-derived exosomes isolated 86 from a subset of the ACTG-384 cohort with and without immune recovery post-cART. We show 87 that Nef+ exosomes persist and can be detected in study participants with undetectable viral 88 loads even after 144 weeks of therapy. This suggests that exNef production is independent of 89 plasma viral load. Interestingly, we demonstrate that Low-IRs have significantly higher levels of 90 exNef compared to High- IRs at 144 weeks post-treatment. Recovery of naive CD4 T-cells 91 (CD45RA+, CD62L+) and total CD4 cells negatively correlated with exNef. 40 Introduction ; https://doi.org/10.1101/708719 doi: bioRxiv preprint 62 T-cells and is released from HIV-infected cells in plasma derived microvesicles that can be 63 detected in HIV-infected patients [13-23]. These studies suggest that Nef does not exist as a 64 soluble protein in vivo but instead non-virion associated Nef is found in microvesicles or 65 exosome-like microvesicles (exNef) [7, 8]. 66 Several in vitro studies have identified pathogenic activities of soluble Nef including its 67 capacity to reduce surface expression of CD4 and MHC class II, to increase HIV infectivity, to 68 stimulate primary macrophages to release pro-inflammatory cytokines and chemokines, and 69 induce apoptosis in CD4 T-cells [13, 18, 24-27]. We have previously shown that expression of nef 70 in HEK293 cells is sufficient to produce exNef, and the resultant exosomes can be absorbed by T- 71 cells and macrophage but only induces apoptosis in T-cells [14]. Taken together, these 72 observations suggest that Nef+ microvesicles/exosome could have an impact on CD4 T-cell 73 recovery. We hypothesize that Nef microvesicles/exosomes are released from HIV infected cells 74 despite successful cART-induced viral suppression and contribute to pathogenesis by impeding 75 CD4-T-cell recovery. 76 Exosomes range in size from 30 -100 nm and are released from hematopoetic (i.e. B-and T- 77 lymphocytes, dendritic cells, monocytes, mast cells, reticulocytes) and non-hematopoetic (i.e. 78 neurons, intestinal epithelial cells, and tumors) cells. Exosomes are detected in physiological 79 fluids such as plasma, urine, malignant effusions, and amniotic fluids. Several functions have 80 been attributed to exosomes/microvesicles including the modulation of cell signaling, cellular 81 homeostasis, intercellular communication, shuttling genetic material, establishment of tissue 82 polarity, regulation of immune responses, and enhancing the site of budding (i.e. HIV and 76 Exosomes range in size from 30 -100 nm and are released from hematopoetic (i.e. B-and T- 77 lymphocytes, dendritic cells, monocytes, mast cells, reticulocytes) and non-hematopoetic (i.e. 78 neurons, intestinal epithelial cells, and tumors) cells. Exosomes are detected in physiological 79 fluids such as plasma, urine, malignant effusions, and amniotic fluids. Several functions have 80 been attributed to exosomes/microvesicles including the modulation of cell signaling, cellular 81 homeostasis, intercellular communication, shuttling genetic material, establishment of tissue 82 polarity, regulation of immune responses, and enhancing the site of budding (i.e. HIV and 4 . CC-BY 4.0 International license a certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. 40 Introduction Overall, we report that 92 Nef+ exosomes are detected in the plasma despite viral suppression and that exNef is negatively 93 associated with changes in naïve and total CD4 count. Taken together these results suggest that 94 exNef may ultimately affect CD4 T-cell recovery and be a biomarker of immune recovery. 96 Material and Methods 97 Study Population. A subset of cryopreserved plasma samples (n = 240) taken from participants 98 in ACTG 384 were used in this study [1] (Robbins, GK 2003; Shafer 2003). As previously 99 described ACTG 384 was a factorial multi-center randomized controlled trial conducted in the 100 United States and Italy that compared sequential three-drug regimens for treatment of HIV 101 infection. Nine hundred eighty ART-naive HIV-1+ subjects were randomized and treated with 102 stavudine/didanosine or zidovudine/lamivudine with nelfinavir, efavirenz, or both nelfinavir 103 and efavirenz. If virological failure occurred, then participants were placed on another regimen 5 . CC-BY 4.0 International license a certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made available under The copyright holder for this preprint (which was not this version posted July 20, 2019. ; https://doi.org/10.1101/708719 doi: bioRxiv preprint . CC-BY 4.0 International license a ertified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made available under The copyright holder for this preprint (which was not this version posted July 20, 2019. ; https://doi.org/10.1101/708719 doi: bioRxiv preprint . CC-BY 4.0 International license a certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made available under The copyright holder for this preprint (which was not this version posted July 20, 2019. ; https://doi.org/10.1101/708719 doi: bioRxiv preprint 104 sequentially. In this study, samples from three distinct groups were obtained and assayed. The 105 groups included 1) Treatment failure (TF) defined as those with a virologic failure with a 106 detectable viral load ( HIV RNA > 50 RNA copies) what at any time point before 144 weeks, 2). 107 Immunologic responders defined as those with high CD4 improvement of > 100 cells/mm3 108 (achieved at any point post study initiation) and suppressed viremia (< 50 RNA copies) (High 109 IRs) and 3) Immunologic non-responders defined as those with CD4 improvement of < 100 110 cells/mm3 and suppressed viremia (Low IRs) (Figure 1). To avoid confounding effects of 111 multiple-treatment regimens, the samples utilized for this sub-cohort were derived from subjects 112 on their first treatment regimen and those with samples from three- or four time intervals (0, 48, 113 96, and/or 144 weeks). . 96 Material and Methods CC-BY 4.0 International license a 104 sequentially. In this study, samples from three distinct groups were obtained and assayed. The 105 groups included 1) Treatment failure (TF) defined as those with a virologic failure with a 106 detectable viral load ( HIV RNA > 50 RNA copies) what at any time point before 144 weeks, 2). 107 Immunologic responders defined as those with high CD4 improvement of > 100 cells/mm3 108 (achieved at any point post study initiation) and suppressed viremia (< 50 RNA copies) (High 109 IRs) and 3) Immunologic non-responders defined as those with CD4 improvement of < 100 110 cells/mm3 and suppressed viremia (Low IRs) (Figure 1). To avoid confounding effects of 111 multiple-treatment regimens, the samples utilized for this sub-cohort were derived from subjects 112 on their first treatment regimen and those with samples from three- or four time intervals (0, 48, 113 96, and/or 144 weeks). . CC-BY 4.0 International license a 114 Isolation of plasma-derived exosomes. Plasma exosomes were isolated as previously described 115 [14, 23, 36]. Plasma was pre-cleared by centrifugation at 10,000g for 30 minutes to remove 116 particulates. Microvesicles were pelleted from pre-cleared plasma via ultra-centrifugation for 1 117 hour at 300,000 x g and then re-suspended in 250 µl of phosphate buffered saline (PBS). Immune 118 complexes within the re-suspended exosomes were removed using acid-dissociation prior to Nef 119 measurement similar to p24 antigen measurements from plasma. Exosomes/high speed pellets 120 (100 µl) were treated with 100 µl of 0.3N hydrochloric (HCl, Sigma) and allowed to incubate for 1 121 hour at 37 C. The acid mixture was neutralized with 100 µl of 0.3N sodium hydroxide (NaCl, 122 Sigma) prior to assaying for Nef. 123 Nef Enzyme-linked Immunosorbent Assay (ELISA). Nef concentration in acid-dissociated 124 microvesicle/exosome preparations was measured using a commercially available anti-Nef 123 Nef Enzyme-linked Immunosorbent Assay (ELISA). Nef concentration in acid-dissociated 6 . CC-BY 4.0 International license a certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made available under The copyright holder for this preprint (which was not this version posted July 20, 2019. ; https://doi.org/10.1101/708719 doi: bioRxiv preprint . CC-BY 4.0 International license a ertified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made available under The copyright holder for this preprint (which was not this version posted July 20, 2019. ; https://doi.org/10.1101/708719 doi: bioRxiv preprint . CC-BY 4.0 International license a certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made available under The copyright holder for this preprint (which was not this version posted July 20, 2019. ; https://doi.org/10.1101/708719 doi: bioRxiv preprint 125 sandwich ELISA kit (Immunodiagnostics, Bedford, MA) according to the manufacturer’s 126 instructions. Briefly, the neutralized microvesicle preparations were diluted 1:1 with sample 127 diluent (Component C) and added to ELISA plates coated with anti-Nef (Component A). 128 Following 1-hour incubation at room temperature the plates were washed three times with wash 129 buffer (Component B), and 100 µl of anti-Nef-HRP labeled antibody solution (component E) was 130 then added to each well. Plates were incubated for 1 hour at room temperature, washed three 131 times with component B, and developed by adding 100 µl of alkaline phosphatase substrate 132 (component F) per well. Plates were developed for approximately 10 minutes after which 100 µl 133 of stop solution (Component G) was added to each well and the absorbance at 450 nm 134 determined using a Spectramax spectrophotometer. 125 sandwich ELISA kit (Immunodiagnostics, Bedford, MA) according to the manufacturer’s 126 instructions. Briefly, the neutralized microvesicle preparations were diluted 1:1 with sample 127 diluent (Component C) and added to ELISA plates coated with anti-Nef (Component A). 128 Following 1-hour incubation at room temperature the plates were washed three times with wash 129 buffer (Component B), and 100 µl of anti-Nef-HRP labeled antibody solution (component E) was 130 then added to each well. Plates were incubated for 1 hour at room temperature, washed three 131 times with component B, and developed by adding 100 µl of alkaline phosphatase substrate 132 (component F) per well. 123 Nef Enzyme-linked Immunosorbent Assay (ELISA). Nef concentration in acid-dissociated Plates were developed for approximately 10 minutes after which 100 µl 133 of stop solution (Component G) was added to each well and the absorbance at 450 nm 134 determined using a Spectramax spectrophotometer. 125 sandwich ELISA kit (Immunodiagnostics, Bedford, MA) according to the manufacturer’s 135 Statistical Analysis. Exosomal Nef concentrations were correlated with various immunologic 136 outcomes (immune recovery, change in cell populations, etc.) in each group using the Spearman 137 rank test. The Kruskal-Wallis test was used to compare continuous outcomes between these three 138 groups. The Mann-Whitney rank-sum test or the Dunn’s multiple comparison tests were used 139 appropriately to compare Nef levels in Treatment failures and immunological responders. 140 Statistical analysis was performed using MYSTAT software version 12 (Systat software, Inc. 141 2007). 7 144 Baseline characteristics of ACTG384 sub-cohort. This sub-cohort consisted primarily of males 145 (88.4%) (Table 1). Plasma HIV RNA levels at baseline were not significantly different between 7 . CC-BY 4.0 International license a certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made available under The copyright holder for this preprint (which was not this version posted July 20, 2019. ; https://doi.org/10.1101/708719 doi: bioRxiv preprint . CC-BY 4.0 International license a ertified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made available under The copyright holder for this preprint (which was not this version posted July 20, 2019. ; https://doi.org/10.1101/708719 doi: bioRxiv preprint . CC-BY 4.0 International license a certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made available under The copyright holder for this preprint (which was not this version posted July 20, 2019. 123 Nef Enzyme-linked Immunosorbent Assay (ELISA). Nef concentration in acid-dissociated ; https://doi.org/10.1101/708719 doi: bioRxiv preprint 150 151 152 153 154 155 156 157 158 159 160 161 Characteristics Complete subcohort Treatment Failures >200 ∆CD4 cell count <200 ∆CD4 cell count sex[n,%] (n=47) (n=14) (n=15) (n=18) Male 41,(88.4%) 10(71%) 13(87%) 18(100%) Female 6,(11.6%) 4(29%) 2(13%) 0 Age (Baseline) 37 36 37 39 Viral load(log10) Baseline 5.447 4.998 5.563 5.425 96 weeks 1.68 3.32 1.699 1.699 CD4 Count *** *** *** * Baseline 116 252 61 87 96 weeks 330 372 409 253.5 Activated CD4/CD38/HLA(%) *** *** *** *** Baseline 28 21 22 31 96 weeks 7 11 7 6.5 Activated CD8/CD38/HLA(%) *** * *** *** Baseline 53 52 57 53 96 weeks 20 29 22.5 18.5 Naïve CD4 (cells/mm3) *** ns *** *** Baseline 6.4 42.7 9.9 5.3 96 weeks 102.3 58.5 151.8 60.9 Memory CD4 *** ns *** *** Baseline 24.5 78.9 22.5 33.9 96 weeks 205.1 161.7 271.4 174.9 161 162 8 . CC-BY 4.0 International license a ertified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made available under The copyright holder for this preprint (which was not this version posted July 20, 2019. ; https://doi.org/10.1101/708719 doi: bioRxiv preprint . CC-BY 4.0 International license a certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made available under The copyright holder for this preprint (which was not this version posted July 20, 2019. ; https://doi.org/10.1101/708719 doi: bioRxiv preprint . CC-BY 4.0 International license a certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made available under The copyright holder for this preprint (which was not this version posted July 20, 2019. ; https://doi.org/10.1101/708719 doi: bioRxiv preprint 163 Table 1. 123 Nef Enzyme-linked Immunosorbent Assay (ELISA). Nef concentration in acid-dissociated Statistical analysis – Intergroup analysis (Column) compared gender and group association using chi- 164 square; ethnicity and group composition at baseline, using Chi-square analysis, #Trend p-value <0.1; Median age of 165 groups compared using ANOVA – Kruskal-Wallis, ns-not significant; Viral load at baseline between groups at 166 baseline and 96 weeks, Kruskall-Wallis, ns- not significant.; Baseline CD4 count, ns-not significantly different 167 between groups; Post-96 weeks CD4 count significantly different between Low and High Immunological responders 168 (IR); Kruskall-Wallis, Dunnett’s Multiple comparisons, ***p-value<.001; CD4 count different between baseline and 96 169 weeks for each group Mann-Whitney ***p-value <.0001; Activated CD4 and CD8 T-cell counts are ns-(not 170 significant) between groups at baseline and 96 weeks, Kruskall-Wallis, Dunnett’s Multiple; Naïve CD4 counts is 171 significantly different between high and low IRs at baseline but at 96 weeks differ significantly, Kruskall-Wallis, 172 Dunnett’s Multiple comparisons ***p-value<.001; Intra-group difference in IR memory differ significantly, Mann- 173 Whitney ***p-value <.0001 174 groups, either at baseline or post 96 weeks of treatment. (Table 1). Other cell subsets B-cells and 175 natural killer (NK) cells were not significantly different between High-IRs and Low-IRs (data not 176 shown). However, naïve and memory CD4 T-cell subsets were significantly different between 177 High-and Low_IRs (Table 1) suggesting that these cell subsets are integral to immune recovery. 178 179 CD4 recovery among immunological responders negatively correlates with plasma exNef 180 concentration. ART-induced decrease in viral load (VL) to <50 RNA copies did not necessarily 181 reduce Nef concentration in plasma or result in CD4 recovery for all HIV+ patients within the 182 sub-cohort. We examined study participants receiving anti-retrovirals with successful viral 183 suppression that had CD4 recovery above or below 300 cells/mm3 at study week 144 (Fig 1A). At 184 study week 144, 40% of the sub-cohort exhibited discordant VL and CD4 cell recovery along with 185 increased exNef level. In the absence of detectable viral replication, exNef could be detected in 186 the plasma and the levels correlated with total CD4 recovery (Fig 1B). 174 groups, either at baseline or post 96 weeks of treatment. (Table 1). Other cell subsets B-cells and 175 natural killer (NK) cells were not significantly different between High-IRs and Low-IRs (data not 176 shown). However, naïve and memory CD4 T-cell subsets were significantly different between 177 High-and Low_IRs (Table 1) suggesting that these cell subsets are integral to immune recovery. 123 Nef Enzyme-linked Immunosorbent Assay (ELISA). Nef concentration in acid-dissociated 179 CD4 recovery among immunological responders negatively correlates with plasma exNef 180 concentration. ART-induced decrease in viral load (VL) to <50 RNA copies did not necessarily 181 reduce Nef concentration in plasma or result in CD4 recovery for all HIV+ patients within the 182 sub-cohort. We examined study participants receiving anti-retrovirals with successful viral 183 suppression that had CD4 recovery above or below 300 cells/mm3 at study week 144 (Fig 1A). At 184 study week 144, 40% of the sub-cohort exhibited discordant VL and CD4 cell recovery along with 185 increased exNef level. In the absence of detectable viral replication, exNef could be detected in 186 the plasma and the levels correlated with total CD4 recovery (Fig 1B). 179 CD4 recovery among immunological responders negatively correlates with plasma exNef 180 concentration. ART-induced decrease in viral load (VL) to <50 RNA copies did not necessarily 181 reduce Nef concentration in plasma or result in CD4 recovery for all HIV+ patients within the 182 sub-cohort. We examined study participants receiving anti-retrovirals with successful viral 183 suppression that had CD4 recovery above or below 300 cells/mm3 at study week 144 (Fig 1A). At 184 study week 144, 40% of the sub-cohort exhibited discordant VL and CD4 cell recovery along with 185 increased exNef level. In the absence of detectable viral replication, exNef could be detected in 186 the plasma and the levels correlated with total CD4 recovery (Fig 1B). 9 9 . CC-BY 4.0 International license a certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made available under The copyright holder for this preprint (which was not this version posted July 20, 2019. ; https://doi.org/10.1101/708719 doi: bioRxiv preprint . CC-BY 4.0 International license a certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made available under The copyright holder for this preprint (which was not this version posted July 20, 2019. ; https://doi.org/10.1101/708719 doi: bioRxiv preprint 187 Figure 1: ARV-treated HIV+ patients exhibit differential CD4 T-cell recovery, which negatively correlates with 188 exNef level despite successful viral suppression. (A) At study weeks 48 (n=38), 96(n=32), and 144 (n=25) the 189 change in CD4 count (as measured by flow cytometry) was tracked in in the participants with VL >50 RNA copies 190 and compared to baseline CD4 count. 123 Nef Enzyme-linked Immunosorbent Assay (ELISA). Nef concentration in acid-dissociated 187 Figure 1: ARV-treated HIV+ patients exhibit differential CD4 T-cell recovery, which negatively correlates with 188 exNef level despite successful viral suppression. (A) At study weeks 48 (n=38), 96(n=32), and 144 (n=25) the 189 change in CD4 count (as measured by flow cytometry) was tracked in in the participants with VL >50 RNA copies 190 and compared to baseline CD4 count. High = change in CD4 count >100; Low= change in CD count <100 cells. 191 Statistical significance determined by Kruskal-Wallis test statistic, ***P<.001, Dunn’s Multiple comparison’s **P<.01. 192 (B) Plasma-derived exNef concentration negatively correlates with the change in CD4 count. *P<0.05, Spearman r. 193 194 Trend in exosomal Nef correlates with immunological response status. cART initiation reduces 195 viral load to undetectable levels over time but the effects of ART on exNef production is 196 unknown. Cross-sectional reports have demonstrated that Nef can be detected in the plasma in 197 the absence and presence of ART [36]. Since exNef may play a role in HIV immunopathogenesis 198 and impact CD4 recovery we examined the longitudinal changes of exNef in High IRs. 199 Interestingly, exNef was detected in the plasma of these patients despite suppression of viral 200 replication (Fig 2A). A retrospective longtitudinal analysis shows that at the initiation of therapy, 201 Nef levels were not significantly different between the Low_IRs and High_IRs (Fig 2A, upper 202 panel). However, by study week 144, the Low-IRs had significantly higher levels of plasma exNef 203 than the High IRs (Fig 2A, lower panel). The median baseline plasma Nef level in the sub- cohort 204 at 48 weeks was approximately 2.5 ng per 1 ml plasma and by 144 weeks Nef levels increased to 205 almost 5 ng per 1 ml plasma in the Low IRs. In fact Low-IRs exhibit an increasing trend in exNef 206 levels from study weeks 48 through 144 while in High-IRs exNef level decreased over the study 207 (Fig 2B). This finding suggests that plasma exNef may play a role immunological recovery. 208 Figure 2: Exosomal Nef is significantly different between low and high immunological responders and 209 trends upward in low immunological responders. (A) exNef concentration as measured by anti-Nef ELISA is 210 increased in Low responders at study weeks 48(Low-n=25, High=5 , 96(Low-n=16; High-n-14), and 144 weeks (Low- 194 Trend in exosomal Nef correlates with immunological response status. 123 Nef Enzyme-linked Immunosorbent Assay (ELISA). Nef concentration in acid-dissociated High = change in CD4 count >100; Low= change in CD count <100 cells 191 Statistical significance determined by Kruskal-Wallis test statistic, ***P<.001, Dunn’s Multiple comparison’s **P<.01 192 (B) Plasma-derived exNef concentration negatively correlates with the change in CD4 count. *P<0.05, Spearman r. 193 194 Trend in exosomal Nef correlates with immunological response status. cART initiation reduces 195 viral load to undetectable levels over time but the effects of ART on exNef production is 196 unknown. Cross-sectional reports have demonstrated that Nef can be detected in the plasma in 197 the absence and presence of ART [36]. Since exNef may play a role in HIV immunopathogenesis 198 and impact CD4 recovery we examined the longitudinal changes of exNef in High IRs 199 Interestingly, exNef was detected in the plasma of these patients despite suppression of viral 200 replication (Fig 2A). A retrospective longtitudinal analysis shows that at the initiation of therapy 201 Nef levels were not significantly different between the Low_IRs and High_IRs (Fig 2A, upper 202 panel). However, by study week 144, the Low-IRs had significantly higher levels of plasma exNef 203 than the High IRs (Fig 2A, lower panel). The median baseline plasma Nef level in the sub- cohort 204 at 48 weeks was approximately 2.5 ng per 1 ml plasma and by 144 weeks Nef levels increased to 205 almost 5 ng per 1 ml plasma in the Low IRs. In fact Low-IRs exhibit an increasing trend in exNef 206 levels from study weeks 48 through 144 while in High-IRs exNef level decreased over the study 207 (Fig 2B). This finding suggests that plasma exNef may play a role immunological recovery. . CC BY 4.0 International license a 187 Figure 1: ARV-treated HIV+ patients exhibit differential CD4 T-cell recovery, which negatively correlates with 188 exNef level despite successful viral suppression. (A) At study weeks 48 (n=38), 96(n=32), and 144 (n=25) the 189 change in CD4 count (as measured by flow cytometry) was tracked in in the participants with VL >50 RNA copies 190 and compared to baseline CD4 count. High = change in CD4 count >100; Low= change in CD count <100 cells. 191 Statistical significance determined by Kruskal-Wallis test statistic, ***P<.001, Dunn’s Multiple comparison’s **P<.01. 192 (B) Plasma-derived exNef concentration negatively correlates with the change in CD4 count. *P<0.05, Spearman r. 123 Nef Enzyme-linked Immunosorbent Assay (ELISA). Nef concentration in acid-dissociated cART initiation reduces 195 viral load to undetectable levels over time but the effects of ART on exNef production is 196 unknown. Cross-sectional reports have demonstrated that Nef can be detected in the plasma in 197 the absence and presence of ART [36]. Since exNef may play a role in HIV immunopathogenesis 198 and impact CD4 recovery we examined the longitudinal changes of exNef in High IRs. 199 Interestingly, exNef was detected in the plasma of these patients despite suppression of viral 200 replication (Fig 2A). A retrospective longtitudinal analysis shows that at the initiation of therapy, 201 Nef levels were not significantly different between the Low_IRs and High_IRs (Fig 2A, upper 202 panel). However, by study week 144, the Low-IRs had significantly higher levels of plasma exNef 203 than the High IRs (Fig 2A, lower panel). The median baseline plasma Nef level in the sub- cohort 204 at 48 weeks was approximately 2.5 ng per 1 ml plasma and by 144 weeks Nef levels increased to 205 almost 5 ng per 1 ml plasma in the Low IRs. In fact Low-IRs exhibit an increasing trend in exNef 206 levels from study weeks 48 through 144 while in High-IRs exNef level decreased over the study 207 (Fig 2B). This finding suggests that plasma exNef may play a role immunological recovery. 208 Figure 2: Exosomal Nef is significantly different between low and high immunological responders and 209 trends upward in low immunological responders. (A) exNef concentration as measured by anti-Nef ELISA is 210 increased in Low responders at study weeks 48(Low-n=25, High=5 , 96(Low-n=16; High-n-14), and 144 weeks (Low- 194 Trend in exosomal Nef correlates with immunological response status. cART initiation reduce 10 . CC-BY 4.0 International license a certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made available under The copyright holder for this preprint (which was not this version posted July 20, 2019. ; https://doi.org/10.1101/708719 doi: bioRxiv preprint . CC-BY 4.0 International license a ertified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made available under The copyright holder for this preprint (which was not this version posted July 20, 2019. ; https://doi.org/10.1101/708719 doi: bioRxiv preprint 211 n=10 ;High n=16). Statistical significance determined via Mann-Whitney, *p-value <0.05, Mann-Whitney. (B) High 212 and low immunological responders defined in sub-cohort defined by change in CD4 count from baseline. High – 213 change 100 (cells/mm3); Low change <100 cells/mm3) (B) Median Nef concentration in exosomes isolated from 214 plasma of subjects with Treatment Failure (n=10) or Success (with high, and low CD4 recovery; n=30) subjects. Nef 215 quantified by anti-Nef ELISA. 216 Naïve CD4 count and recovery inversely correlate with exNef. W 216 Naïve CD4 count and recovery inversely correlate with exNef. We then investigated whether 217 Nef+ exosome levels correlated with recovery of naïve CD4 cell counts specifically. 218 Immunological recovery (as defined in Methods) 48 weeks post treatment and close to 90% (18 219 out of 20) by week 96 of treatment (Fig 3A). However, none of the Low-IRs exhibited increases in 220 CD4 T-cell count close to 350 cells/mm3 by 144 weeks (Fig 3A). Although naïve T-cells do recover 221 in both High- or Low-IR the IRs have appreciably less naïve CD4 cells than the IRs 96- and 144 222 weeks post treatment initiation (Fig 3C). Notably the changes in both CD4 T-cell count and CD4 223 naïve T-cells negatively correlated with the Nef concentration in plasma-derived microvesicles 224 (Fig 3, B and D) suggesting that in vivo microvesicular Nef may be associated with immune 225 recovery/ CD4 T-cell rebound. 11 226 Figure #3: Naïve CD4 counts inversely correlate with exNef. (A) Naïve counts (as measured by flow cytometry) 227 significantly higher in the High_IR group at study week 48 and (B) study week 96. (C ) Nef-level inversely correlates 228 with both Naïve CD T-cell count and the (D)change in Naïve CD4 count post ARV-treatment. Correlation determined 229 by Spearman r, *p-value<.05, #p-value <.1 (trend). Statistical significance determined by One-Way ANOVA Kruskal- 230 Wallis test statistic and Dunn’s Multiple comparison * P-value<0.05, **p-value<.01, and ***p-value<.001. 231 232 Memory CD4 T-cells are significantly different in high low and responders. Given the role of 233 memory cells in immunological recovery, we sought to determine whether exNef also impacted 234 CD4 memory cell recovery. Interestingly at weeks 48 and 96 post-therapy High-IRs exhibited 226 Figure #3: Naïve CD4 counts inversely correlate with exNef. (A) Naïve counts (as measured by flow cytometry) 227 significantly higher in the High_IR group at study week 48 and (B) study week 96. (C ) Nef-level inversely correlates 228 with both Naïve CD T-cell count and the (D)change in Naïve CD4 count post ARV-treatment. Correlation determined 229 by Spearman r, *p-value<.05, #p-value <.1 (trend). Statistical significance determined by One-Way ANOVA Kruskal- 230 Wallis test statistic and Dunn’s Multiple comparison * P-value<0.05, **p-value<.01, and ***p-value<.001. 226 Figure #3: Naïve CD4 counts inversely correlate with exNef. 216 Naïve CD4 count and recovery inversely correlate with exNef. W (A) Naïve counts (as measured by flow cytometry) 227 significantly higher in the High_IR group at study week 48 and (B) study week 96. (C ) Nef-level inversely correlates 228 with both Naïve CD T-cell count and the (D)change in Naïve CD4 count post ARV-treatment. Correlation determined 229 by Spearman r, *p-value<.05, #p-value <.1 (trend). Statistical significance determined by One-Way ANOVA Kruskal- 230 Wallis test statistic and Dunn’s Multiple comparison * P-value<0.05, **p-value<.01, and ***p-value<.001. 238 Figure #4: Memory CD4 T-cells are significantly different in high low and responders. (A) CD4 memory cell 238 Figure #4: Memory CD4 T-cells are significantly different in high low and responders. (A) CD4 memory cell 239 counts (as determined by flow cytometry) in high-immunological responders are significantly increased compared to 240 low responders at study weeks 48 (Low-n=25; High-n=5); and 96 (High- n=14; Low-n=16). Significance from baseline 241 determined via Kruskal-Wallis, intergroup differences compared via Dunn’s Multiple comparison *p-value<0.05, **p- 242 value<.01, and ***p-value<.001. (B) The change in CD4 memory count from baseline(n=33) to study week 96 (n=30) 243 directly correlates with exNef level in low- immunological responders (n=18). Correlation determined using 244 Spearman r, *p-value<0.05, ns=not significant. 238 Figure #4: Memory CD4 T-cells are significantly different in high low and responders. (A) CD4 memory cell 239 counts (as determined by flow cytometry) in high-immunological responders are significantly increased compared to 240 low responders at study weeks 48 (Low-n=25; High-n=5); and 96 (High- n=14; Low-n=16). Significance from baseline 241 determined via Kruskal-Wallis, intergroup differences compared via Dunn’s Multiple comparison *p-value<0.05, **p- 242 value<.01, and ***p-value<.001. (B) The change in CD4 memory count from baseline(n=33) to study week 96 (n=30) 243 directly correlates with exNef level in low- immunological responders (n=18). Correlation determined using 244 Spearman r, *p-value<0.05, ns=not significant. 232 Memory CD4 T-cells are significantly different in high low and responders. Given the role of 11 . CC-BY 4.0 International license a certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made available under The copyright holder for this preprint (which was not this version posted July 20, 2019. ; https://doi.org/10.1101/708719 doi: bioRxiv preprint . CC-BY 4.0 International license a ertified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made available under The copyright holder for this preprint (which was not this version posted July 20, 2019. ; https://doi.org/10.1101/708719 doi: bioRxiv preprint . CC-BY 4.0 International license a certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made available under The copyright holder for this preprint (which was not this version posted July 20, 2019. ; https://doi.org/10.1101/708719 doi: bioRxiv preprint 235 appreciably more CD4 memory cells than the Low_IRs (Fig 4, panel A). The degree of CD4 236 increase directly correlated with exNef in the Low_IRs (Fig 4, panel B), suggesting that CD4 237 memory cells could be one of the sources of exNef during anti-viral suppression. 245 PI-sparing regimen significantly reduced plasma exNef. ACTG 384 was a prospective double- 245 PI-sparing regimen significantly reduced plasma exNef. ACTG 384 was a prospective double- 246 blinded study using a factorial design to compare sequential three-drug regimens. Study arms 247 are depicted in Table 2. Basically, two NRTIs zidovudine and lamuvidine or didanosine and 248 stauvidine followed by either efavirenz or nelfinavir were compared (Fig 5A, upper panel). 249 Previous results from the ACTG-384 cohort demonstrated that the combination of zidovudine, 250 lamivudine, and efavirenz lead to the shortest time to viral suppression suggesting that this 251 combination was the most efficacious combination [2]. However, we are still able to detect exNef 252 in the sub-cohort of ACTG384 participants with successful viral suppression. If exNef negatively 253 affects immunological recovery, then we must identify a treatment regimen that suppresses both 254 viral replication and exNef release. To determine how treatment regimen impacts exNef 255 production we stratified participants by treatment regimen and compared their respective exNef 256 level. By weeks 96 and 144 exNef was reduced 2-fold only in participants (with undetectable 257 viral load) that received regimen A (Didanosine, Stauvidine and Efavirenz) (Fig 5, lower panel). 12 12 . CC-BY 4.0 International license a certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made available under The copyright holder for this preprint (which was not this version posted July 20, 2019. ; https://doi.org/10.1101/708719 doi: bioRxiv preprint . CC-BY 4.0 International license a certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made available under The copyright holder for this preprint (which was not this version posted July 20, 2019. ; https://doi.org/10.1101/708719 doi: bioRxiv preprint his suggests that drug regimen may also dictate exNef levels and that PI-sparing regimens duce both viral load and exNef level. . CC-BY 4.0 International license a ertified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made available under The copyright holder for this preprint (which was not this version posted July 20, 2019. ; https://doi.org/10.1101/708719 doi: bioRxiv preprint 258 This suggests that drug regimen may also dictate exNef levels and that PI-sparing regimens 259 reduce both viral load and exNef level. 245 PI-sparing regimen significantly reduced plasma exNef. ACTG 384 was a prospective double- It is made available under The copyright holder for this preprint (which was not this version posted July 20, 2019. ; https://doi.org/10.1101/708719 doi: bioRxiv preprint . CC-BY 4.0 International license a ertified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made available under The copyright holder for this preprint (which was not this version posted July 20, 2019. ; https://doi.org/10.1101/708719 doi: bioRxiv preprint 276 immunological recovery. We posit that exNef may represent a novel mechanism utilized by HIV 277 to promote viral replication in resting CD4 T-cells. The impact of exNef on HIV pathogenesis 278 and CD4 T-cell recovery during cART is unknown. Additionally, we demonstrate that exNef 279 level was not only elevated in cART-treated HIV+ patients with low immunological recovery but 280 also negatively correlated with CD4 count recovery in these participants. This clearly suggests a 281 role for exNef in immunological recovery. 276 immunological recovery. We posit that exNef may represent a novel mechanism utilized by HIV 277 to promote viral replication in resting CD4 T-cells. The impact of exNef on HIV pathogenesis 278 and CD4 T-cell recovery during cART is unknown. Additionally, we demonstrate that exNef 279 level was not only elevated in cART-treated HIV+ patients with low immunological recovery but 280 also negatively correlated with CD4 count recovery in these participants. This clearly suggests a 281 role for exNef in immunological recovery. 282 Plasma exNef could theoretically determine immune recovery potential. Detection of Nef 283 microvesicles is not unexpected since several studies have shown that in order to lower surface 284 expression of CD4 and MHC class II Nef interact with component of the endocytic and exocytic 285 machinery [13, 22]. Nef has no enzymatic activity and functions primarily as an adaptor within 286 an infected cell. The function of extracellular Nef is unclear. However, several in vitro studies 287 have demonstrated that extracellular soluble Nef is cytotoxic to cells, induces cytokine and 288 chemokine release from macrophage activation, increases viral infectivity and alters innate 289 immune signaling pathways [13, 18, 27, 37-40]. We know from in vitro and ex vivo studies that 290 Nef is released in exosome-like vesicles from nef-transfected and HIV-infected cells and that these 291 vesicles are detected in the plasma of HIV+ patients [14, 23]. 245 PI-sparing regimen significantly reduced plasma exNef. ACTG 384 was a prospective double- 260 Initial Initial NNRTI Initial PI Initial PI +NNRTI Efavirenz Nelfinavir NFV + EFV NRTI Factor (EFV) (NFV) DDI+D4T A (5) B (3) E (11) ZDV /3TC C (10) D (7) F(14) 261 13 261 262 Table 2: Study arms of ACTG384. Drug regimen groupings as described by Smeaton et al 2001 263 followed by the number of participants (in parentheses) within regimen group. 264 Figure #5: PI-sparing regimen significantly reduced plasma exNef by study weeks 96 and 144. (Upper panel) 265 Schematic of drug regimens administered to sub-cohort in the first tier of the ACTG384 study. Participants on 266 regimens A (n=5) or C (n=10) received efavirenz with DDI/4TC or ZDV/4TC while those given regimen D(n=7) 267 received ZDV/4TC and Nelfinavir(Lower Panel). Longtitudinal comparison of plasma exNef level as measured by 268 anti-Nef ELISA in participants taking regimen A,C, and D. Statistical significance determined via Kruskal-Wallis, and 269 Dunn’s multiple comparison *p-value <0.05, **p-value<.01, ***p-value<.001, and ns=not significant. 270 271 Discussion 272 This study shows that exNef is detected in the plasma of cART-treated HIV+ patients with 273 successful viral suppression. Immunological recovery does not occur in 40% of cART-treated 274 HIV+ patients. Understanding the factors involved in poor immunological recovery could lead to 275 the development of novel therapeutics that inhibit viral replication while promoting 261 262 Table 2: Study arms of ACTG384. Drug regimen groupings as described by Smeaton et al 2001 263 followed by the number of participants (in parentheses) within regimen group. 262 Table 2: Study arms of ACTG384. Drug regimen groupings as described by Smeaton et al 2001 263 followed by the number of participants (in parentheses) within regimen group. 264 Figure #5: PI-sparing regimen significantly reduced plasma exNef by study weeks 96 and 144. (Upper panel) 265 Schematic of drug regimens administered to sub-cohort in the first tier of the ACTG384 study. Participants on 266 regimens A (n=5) or C (n=10) received efavirenz with DDI/4TC or ZDV/4TC while those given regimen D(n=7) 267 received ZDV/4TC and Nelfinavir(Lower Panel). Longtitudinal comparison of plasma exNef level as measured by 268 anti-Nef ELISA in participants taking regimen A,C, and D. Statistical significance determined via Kruskal-Wallis, and 269 Dunn’s multiple comparison *p-value <0.05, **p-value<.01, ***p-value<.001, and ns=not significant. 13 . CC-BY 4.0 International license a certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. 245 PI-sparing regimen significantly reduced plasma exNef. ACTG 384 was a prospective double- Although the biological role of Nef+ 292 exosomes is still unknown we expected exNef to have similar cellular and functional affects 293 ascribed to soluble Nef. 294 It has recently been reported that released Nef microvesicles/exosomes similar to soluble Nef 295 can trigger activation induced cell death (apoptosis) in peripheral blood leukocytes thus 296 promoting the depletion of CD4+ T-cells [21]. Another report indicated that Nef induces massive 294 It has recently been reported that released Nef microvesicles/exosomes similar to soluble Nef 295 can trigger activation induced cell death (apoptosis) in peripheral blood leukocytes thus 296 promoting the depletion of CD4+ T-cells [21]. Another report indicated that Nef induces massive 14 14 14 . CC-BY 4.0 International license a certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made available under The copyright holder for this preprint (which was not this version posted July 20, 2019. ; https://doi.org/10.1101/708719 doi: bioRxiv preprint . CC-BY 4.0 International license a ertified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made available under The copyright holder for this preprint (which was not this version posted July 20, 2019. ; https://doi.org/10.1101/708719 doi: bioRxiv preprint 297 secretion of microvesicle clusters in HIV-infected T-cells and that extracellular Nef is then passed 298 to uninfected bystander by cell-to-cell contact via an ERK-1/2 dependent mechanism [41]. Other 299 studies have shown that extracellular Nef vesicles are taken-up/absorbed by T-cells and that 300 these released Nef microvesicles cause activation-induced cell death in primary peripheral blood 301 leukocytes [14, 21]. Primary leukocytes exposed to exNef as observed with soluble Nef release 302 chemokines MIP-1a and MIP-1b (unpublished result). 297 secretion of microvesicle clusters in HIV-infected T-cells and that extracellular Nef is then passed 298 to uninfected bystander by cell-to-cell contact via an ERK-1/2 dependent mechanism [41]. Other 299 studies have shown that extracellular Nef vesicles are taken-up/absorbed by T-cells and that 300 these released Nef microvesicles cause activation-induced cell death in primary peripheral blood 301 leukocytes [14, 21]. Primary leukocytes exposed to exNef as observed with soluble Nef release 302 chemokines MIP-1a and MIP-1b (unpublished result). 245 PI-sparing regimen significantly reduced plasma exNef. ACTG 384 was a prospective double- 303 Here we show that plasma-derived microvesicles/exosomes from participants within the 304 ACTG384 cohort contain Nef even in the presence of successful HAART therapy and are elevated 305 in low immunological responders. This suggests that exNef may play a role in preventing 306 sufficient CD4 T-cell recovery thereby promoting immunodeficiency despite successful HAART 307 outcomes. Taken together, these studies show that extracellular Nef exosomes function similar to 308 soluble Nef in that exNef is absorbed by T-cells, induce apoptosis and may play a role in 309 discordant successful viral suppression and CD4 T-cell recovery. 310 Most interestingly Nef+ microvesicles/exosomes persist and are detectable over a 144-week 311 period suggesting that exNef may simply be a product of chronic HIV infection. We observed 312 that significant reduction of viral load correlated with an increase in exNef level suggesting that 313 exNef may regulate viral replication and/or cell function. These findings are novel in HIV- 314 pathobiology but may not be a unique to HIV. Indeed, recent reports demonstrated that EBV- 315 infected nasopharyngeal cells release inhibitory exosomes containing the EBV-encoded protein 316 latent membrane protein-1 (LMP-1) [42]. These LMP-1+ exosomes inhibited T-cell activation and 15 . CC-BY 4.0 International license a certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made available under The copyright holder for this preprint (which was not this version posted July 20, 2019. ; https://doi.org/10.1101/708719 doi: bioRxiv preprint . CC-BY 4.0 International license a certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made available under The copyright holder for this preprint (which was not this version posted July 20, 2019. ; https://doi.org/10.1101/708719 doi: bioRxiv preprint anti-EBV immune responses [42] So exNef released from HIV-infected cells may have a similar . CC-BY 4.0 International license a certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made available under The copyright holder for this preprint (which was not this version posted July 20, 2019. ; https://doi.org/10.1101/708719 doi: bioRxiv preprint nti-EBV immune responses [42]. So exNef released from HIV-infected cells may have a similar fect on anti-HIV immune responses. . 245 PI-sparing regimen significantly reduced plasma exNef. ACTG 384 was a prospective double- CC-BY 4.0 International license a ertified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made available under The copyright holder for this preprint (which was not this version posted July 20, 2019. ; https://doi.org/10.1101/708719 doi: bioRxiv preprint . CC-BY 4.0 International license a certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made available under The copyright holder for this preprint (which was not this version posted July 20, 2019. ; https://doi.org/10.1101/708719 doi: bioRxiv preprint 317 anti-EBV immune responses [42]. So exNef released from HIV-infected cells may have a similar 318 effect on anti-HIV immune responses. 317 anti-EBV immune responses [42]. So exNef released from HIV-infected cells may have a similar 319 Aside from the ACTG384, several studies have shown HIV+ patients with successful virological 320 responses to HAART yet incomplete CD4 recovery have increased mortality [43-45]. Age and 321 prolonged periods of immunodeficiency prior to successful HAART are risk factors for 322 incomplete/insufficient CD4 T-cell recovery [46]. We provide evidence in this study that plasma 323 derived Nef+ microvesicles may be associated with immune recovery. We detected increased in 324 exNef only in Low_IRs. In these Low-IRs exNef concentration correlated negatively and positively 325 with the recovery of CD4 Naïve and memory T-cell counts, respectively. This result suggests that 326 naïve CD4 cells are a potential exNef targets while the CD4 memory cells could be a source of the 327 Nef+ exosomes. 328 Treatment regimens not only directly impact viral replication but also appear to affect the 329 generation of Nef+ exosomes. PI-sparing HAART regimens (e.g. Regimen A: Didanosine, 330 Stauvidine, and Efavirenz) reduced both viral load and exNef level. This result suggests that 331 treatment regimen may dictate exNef level and exNef in turn may be developed as a prognostic 332 indicator of CD4 immune recovery during HAART. 333 We posit that the increased level of Nef+ exosomes early in High_IRs (by week 48) are 334 indicative of reduced virus production and suggests that in the absence of productive viral 335 replication HIV-infected cells release more Nef+ exosomes. If these exosomes affect T-cell 336 activation or viability, then this may impact immune recovery. By 144 weeks however, exNef is 337 significantly reduced in High-IRs participants relative to Low-IRs participants. Taken together 16 16 . 245 PI-sparing regimen significantly reduced plasma exNef. ACTG 384 was a prospective double- CC-BY 4.0 International license a certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made available under The copyright holder for this preprint (which was not this version posted July 20, 2019. ; https://doi.org/10.1101/708719 doi: bioRxiv preprint . CC-BY 4.0 International license a certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made available under The copyright holder for this preprint (which was not this version posted July 20, 2019. ; https://doi.org/10.1101/708719 doi: bioRxiv preprint ur study suggests that extended use of combination therapy lacking a HIV protease inhibitor mpairs the release of Nef+ exosomes while NRTI/NNRTIs have no long-term effects on exNef. . CC-BY 4.0 International license a ertified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made available under The copyright holder for this preprint (which was not this version posted July 20, 2019. ; https://doi.org/10.1101/708719 doi: bioRxiv preprint 338 our study suggests that extended use of combination therapy lacking a HIV protease inhibitor 339 impairs the release of Nef+ exosomes while NRTI/NNRTIs have no long-term effects on exNef. 340 Overall, these data also suggest that increased Nef levels maybe a double-edged sword – in 341 terms of viral suppression- high exNef is associated with decreased viral load but in regard to 342 immune recovery- elevated exNef is associated with reduced CD4 T-cell rebound/immune 343 recovery. Recently, regions within Nef important for secretion have been identified. Disruption 344 of the secretion modification region (SMR) within the Nef gene appears to abolish its release in 345 microvesicles/exosomes [61]. Peptide-targeted disruption of the SMR also inhibited Nef release 346 suggesting that SMR peptides could be developed as a therapeutic agent. . CC-BY 4.0 International license a 338 our study suggests that extended use of combination therapy lacking a HIV protease inhibitor 339 impairs the release of Nef+ exosomes while NRTI/NNRTIs have no long-term effects on exNef. 338 our study suggests that extended use of combination therapy lacking a HIV protease inhibitor 339 impairs the release of Nef+ exosomes while NRTI/NNRTIs have no long-term effects on exNef. 245 PI-sparing regimen significantly reduced plasma exNef. ACTG 384 was a prospective double- 340 Overall, these data also suggest that increased Nef levels maybe a double-edged sword – in 341 terms of viral suppression- high exNef is associated with decreased viral load but in regard to 342 immune recovery- elevated exNef is associated with reduced CD4 T-cell rebound/immune 343 recovery. Recently, regions within Nef important for secretion have been identified. Disruption 344 of the secretion modification region (SMR) within the Nef gene appears to abolish its release in 345 microvesicles/exosomes [61]. Peptide-targeted disruption of the SMR also inhibited Nef release 346 suggesting that SMR peptides could be developed as a therapeutic agent. 347 Ultimately, we provide evidence of exNef as a novel virological factor contributing to the 348 dissociation of viral load and immunological recovery. Since, drug regimens can alter exNef 349 levels, therapies need to be developed that both successfully lower viral load and Nef+ exosomes 350 levels. Our findings also suggest that clinicians could monitor exNef level along with CD4 T-cell 351 count in patients undergoing ARV-treatment to assess the effectiveness of therapeutic regimens. 17 . CC-BY 4.0 International license a ertified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made available under The copyright holder for this preprint (which was not this version posted July 20, 2019. ; https://doi.org/10.1101/708719 doi: bioRxiv preprint . CC-BY 4.0 International license a certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made available under The copyright holder for this preprint (which was not this version posted July 20, 2019. ; https://doi.org/10.1101/708719 doi: bioRxiv preprint . CC-BY 4.0 International license a certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. 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Virologic and immunologic characterization of long-term 398 survivors of human immunodeficiency virus type 1 infection. N Engl J Med 1995; 332(4):201- 399 208 365 1. Smeaton LM, DeGruttola V, Robbins GK, Shafer RW. ACTG (AIDS Clinical Trials Group) 384: a 366 strategy trial comparing consecutive treatments for HIV-1. Control Clin Trials 2001; 22(2):142- 367 159. 68 2. Robbins GK, De G, V, Shafer RW, Smeaton LM, Snyder SW, Pettinelli C, et al. Comparison of 69 sequential three-drug regimens as initial therapy for HIV-1 infection. N Engl J Med 2003; 70 349(24):2293-2303. 372 treatment-related factors on immunologic recovery after initiation of antiretroviral therapy in 373 HIV-1-positive subjects: results from ACTG 384. J Acquir Immune Defic Syndr 2006; 42(4):426- 374 434. 375 4. Greub G, Ledergerber B, Battegay M, Grob P, Perrin L, Furrer H, et al. Clinical progression, survival, 376 and immune recovery during antiretroviral therapy in patients with HIV-1 and hepatitis C 377 virus coinfection: the Swiss HIV Cohort Study. Lancet 2000; 356(9244):1800-1805. 378 5. Jevtovic D, Salemovic D, Ranin J, Pesic I, Zerjav S, Djurkovic-Djakovic O. The dissociation between 379 virological and immunological responses to HAART. Biomed Pharmacother 2005; 59(8):446-451. 380 6. Pakker NG, Kroon ED, Roos MT, Otto SA, Hall D, Wit FW, et al. Immune restoration does not 381 invariably occur following long-term HIV-1 suppression during antiretroviral therapy. 382 INCAS Study Group. AIDS 1999; 13(2):203-212. 375 4. Greub G, Ledergerber B, Battegay M, Grob P, Perrin L, Furrer H, et al. Clinical progression, survival, 376 and immune recovery during antiretroviral therapy in patients with HIV-1 and hepatitis C 377 virus coinfection: the Swiss HIV Cohort Study. Lancet 2000; 356(9244):1800-1805. 378 5. Jevtovic D, Salemovic D, Ranin J, Pesic I, Zerjav S, Djurkovic-Djakovic O. The dissociation between 379 virological and immunological responses to HAART. Biomed Pharmacother 2005; 59(8):446-451. 380 6. Pakker NG, Kroon ED, Roos MT, Otto SA, Hall D, Wit FW, et al. Immune restoration does not 381 invariably occur following long-term HIV-1 suppression during antiretroviral therapy. 382 INCAS Study Group. AIDS 1999; 13(2):203-212. 383 7. Piketty C, Castiel P, Belec L, Batisse D, Si MA, Gilquin J, et al. Discrepant responses to triple 384 combination antiretroviral therapy in advanced HIV disease. AIDS 1998; 12(7):745-750. 385 8. 353 Acknowledgements 354 We would like to acknowledge the assistance of Dr. Gale Newman (Morehouse School of 355 Medicine). We also acknowledge the support of RCMI core facilities at Morehouse School of 356 Medicine (U54MD007602), the Emory Center for AIDS Research (P30AI050409), and the NIH 357 AIDS Reagent program. We are grateful for funding support from grants S06-GM08428, R21 358 A1060370, and R21 NS105577). Research reported in this publication was supported by the 359 National Institute of Allergy and Infectious Diseases of the National Institutes of Health under 360 Award Number UM1AI068634, UM1 AI068636 and UM1 AI106701. The content is solely the 361 responsibility of the authors and does not necessarily represent the official views of the National 362 Institutes of Health”. 18 . CC-BY 4.0 International license a certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made available under The copyright holder for this preprint (which was not this version posted July 20, 2019. ; https://doi.org/10.1101/708719 doi: bioRxiv preprint . CC-BY 4.0 International license a certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. 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Long-term mortality in HIV 481 patients virally suppressed for more than three years with incomplete CD4 recovery: a cohort 482 study. BMC Infect Dis 2010; 10:318. 483 47. Ahuja SK, Kulkarni H, Catano G, Agan BK, Camargo JF, He W, et al. CCL3L1-CCR5 genotype 484 influences durability of immune recovery during antiretroviral therapy of HIV-1-infected 485 individuals. Nat Med 2008; 14(4):413-420. 486 48. 364 References Role of CCL3L1- 487 genotypes in the epidemic spread of HIV-1 and evaluation of vaccine efficacy. PLoS One 488 3(11):e3671. 489 49. Kulkarni H, Agan BK, Marconi VC, O'Connell RJ, Camargo JF, He W, et al. CCL3L1-CCR5 geno 490 improves the assessment of AIDS Risk in HIV-1-infected individuals. PLoS One 2008; 491 3(9):e3165. 492 50. Liang JS, Distler O, Cooper DA, Jamil H, Deckelbaum RJ, Ginsberg HN, et al. HIV protease inhi 493 protect apolipoprotein B from degradation by the proteasome: a potential mechanism fo 494 protease inhibitor-induced hyperlipidemia. Nat Med 2001; 7(12):1327-1331. 495 51. Hui DY. Effects of HIV protease inhibitor therapy on lipid metabolism. Prog Lipid Res 2003; 42(2 496 92. 497 52. Hamel FG, Fawcett J, Tsui BT, Bennett RG, Duckworth WC. Effect of nelfinavir on insulin 498 metabolism, proteasome activity and protein degradation in HepG2 cells. Diabetes Obes 499 2006; 8(6):661-668. 500 53. De BS, Zakaroff-Girard A, Lafontan M, Galitzky J, Bourlier V. Inhibition of human preadipocyt 501 proteasomal activity by HIV protease inhibitors or specific inhibitor lactacystin leads to 502 defect in adipogenesis, which involves matrix metalloproteinase-9. J Pharmacol Exp Ther 503 320(1):291-299. 504 54. Andre P, Groettrup M, Klenerman P, de GR, Booth BL, Jr., Cerundolo V, et al. An inhibitor of H 505 protease modulates proteasome activity, antigen presentation, and T cell responses. Pro 506 Acad Sci U S A 1998; 95(22):13120-13124. 0 k l b h A d h h d f 471 43. Gutierrez F, Padilla S, Masia M, Iribarren JA, Moreno S, Viciana P, et al. Patients' characteristics and 472 clinical implications of suboptimal CD4 T-cell gains after 1 year of successful antiretroviral 473 therapy. Curr HIV Res 2008; 6(2):100-107. 474 44. Moore RD, Keruly JC. CD4+ cell count 6 years after commencement of highly active antiretroviral 475 therapy in persons with sustained virologic suppression. Clin Infect Dis 2007; 44(3):441-446. 476 45. Piketty C, Weiss L, Thomas F, Mohamed AS, Belec L, Kazatchkine MD. Long-term clinical outcome 477 of human immunodeficiency virus-infected patients with discordant immunologic and 478 virologic responses to a protease inhibitor-containing regimen. J Infect Dis 2001; 183(9):1328- 479 1335. 480 46. Engsig FN, Gerstoft J, Kronborg G, Larsen CS, Pedersen G, Roge B, et al. Long-term mortality in HIV 481 patients virally suppressed for more than three years with incomplete CD4 recovery: a cohort 482 study. BMC Infect Dis 2010; 10:318. 483 47. 364 References Kulkarni H, Marconi VC, Agan BK, McArthur C, Crawford G, Clark RA, et al. Role of CCL3L1-CCR5 487 genotypes in the epidemic spread of HIV-1 and evaluation of vaccine efficacy. PLoS One 2008; 488 3(11):e3671. 489 49. Kulkarni H, Agan BK, Marconi VC, O'Connell RJ, Camargo JF, He W, et al. CCL3L1-CCR5 genotype 490 improves the assessment of AIDS Risk in HIV-1-infected individuals. PLoS One 2008; 491 3(9):e3165. 22 . CC-BY 4.0 International license a certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made available under The copyright holder for this preprint (which was not this version posted July 20, 2019. ; https://doi.org/10.1101/708719 doi: bioRxiv preprint . CC-BY 4.0 International license a certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made available under The copyright holder for this preprint (which was not this version posted July 20, 2019. ; https://doi.org/10.1101/708719 doi: bioRxiv preprint . CC-BY 4.0 International license a certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made available under The copyright holder for this preprint (which was not this version posted July 20, 2019. ; https://doi.org/10.1101/708719 doi: bioRxiv preprint 509 radiosensitization in non-HIV-associated human cancer cells. Cancer Res 2002; 62(18):5230 510 5235. 511 56. Piccinini M, Rinaudo MT, Chiapello N, Ricotti E, Baldovino S, Mostert M, et al. The human 26S 512 proteasome is a target of antiretroviral agents. AIDS 2002; 16(5):693-700. 513 57. Piccinini M, Rinaudo MT, Anselmino A, Buccinna B, Ramondetti C, Dematteis A, et al. The HIV 514 protease inhibitors nelfinavir and saquinavir, but not a variety of HIV reverse transcripta 515 inhibitors, adversely affect human proteasome function. Antivir Ther 2005; 10(2):215-223. 516 58. Schmidtke G, Holzhutter HG, Bogyo M, Kairies N, Groll M, de GR, et al. How an inhibitor of the 517 HIV-I protease modulates proteasome activity. J Biol Chem 1999; 274(50):35734-35740. 518 59. Kisselev AF, Goldberg AL. Proteasome inhibitors: from research tools to drug candidates. Chem 519 2001; 8(8):739-758. 520 60. Mathews JA, Gibb DR, Chen BH, Scherle P, Conrad DH. CD23 Sheddase A disintegrin and 521 metalloproteinase 10 (ADAM10) is also required for CD23 sorting into B cell-derived 522 microvesicles. J Biol Chem 2010; 285(48):37531-37541. 523 61. 364 References Ali SA, Huang MB, Campbell PE, Roth WW, Campbell T, Khan M, et al. Genetic characterization 524 HIV type 1 Nef-induced vesicle secretion. AIDS Res Hum Retroviruses 2010; 26(2):173-192. 525 526 527 . CC-BY 4.0 International license a certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made available un 509 radiosensitization in non-HIV-associated human cancer cells. Cancer Res 2002; 62(18):5230- 510 5235. 511 56. Piccinini M, Rinaudo MT, Chiapello N, Ricotti E, Baldovino S, Mostert M, et al. The human 26S 512 proteasome is a target of antiretroviral agents. AIDS 2002; 16(5):693-700. 513 57. Piccinini M, Rinaudo MT, Anselmino A, Buccinna B, Ramondetti C, Dematteis A, et al. The HIV 514 protease inhibitors nelfinavir and saquinavir, but not a variety of HIV reverse transcriptase 515 inhibitors, adversely affect human proteasome function. Antivir Ther 2005; 10(2):215-223. 516 58. Schmidtke G, Holzhutter HG, Bogyo M, Kairies N, Groll M, de GR, et al. How an inhibitor of the 517 HIV-I protease modulates proteasome activity. J Biol Chem 1999; 274(50):35734-35740. 518 59. Kisselev AF, Goldberg AL. Proteasome inhibitors: from research tools to drug candidates. Chem Biol 519 2001; 8(8):739-758. 520 60. Mathews JA, Gibb DR, Chen BH, Scherle P, Conrad DH. CD23 Sheddase A disintegrin and 521 metalloproteinase 10 (ADAM10) is also required for CD23 sorting into B cell-derived 522 microvesicles. J Biol Chem 2010; 285(48):37531-37541. 523 61. Ali SA, Huang MB, Campbell PE, Roth WW, Campbell T, Khan M, et al. Genetic characterization of 524 HIV type 1 Nef-induced vesicle secretion. AIDS Res Hum Retroviruses 2010; 26(2):173-192. 525 23
https://openalex.org/W2130952443
https://www.cdc.gov/pcd/issues/2015/pdf/15_0290.pdf
English
null
Consumption of Alcoholic Beverages and Liquor Consumption by Michigan High School Students, 2011
Preventing chronic disease
2,015
public-domain
7,895
Katherine R. Gonzales, MPH; Thomas W. Largo, MPH; Corinne Miller, PhD, DDS; Dafna Kanny, PhD; Robert D. Brewer, MD, MSPH Katherine R. Gonzales, MPH; Thomas W. Largo, MPH; Corinne Miller, PhD, DDS; Dafna Kanny, PhD; Robert D. Brewer, MD, MSPH Katherine R. Gonzales, MPH; Thomas W. Largo, MPH; Corinne Miller, PhD, DDS; Dafna Kanny, PhD; Robert D. Brewer, MD, MSPH antly more likely than nonbinge drinkers to consume vodka and to mix alcohol with energy drinks (49.0% vs 18.2%, respectively). Suggested citation for this article: Gonzales KR, Largo TW, Miller C, Kanny D, Brewer RD. Consumption of Alcoholic Beverages and Liquor Consumption by Michigan High School Students, 2011. Prev Chronic Dis 2015;12:150290. DOI: http:// dx.doi.org/10.5888/pcd12.150290. Introduction Excessive alcohol consumption was responsible for approxim- ately 4,300 annual deaths in the United States among people younger than 21 from 2006 through 2010. Underage drinking cost the United States $24.6 billion in 2006. Previous studies have shown that liquor is the most common type of alcohol consumed by high school students. However, little is known about the types of liquor consumed by youth or about the mixing of alcohol with energy drinks. Annually from 2006 through 2010, excessive alcohol consump- tion was responsible for approximately 4,300 deaths and 260,000 years of potential life lost in the United States among people younger than 21 years (1). In 2006, underage drinking cost the United States approximately $24.6 billion (2). More than 90% of the alcohol consumed by underage drinkers occurs during binge drinking episodes (3). Underage drinking is also associated with many health and social consequences, such as poor academic per- formance, interpersonal violence, injuries, risky sexual behavior, and unplanned pregnancies (4–6). Alcohol consumption by youth is also strongly correlated with alcohol consumption by adults at the state level (7), and youth often obtain the alcohol they con- sume from adults (8). Conclusions Liquor is the most common type of alcoholic beverage consumed by Michigan high school students; vodka is the most common type of liquor consumed. Mixing alcohol and energy drinks is common, particularly among binge drinkers. Community Guide strategies for reducing excessive drinking (eg, increasing alcohol taxes) can reduce underage drinking. Methods The 2011 Michigan Youth Tobacco Survey was used to assess usual alcohol beverage consumption and liquor consumption and the mixing of alcohol with energy drinks by Michigan high school students. Beverage preferences were analyzed by demographic characteristics and drinking patterns. Recent studies identified liquor (eg, vodka, rum, scotch, bourbon, whiskey) as the usual type of alcohol consumed by high school students (9–13). Students who reported usually consuming liquor were also more likely to report frequent alcohol consumption and binge drinking than students who reported consuming other types of alcoholic beverages (11). However, limited information is avail- able on the type of liquor that is consumed by underage youth and whether specific types of alcohol are more likely to be mixed with other types of beverages, such as energy drinks (eg, Monster, Red Bull). Understanding the type of alcohol usually consumed by Results Overall, 34.2% of Michigan high school students consumed alco- hol in the past month, and 20.8% reported binge drinking. Among current drinkers, liquor was the most common type of alcohol con- sumed (51.2%), and vodka was the most prevalent type of liquor consumed by those who drank liquor (53.0%). The prevalence of liquor consumption was similar among binge drinkers and non- binge drinkers, but binge drinkers who drank liquor were signific- www.cdc.gov/pcd/issues/2015/15_0290.htm • Centers for Disease Control and Prevention 1 VOLUME 12, E194 NOVEMBER 2015 vey of beverage-specific alcohol consumption (12). Students were asked about their 30-day consumption of alcohol; binge drinking; the largest number of drinks consumed on any occasion; the usual type of alcohol consumed (ie, students could select one alcoholic beverage type that they usually consumed in the past 30 days); the usual type of liquor consumed; the usual brand of liquor, beer, or flavored alcoholic beverage consumed; how frequently they mixed alcohol with energy drinks; their usual drinking location; and vari- ous direct consequences that could have resulted from alcohol consumption (eg, getting into a fight, riding with a driver who had been drinking). youth is important for developing and targeting interventions to prevent underage and binge drinking, because alcohol control policies (eg, alcohol taxes) and alcohol advertising vary by bever- age type (9,10). In addition, previous studies suggested that youth who mix alcohol with energy drinks are more likely to binge drink, consume more total drinks, have higher levels of alcohol in- toxication, and experience alcohol-attributable harms (eg, sexual assault) than those who do not mix energy drinks with alcohol (14,15). As part of its efforts to reduce underage drinking, the Michigan Department of Health and Human Services added supplemental questions on alcohol to its 2011 Youth Tobacco Survey. The pur- pose of this study was to use these survey findings to assess the type of alcoholic beverage (eg, beer, wine, liquor) usually con- sumed by Michigan high school students, the type of liquor con- sumed by students who drank liquor, the prevalence of mixing al- cohol with energy drinks, and how these drinking behaviors var- ied by the demographic characteristics and drinking patterns (eg, binge drinking) of these students. Current drinkers were defined as students who reported consum- ing at least 1 alcoholic drink in the 30 days before survey adminis- tration. Binge drinkers were defined as current drinkers who re- ported consuming 5 or more alcoholic drinks in a row (ie, within a couple of hours) during the 30 days before survey administration. Nonbinge drinkers were students who identified themselves as current drinkers but did not report consuming 5 or more alcoholic drinks in a row during the past 30 days. Methods Analyses were conducted using SAS, version 9.2 (SAS Institute, Inc) to account for the complex survey design and to produce es- timates that were representative of Michigan’s high school stu- dent population. Prevalence estimates with 95% confidence inter- vals were calculated. Pearson’s χ2was used to test for differences in the prevalence of beverage-specific alcohol consumption between groups defined by various demographic factors (ie, sex, grade, and race/ethnicity) and by drinking patterns (eg, drinking frequency, drinking intensity, usual place of consumption, usual alcohol type consumed, usual liquor type consumed, mixing alco- hol and energy drinks). The Michigan Youth Tobacco Survey (Michigan YTS) is a school-based survey of a random sample of public-school stu- dents in grades 9 through 12. Students surveyed complete an an- onymous, self-administered questionnaire that consists of ques- tions on tobacco use and on various environmental factors related to tobacco use, such as access to tobacco products in retail set- tings. In 2011, 4,142 Michigan high school students from 42 schools completed the Michigan YTS. The school response rate was 68%, the student response rate was 93%, and the overall re- sponse rate was 63%. Data were weighted by school, student non- response, and selected demographic characteristics (ie, the sex, race/ethnicity, and grade of students). Because the Michigan YTS is an established public health surveillance system, it does not re- quire review of the Michigan Department of Health and Human Services Institutional Review Board. PREVENTING CHRONIC DISEASE PUBLIC HEALTH RESEARCH, PRACTICE, AND POLICY VOLUME 12, E194 NOVEMBER 2015 Centers for Disease Control and Prevention • www.cdc.gov/pcd/issues/2015/15_0290.htm The opinions expressed by authors contributing to this journal do not necessarily reflect the opinions of the U.S. Department of Health and Human Services, the Public Health Service, the Centers for Disease Control and Prevention, or the authors’ affiliated institutions. 2 Centers for Disease Control and Prevention • www cdc gov/pcd/issues/2015/15 0290 htm The opinions expressed by authors contributing to this journal do not necessarily reflect the opinions of the U.S. Department of Health and Human Services, the Public Health Service, the Centers for Disease Control and Prevention, or the authors’ affiliated institutions. VOLUME 12, E194 NOVEMBER 2015 had more than twice the prevalence of mixing energy drinks with alcohol compared with nonbinge drinkers (49.0% vs 18.2%; P < .001). ergy drinks by high school students in a state. We found that more than half of the Michigan high school students who drank alcohol consumed liquor and that approximately half of the students who drank liquor usually consumed vodka. Liquor consumption was also associated with other dangerous drinking behaviors, includ- ing binge drinking and the mixing of alcohol and energy drinks. This finding is concerning because binge drinking increases the risk of alcohol-attributable harms, including sexual assault, and mixing of alcohol and energy drinks can increase binge drinking intensity, further increasing the risk of alcohol-attributable injur- ies and alcohol poisoning (15). Most Michigan high school students who drank alcohol reported usually consuming liquor (51.2%) (Table 3). The prevalence of li- quor consumption was slightly higher for binge drinkers (54.2%) than for nonbinge drinkers (46.9%). Liquor was the most fre- quently reported usual type of alcohol consumed by current drink- ers across all demographic groups and drinking patterns and was reported by most students who reported mixing alcohol and en- ergy drinks (52.7%). Girls reported a significantly higher preval- ence of drinking flavored alcoholic beverages than boys (21.0% vs 8.1%; P < .001), whereas boys reported a significantly higher pre- valence of drinking beer than girls (24.3% vs 11.1%; P < .001). Other types of alcohol (wine and cordials) accounted for a small proportion (11.2%) of the usual type of alcohol consumed by Michigan high school students. Only 5.2% of current drinkers re- ported not having a usual beverage type. The finding that liquor is the usual type of alcohol consumed by Michigan high school students who drink is consistent with the findings of other studies (10–13). This is likely due to the fact that liquor has a higher alcohol concentration, is more portable and easily concealed than other alcoholic beverages, and can easily be mixed with other beverages, thereby making liquor more palat- able to youth (16). Youth exposure to alcohol advertising in the United States increased by 71% from 2001 through 2009, largely because of an increase in liquor advertising on television (17), and youth exposure to alcohol advertising is associated with both the initiation of alcohol consumption by youth and the amount con- sumed per drinking occasion (18). VOLUME 12, E194 NOVEMBER 2015 The high prevalence of vodka consumption among high school students who drank liquor is also consistent with the reported increase in vodka sales in the United States and in Michigan (19,20), which has likely made vodka more accessible to youth, as well, particularly given that most high school students obtain the alcohol they consume from someone else (10). Vodka consumption was reported by 37.3% of current drinkers, making it by far the most common type of liquor consumed by Michigan high school students (Table 4). Vodka consumption was more common among binge drinkers (42.1%) than among non- binge drinkers (30.7%) and was reported by almost half of the stu- dents who reported mixing alcohol and energy drinks. Vodka was the most common type of liquor consumed by current drinkers across all demographic groups, but it was somewhat more com- mon among girls (42.8%) than boys (31.7%). Students who drank in someone else’s home also had a significantly higher prevalence of vodka consumption (49.7%) than those who usually drank in their own home (30.9%) or at some public place (30.9%). However, the prevalence of vodka consumption by current drink- ers did not vary significantly by drinking frequency (the number of drinking days) or intensity (the largest number of drinks con- sumed on 1 occasion). Rum consumption was somewhat more common among boys than girls (15.4% vs 7.8%), as was the con- sumption of whiskey (13.0% vs 4.4%,). However, the prevalence of tequila consumption did not vary significantly by sex. The high prevalence of mixing liquor, such as vodka, with energy drinks, particularly among binge drinkers, is especially concern- ing because caffeine can mask some of the sensory cues that alert drinkers to their level of intoxication. This may lead youth to binge drink at higher intensity levels, increasing the risk of alco- hol-attributable harms, including alcohol poisoning, motor vehicle crashes, and sexual assault (21). In fact, one study involving col- lege students found that those who mixed energy drinks and alco- hol were twice as likely to report being hurt or injured, riding in a car with an intoxicated driver, or requiring medical treatment than those who did not mix energy drinks and alcohol, even after ad- justing for the amount of alcohol consumed (15). Vodka was the most common liquor type among those who repor- ted usually consuming liquor in the past 30 days (53.0%). Results Overall, 34.2% of Michigan high school students reported current alcohol use; 20.8% of students were binge drinkers (Table 1). Binge drinking prevalence was higher in each successive grade, ranging from 14.3% among 9th-grade students to 35.9% among 12th-grade students. However, there were no significant differ- ences in binge drinking prevalence by sex or by race/ethnicity. To assess beverage-specific alcohol consumption by youth, 11 state-added questions on alcohol consumption were included in the 2011 Michigan YTS. These questions were developed in col- laboration with the Excessive Alcohol Use Prevention Program of the National Center for Chronic Disease Prevention and Health Promotion at the Centers for Disease Control and Prevention, the Center on Alcohol Marketing and Youth at the Johns Hopkins Bloomberg School of Public Health, the Michigan Liquor Control Commission, and other experts in alcohol and public health. The questions added were based on questions that were used in other surveys, such as the Youth Risk Behavior Survey and a panel sur- Among students who reported drinking in the past 30 days, binge drinkers had a significantly higher prevalence of drinking in someone else’s home than did nonbinge drinkers (61.6% vs 44.0%; P < .001) and had more than 5 times the prevalence of drinking on 10 or more days in the past 30 days than nonbinge drinkers (24.4% vs 4.5%; P < .001) (Table 2). Binge drinkers also The opinions expressed by authors contributing to this journal do not necessarily reflect the opinions of the U.S. Department of Health and Human Services, the Public Health Service, the Centers for Disease Control and Prevention, or the authors’ affiliated institutions. or Disease Control and Prevention • www.cdc.gov/pcd/issues/2015/15_0290.htm VOLUME 12, E194 NOVEMBER 2015 Girls who reported usually consuming liquor had a higher prevalence of vodka consumption (60.3%) than boys (44.8%), but the difference between the 2 was not significant (data not shown). Although liquor consumption was common among all Michigan high school students who were current drinkers, girls were some- what more likely than boys to report consuming liquor. This is particularly concerning because women tend to achieve higher blood alcohol concentrations than men at the same consumption level, even taking into account differences in body size, food con- PREVENTING CHRONIC DISEASE PUBLIC HEALTH RESEARCH, PRACTICE, AND POLICY VOLUME 12, E194 NOVEMBER 2015 VOLUME 12, E194 NOVEMBER 2015 VOLUME 12, E194 NOVEMBER 2015 shown that reducing the threshold for defining binge drinking from 5 drinks to 4 drinks increases the relative prevalence of binge drinking by more than one-third (27). sumption, and other factors, thus increasing the risk of their exper- iencing alcohol-attributable harms, including unintended and alco- hol-exposed pregnancies and adverse reproductive outcomes (22). These data highlight the need for additional and targeted efforts to prevent binge drinking among girls, including increased efforts to monitor and reduce youth exposure to alcohol marketing, particu- larly given that underage girls are overexposed to alcohol market- ing relative to women to an even greater extent than underage boys are overexposed to alcohol marketing relative to men (23). This study shows that current drinking, particularly binge drink- ing, by high school students in Michigan remains a serious public health problem and that high school students who drink are most likely to drink liquor, particularly vodka. Surveillance on bever- age-specific alcohol consumption among youth can be useful for planning prevention strategies that target specific beverage types (eg, retail access to liquor) (9–11). Furthermore, because alcohol taxes are beverage-specific, knowing the type of alcoholic bever- age usually consumed by youth can help inform alcohol tax policies in states and communities to prevent underage drinking (9–11). The Task Force on Community Preventive Services re- commends several population-level, evidence-based strategies for preventing excessive alcohol use, including underage drinking, that can help reduce beverage-specific alcohol consumption by youth. These include increasing alcohol excise taxes, regulating al- cohol outlet density, establishing commercial host liability, en- hanced enforcement of the age 21 minimum legal drinking-age (eg, through compliance checks in which minors or youthful-look- ing adults attempt to purchase alcohol from retail establishments), and avoiding privatization of alcohol sales (28). Previous research has demonstrated that teenagers and young adults are especially responsive to increases in the price of alcohol (29). Improved compliance with the voluntary industry threshold for the place- ment of alcohol advertising in television programs and in other media venues (ie, 28.4% youth audience composition), particu- larly advertising that is placed on cable nonsports television pro- grams, could also help reduce the risk of underage drinking (30). Finally, states can routinely assess beverage-specific alcohol con- sumption using the YTS and use these data to help guide efforts to reduce youth exposure to alcohol marketing. VOLUME 12, E194 NOVEMBER 2015 For example, the Michigan Department of Health and Human Services has worked closely with state and local coalitions to collect and report inform- ation on excessive alcohol use and related harms and to develop a statewide plan to reduce underage drinking. This study also demonstrated the potential usefulness of the YTS for assessing alcohol consumption among high school students. Although primarily focused on tobacco consumption among youth, the YTS has the flexibility to accommodate state-added questions on alcohol consumption, including questions on bever- age-specific alcohol consumption, and is relatively inexpensive to administer. Furthermore, the collection of alcohol information in the YTS facilitates public health surveillance by providing a more specific assessment of the relationship between alcohol consump- tion and smoking by youth, including the potential impact of to- bacco control measures and alcohol interventions on the drinking and smoking behaviors of high school students. The results are subject to at least 7 limitations. First, this study as- sessed only the alcohol consumption patterns of Michigan stu- dents in 9th through 12th grade; therefore, these findings may not be generalizable to high school students in other states. Second, the YTS sample population was not large enough to assess signi- ficant differences between the demographic characteristics of li- quor drinkers by type of liquor consumed (eg, vodka, rum, whis- key). Third, the YTS sample includes only students in public high schools, and although more than 90% of Michigan youth attend public schools, the results may not be representative of those who attend private or other nonpublic schools or students who do not attend school; previous research has demonstrated that students who attend alternative schools may have even higher rates of alco- hol use than public school students (24). Fourth, all prevalence es- timates are based on self-report, which are likely to underestimate alcohol consumption because of social desirability and recall bi- ases (25). Fifth, about one-third of Michigan public high schools declined to participate in the YTS. The exclusion of the students in these schools from the survey may have affected the representat- iveness of the survey findings. Sixth, the data in this study were collected in 2011, and usual beverage consumption may have changed since then. However, liquor has consistently been repor- ted to be the usual type of alcohol consumed by high school stu- dents in other studies (10–13), and it therefore seems unlikely this has changed since 2011. Discussion To our knowledge, this is the first study to use the YTS to assess the types of alcohol consumed and the mixing of alcohol with en- www.cdc.gov/pcd/issues/2015/15_0290.htm • Centers for Disease Control and Prevention 3 VOLUME 12, E194 NOVEMBER 2015 Finally, the definition of binge drinking that was used in the YTS (ie, 5 or more drinks within a couple of hours) was not sex-specific (26), and studies among women have The opinions expressed by authors contributing to this journal do not necessarily reflect the opinions of the U.S. Department of Health and Human Services, the Public Health Service, the Centers for Disease Control and Prevention, or the authors’ affiliated institutions. 4 Centers for Disease Control and Prevention • www.cdc.gov/pcd/issues/2015/15_0290.htm Centers for Disease Control and Prevention • www.cdc.gov/pcd/issues/2015/15_0290.htm The opinions expressed by authors contributing to this journal do not necessarily reflect the opinions of the U.S. Department of Health and Human Services, the Public Health Service, the Centers for Disease Control and Prevention, or the authors’ affiliated institutions. 4 C f Di C l d P i d / d/i /2015/15 0290 h PREVENTING CHRONIC DISEASE PUBLIC HEALTH RESEARCH, PRACTICE, AND POLICY VOLUME 12, E194 NOVEMBER 2015 VOLUME 12, E194 NOVEMBER 2015 References Thombs DL, O’Mara RJ, Tsukamoto M, Rossheim ME, Weiler RM, Merves ML, et al. Event-level analyses of energy drink consumption and alcohol intoxication in bar patrons. Addict Behav 2010;35(4):325–30. 14. Stahre M, Roeber J, Kanny D, Brewer RD, Zhang X. Contribution of excessive alcohol consumption to deaths and years of potential life lost in the United States. Prev Chronic Dis 2014;11:E109. 1. O’Brien MC, McCoy TP, Rhodes SD, Wagoner A, Wolfson M. Caffeinated cocktails: energy drink consumption, high-risk drinking, and alcohol-related consequences among college students. Acad Emerg Med 2008;15(5):453–60. 15. Bouchery EE, Harwood HJ, Sacks JJ, Simon CJ, Brewer RD. Economic costs of excessive alcohol consumption in the US, 2006. Am J Prev Med 2011;41(5):516–24. 2. Office of Juvenile Justice and Delinquency Prevention. Drinking in America: myths, realities, and prevention policy. Washington (DC): US Department of Justice, Office of Justice Programs, Office of Juvenile Justice and Delinquency Prevention, 2005. 3. Centers for Disease Control and Prevention (CDC). Alcohol use among high school students — Georgia, 2007. MMWR Morb Mortal Wkly Rep 2009;58(32):885–90. 16. Center on Alcohol Marketing and Youth. Youth exposure to alcohol advertising on television, 2001–2009. Washington, D C , 2 0 1 2 . h t t p : / / w w w . c a m y . o r g / r e s e a r c h / Youth_Exposure_to_Alcohol_Ads_on_TV_Growing_Faster_T han_Adults/_includes/TVReport01-09_Revised_7-12.pdf. Accessed August 28, 2015. 17. US Department of Health and Human Services. The Surgeon General’s Call to Action To Prevent and Reduce Underage Drinking. Washington (DC): US Department of Health and Human Services, Office of the Surgeon General; 2007. 4. Bonnie RJ, O’Connell ME, editors. Reducing underage drinking: a collective responsibility. Washington (DC): The National Academies Press; 2004. http://www.nap.edu/ openbook.php?isbn=0309089352. Accessed August 21, 2015. 5. Anderson P, de Bruijn A, Angus K, Gordon R, Hastings G. Impact of alcohol advertising and media exposure on adolescent alcohol use: a systematic review of longitudinal studies. Alcohol Alcohol 2009;44(3):229–43. 18. Miller JW, Naimi TS, Brewer RD, Jones SE. Binge drinking and associated health risk behaviors among high school students. Pediatrics 2007;119(1):76–85. 6. Distilled Spirits Council of the United States. Vodka: the spirit of the industry. http://www.discus.org/assets/1/7/ Vodka2010.pdf. Accessed August 28, 2015. 19. Nelson DE, Naimi TS, Brewer RD, Nelson HA. State alcohol- use estimates among youth and adults, 1993–2005. Am J Prev Med 2009;36(3):218–24. 7. Michigan Liquor Control Commission. Annual financial report; 2011. http://www.michigan.gov/documents/lara/ statbook2011_new_397864_7.pdf. Acknowledgments We thank David Jernigan, PhD, Department of Health, Behavior, and Society, Johns Hopkins Bloomberg School of Public Health, Baltimore, Maryland, for his assistance in developing the ques- tions and providing comments on this manuscript. None of the au- thors have any conflicts of interest or financial disclosures. The findings and conclusions in this article are those of the authors and do not necessarily represent the official position of the Centers for Disease Control and Prevention. or Disease Control and Prevention • www.cdc.gov/pcd/issues/2015/15_0290.htm Author Information Corresponding Author: Dafna Kanny, PhD, Division of Population Health, National Center for Chronic Disease Prevention and Health Promotion, Centers for Disease Control and Prevention, 4770 Buford Hwy NE, MS F-78, Atlanta, GA 30341-3717. Telephone: 770-488-5411. E-mail: dkanny@cdc.gov. Cremeens JL, Miller JW, Nelson DE, Brewer RD. Assessment of source and type of alcohol consumed by high school students: analyses from four States. J Addict Med 2009; 3(4):204–10. 10. Siegel MB, Naimi TS, Cremeens JL, Nelson DE. Alcoholic beverage preferences and associated drinking patterns and risk behaviors among high school youth. Am J Prev Med 2011; 40(4):419–26. 11. Author Affiliations: Katherine R. Gonzales, Thomas W. Largo, Corinne Miller, Bureau of Disease Control, Prevention and Epidemiology, Michigan Department of Health and Human Services, Lansing, Michigan; Robert D. Brewer, Alcohol Program, Epidemiology and Surveillance Branch, Division of Population Health, National Center for Chronic Disease Prevention and Health Promotion, Centers for Disease Control and Prevention, Atlanta, Georgia. Siegel M, DeJong W, Naimi TS, Heeren T, Rosenbloom DL, Ross C, et al. Alcohol brand preferences of underage youth: results from a pilot survey among a national sample. Subst Abus 2011;32(4):191–201. 12. Tanski SE, McClure AC, Jernigan DH, Sargent JD. Alcohol brand preference and binge drinking among adolescents. Arch Pediatr Adolesc Med 2011;165(7):675–6. 13. VOLUME 12, E194 NOVEMBER 2015 Centers for Disease Control and Prevention. Types of alcoholic beverages usually consumed by students in 9th–12th grades — four states, 2005. MMWR Morb Mortal Wkly Rep 2007; 56(29):737–40. 9. PREVENTING CHRONIC DISEASE PUBLIC HEALTH RESEARCH, PRACTICE, AND POLICY VOLUME 12, E194 NOVEMBER 2015 References Accessed August 28, 2015. 20. Wagenaar AC, Toomey TL, Murray DM, Short BJ, Wolfson M, Jones-Webb R. Sources of alcohol for underage drinkers. J Stud Alcohol 1996;57(3):325–33. 8. Ferreira SE, de Mello MT, Pompéia S, de Souza-Formigoni ML. Effects of energy drink ingestion on alcohol intoxication. Alcohol Clin Exp Res 2006;30(4):598–605. 21. g to this journal do not necessarily reflect the opinions of the U.S. Department of Health and Human Services, e, the Centers for Disease Control and Prevention, or the authors’ affiliated institutions. The opinions expressed by authors contributing to this journal do not necessarily reflect the opinions of the U.S. Department of Health and Human Services, the Public Health Service, the Centers for Disease Control and Prevention, or the authors’ affiliated institutions. www.cdc.gov/pcd/issues/2015/15_0290.htm • Centers for Disease Control and Prevention 5 5 Centers for Disease Control and Prevention • www.cdc.gov/pcd/issues/2015/15_0290.htm The opinions expressed by authors contributing to this journal do not necessarily reflect the opinions of the U.S. Department of Health and Human Services, the Public Health Service, the Centers for Disease Control and Prevention, or the authors’ affiliated institutions. PREVENTING CHRONIC DISEASE PUBLIC HEALTH RESEARCH, PRACTICE, AND POLICY VOLUME 12, E194 NOVEMBER 2015 PREVENTING CHRONIC DISEASE PUBLIC HEALTH RESEARCH, PRACTICE, AND POLICY VOLUME 12, E194 NOVEMBER 2015 Naimi TS, Lipscomb LE, Brewer RD, Gilbert BC. Binge drinking in the preconception period and the risk of unintended pregnancy: implications for women and their children. Pediatrics 2003;111(5 Pt 2):1136–41. 22. Jernigan DH, Ostroff J, Ross C, O’Hara JA 3d. Sex differences in adolescent exposure to alcohol advertising in magazines. Arch Pediatr Adolesc Med 2004;158(7):629–34. 23. Grunbaum JA, Lowry R, Kann L. Prevalence of health-related behaviors among alternative high school students as compared with students attending regular high schools. J Adolesc Health 2001;29(5):337–43. 24. ( ) Davis CG, Thake J, Vilhena N. Social desirability biases in self-reported alcohol consumption and harms. Addict Behav 2010;35(4):302–11. 25. National Institute on Alcohol Abuse and Alcoholism. NIAAA Council approves binge drinking defınition. NIAAA Newsletter 2004;(3):3. Alcohol Abuse and Alcoholism. http:// pubs.niaaa.nih.gov/publications/Newsletter/winter2004/ Newsletter_Number3.pdf. Accessed August 28, 2015. 26. Chavez PR, Nelson DE, Naimi TS, Brewer RD. Impact of a new gender-specific definition for binge drinking on prevalence estimates for women. Am J Prev Med 2011; 40(4):468–71. 27. Task Force on Community Preventive Services. Preventing excessive alcohol consumption. In: The guide to community preventive services. New York (NY): Oxford University Press; 2005. http://www.thecommunityguide.org/alcohol/index.html. Accessed on April 28, 2014. 28. Xu X, Chaloupka FJ. The effects of prices on alcohol use and its consequences. Alcohol Res Health 2011;34(2):236–45. 29. Centers for Disease Control and Prevention. Youth exposure to alcohol advertising on television — 25 markets, United States, 2010. MMWR Morb Mortal Wkly Rep 2013;62(44):877–80. 30. Abbreviation: CI, confidence interval. a Binge drinkers were defined as students who had 5 or more drinks of alcohol in a row within a couple of hours on at least 1 day during the 30 days before the survey. b Nonbinge drinkers were defined as students who had at least 1 drink of alcohol on at least 1 day during the 30 days before the survey who did not report binge drinking. c Nondrinkers were defined as students who reported consuming no alcoholic drinks in the 30 days before the survey. Tables Table 1. Prevalence of Binge, Nonbinge, or Nondrinking Among Michigan 9th Through 12th Grade Students, by Demographic Char- acteristics, Michigan Youth Tobacco Survey, 2011 Demographic Characteristic Binge Drinkersa, % (95% CI) Nonbinge Drinkersb, % (95% CI) Nondrinkersc, % (95% CI) Overall (n = 3,985) 20.8 (17.5–24.1) 13.4 (11.9–15.0) 65.8 (62.1–69.4) Sex Female (n = 2,009) 19.4 (16.2–22.7) 15.0 (13.2–16.9) 65.5 (61.8–69.3) Male (n = 1,947) 22.2 (17.9–26.5) 11.8 (9.6–14.0) 66.0 (61.1–70.9) Grade 9th (n = 1,612) 14.3 (10.9–7.7) 11.9 (9.7–14.2) 73.8 (69.5–78.1) 10th (n = 1,308) 16.2 (12.8–19.6) 12.9 (10.8–14.9) 71.0 (66.7–75.2) 11th (n = 663) 18.7 (14.3–23.2) 14.4 (10.8–18.0) 66.9 (61.5–72.2) 12th (n = 394) 35.9 (27.7–44.0) 14.8 (11.2–18.4) 49.3 (39.7–58.9) Race/ethnicity White (n = 2,988) 21.3 (17.2–25.4) 13.4 (11.8–15.1) 65.2 (60.5–70.0) Black (n = 478) 18.4 (11.4–25.4) 15.5 (11.0–20.0) 66.1 (58.7–73.5) Hispanic (n = 182) 24.2 (15.6–32.8) 14.6 (9.0–20.3) 61.1 (52.9–69.4) Other (n = 285) 18.4 (10.5–26.2) 6.2 (4.2–8.3) 75.4 (67.3–83.5) Abbreviation: CI, confidence interval. a Binge drinkers were defined as students who had 5 or more drinks of alcohol in a row within a couple of hours on at least 1 day during the 30 days before the survey. b Nonbinge drinkers were defined as students who had at least 1 drink of alcohol on at least 1 day during the 30 days before the survey who did not report binge drinking. c Nondrinkers were defined as students who reported consuming no alcoholic drinks in the 30 days before the survey. e, or Nondrinking Among Michigan 9th Through 12th Grade Students, by Demographic Char- urvey, 2011 The opinions expressed by authors contributing to this journal do not necessarily reflect the opinions of the U.S. Department of Health and Human Services, the Public Health Service, the Centers for Disease Control and Prevention, or the authors’ affiliated institutions. g to this journal do not necessarily reflect the opinions of the U.S. Department of Health and Human Services, ce, the Centers for Disease Control and Prevention, or the authors’ affiliated institutions. www.cdc.gov/pcd/issues/2015/15_0290.htm • Centers for Disease Control and Prevention 7 www.cdc.gov/pcd/issues/2015/15_0290.htm • Centers for Disease Control and Preventio Centers for Disease Control and Prevention • www.cdc.gov/pcd/issues/2015/15_0290.htm The opinions expressed by authors contributing to this journal do not necessarily reflect the opinions of the U.S. Department of Health and Human Services, the Public Health Service, the Centers for Disease Control and Prevention, or the authors’ affiliated institutions. PREVENTING CHRONIC DISEASE PUBLIC HEALTH RESEARCH, PRACTICE, AND POLICY VOLUME 12, E194 NOVEMBER 2015 PREVENTING CHRONIC DISEASE PUBLIC HEALTH RESEARCH, PRACTICE, AND POLICY VOLUME 12, E194 NOVEMBER 2015 Table 3. PREVENTING CHRONIC DISEASE PUBLIC HEALTH RESEARCH, PRACTICE, AND POLICY Distribution of Usual Types of Alcohol Consumeda Among Current (Binge and Nonbinge) Drinkers, by Sociodemographic Characteristics and Consumption Patterns, Michigan Youth Tobacco Survey, 2011 Characteristic Liquor, % (95% CI) Beer, % (95% CI) Flavored Alcoholic Beverages, % (95% CI) No Usual Type, % (95% CI) Other Typeb, % (95% CI) Overall (n = 963) 51.2 (47.5–54.8) 17.9 (14.4–21.5) 14.5 (11.0–18.1) 5.2 (3.2–7.3) 11.2 (7.0–15.3) Sex Female (n = 485) 53.6 (46.2–61.0) 11.1 (7.2–15.1) 21.0 (15.3–26.6) 5.8 (2.6–8.9) 8.5 (3.9–13.1) Male (n = 473) 49.0 (45.4–52.6) 24.3 (18.6–29.9) 8.1 (4.8–11.5) 4.7 (2.3–7.1) 13.9 (8.7–19.0) Grade 9th (n = 324) 51.4 (44.6–58.1) 16.2 (10.7–21.8) 15.1 (11.0–19.2) 6.0 (3.3–8.6) 11.4 (7.1–15.6) 10th (n = 294) 53.7 (47.6–59.7) 13.5 (9.6–17.5) 16.5 (10.9–22.2) 7.1 (4.0–10.1) 9.2 (5.5–13.0) 11th (n = 174) 49.9 (42.5–57.3) 13.4 (7.3–19.5) 18.6 (11.0–26.3) 2.4 (0.0–4.7) 15.7 (5.5–25.9) 12th (n = 170) 50.5 (41.6–59.4) 24.6 (18.6–30.6) 10.3 (4.5–16.1) 5.6 (0.8–10.4) 9.0 (4.1–13.9) Race/ethnicity White (n = 746) 52.1 (48.5–55.7) 19.6 (15.3–23.9) 12.8 (9.0–16.7) 5.8 (3.7–7.9) 9.7 (5.5–13.8) Black (n = 110) 48.9 (35.7–62.0) 10.1 (1.9–18.3) 23.4 (10.9–35.9) 3.1 (0.0–8.2) 14.5 (6.1–22.9) Hispanic (n = 50) 55.1 (36.8–73.4) 16.0 (1.4–30.7) 11.7 (2.4–21.0) 4.0 (0.0–9.1) 13.1 (0.1–26.2) Drinking status Nonbinge drinkersc (n = 406) 46.9 (39.7–54.1) 17.1 (13.2–20.9) 17.6 (12.4–22.9) 5.5 (1.5–9.6) 12.9 (5.3–20.5) Binge drinkersd (n = 551) 54.2 (49.1–59.3) 18.6 (13.5–23.7) 12.1 (8.2–16.1) 5.1 (2.7–7.4) 10.0 (6.4–13.7) Usual place of consumption My home (n = 340) 37.9 (30.5–45.3) 21.2 (15.5–26.9) 18.3 (12.6–24.0) 6.8 (3.9–9.8) 15.7 (9.2–22.2) Someone else’s home (n = 546) 60.2 (54.7–65.7) 15.4 (9.8–21.1) 13.4 (8.0–18.7) 5.0 (3.3–6.6) 6.0 (2.8–9.2) Other placee (n = 163) 41.7 (31.6–51.7) 20.3 (12.7–27.9) 13.1 (6.0–20.2) 6.5 (0.3–12.7) 18.4 (11.9–24.9) Number of drinking days 1 or 2 (n = 463) 47.8 (41.5–54.1) 17.8 (13.6–22.0) 18.1 (12.5–23.7) 5.0 (1.6–8.4) 11.4 (5.1–17.7) 3–9 (n = 355) 57.6 (51.8–63.5) 16.2 (10.7–21.6) 11.7 (7.8–15.5) 4.9 (2.8–6.9) 9.7 (5.4–13.9) 10–30 (n = 145) 45.9 (36.6–55.2) 22.6 (14.9–30.4) 10.7 (2.4–19.0) 6.7 (0.5–13.0) 14.0 (6.8–21.3) Largest number of drinks on any occasion 1–3 (n = 474) 46.2 (41.0–51.3) 16.7 (11.9–21.5) 18.7 (14.2–23.1) 4.8 (1.3–8.3) 13.7 (7.7–19.7) 4 or 5 (n = 176) 56.9 (48.1–65.7) 12.3 (5.5–19.1) 18.3 (11.3–25.4) 5.2 (2.4–8.0) 7.3 (2.4–12.2) ≥6 (n = 328) 55.1 (49.9–60.3) 23.4 (17.0–29.8) 7.1 (4.0–10.3) 5.2 (1.3–9.1) 9.1 (5.5–12.8) Mixed energy drinks and alcohol (n = 432) 52.7 (45.1–60.3) 18.0 (12.1–23.9) 12.2 (7.0–17.4) 5.2 (2.8–7.6) 12.0 (6.7–17.2) Abbreviation: CI, confidence interval. VOLUME 12, E194 NOVEMBER 2015 Table 2. Distribution of Alcohol Consumption Characteristics Among Students Who Were Binge or Nonbinge Drinkers, Michigan Youth Tobacco Survey, 2011 Characteristic Binge Drinkersa, n = 698, % (95% CI) Nonbinge Drinkersb, n = 504, % (95% CI) Usual place of consumption My home (n = 307) 22.7 (18.0–27.5) 37.7 (32.2–43.2) Someone else’s home (n = 529) 61.6 (55.4–67.7) 44.0 (37.6–50.4) Restaurant/bar (n = 30) 3.6 (1.9–5.3) 2.7 (0.6–4.9) Other placec (n = 123) 12.1 (8.5–15.7) 15.6 (10.3–20.8) Number of drinking days 1 or 2 (n = 577) 27.3 (24.0–30.6) 75.3 (71.2–79.3) 3–9 (n = 434) 48.3 (44.2–52.4) 20.3 (16.4–24.1) 10–30 (n = 191) 24.4 (21.1–27.7) 4.5 (1.9–7.0) Mixed energy drinks and alcohol (n = 431) 49.0 (43.7–54.2) 18.2 (13.1–23.3) Abbreviation: CI, confidence interval. a Binge drinkers were defined as students who had 5 or more drinks of alcohol in a row within a couple of hours on at least 1 day during the 30 days before the survey. b Nonbinge drinkers were defined as students who had at least 1 drink of alcohol on at least 1 day during the 30 days before the survey who did not report binge drinking. c Other place was defined as riding or driving in a car; at a beach, park, concert, or sporting event; or on school property. Abbreviation: CI, confidence interval. a Binge drinkers were defined as students who had 5 or more drinks of alcohol in a row within a couple of hours on at least 1 day during the 30 days before the survey. b Nonbinge drinkers were defined as students who had at least 1 drink of alcohol on at least 1 day during the 30 days before the survey who did not report binge drinking. c Other place was defined as riding or driving in a car; at a beach, park, concert, or sporting event; or on school property. 8 www.cdc.gov/pcd/issues/2015/15_0290.htm • Centers for Disease Control and Prevention 9 PREVENTING CHRONIC DISEASE PUBLIC HEALTH RESEARCH, PRACTICE, AND POLICY a Survey question to assess usual types of alcohol was, “During the past 30 days, what type of alcohol did you usually drink? (Select only one response)”. b Other type of alcohol includes wine and cordials. c Nonbinge drinkers were defined as students who had at least 1 drink of alcohol on at least 1 day during the 30 days before the survey, but did not report binge drinking. d Binge drinkers were defined as students who had 5 or more drinks of alcohol in a row within a couple of hours on at least 1 day during the 30 days before the survey. e Other place was defined as a bar or restaurant; riding or driving in a car; at a park, beach, concert, or sporting event; or on school property. The opinions expressed by authors contributing to this journal do not necessarily reflect the opinions of the U.S. Department of Health and Human Services, the Public Health Service, the Centers for Disease Control and Prevention, or the authors’ affiliated institutions. Abbreviation: CI, confidence interval. www.cdc.gov/pcd/issues/2015/15_0290.htm • Centers for Disease Control and Prevention 9 9 0 Centers for Disease Control and Prevention • www.cdc.gov/pcd/issues/2015/15_0290.htm pes of alcohol was, “During the past 30 days, what type of liquor did you usually drink? (Select only one response)”. Survey question to assess usual types of alcohol was, During the past 30 days, what type of liquor did you usually drink? (Select only one response) . b Nonbinge drinkers were defined as students who had at least 1 drink of alcohol on at least 1 day during the 30 days before the survey, but did not report binge drinking. c Binge drinkers were defined as students who had 5 or more drinks of alcohol in a row within a couple of hours on at least 1 day during the 30 days before the sur- vey. d Other place was defined as bar or restaurant; while riding or driving in a car; at a park, beach, concert, or sporting event; or on school property. (continued on next page) The opinions expressed by authors contributing to this journal do not necessarily reflect the opinions of the U.S. Department of Health and Human Services, the Public Health Service, the Centers for Disease Control and Prevention, or the authors’ affiliated institutions. PREVENTING CHRONIC DISEASE PUBLIC HEALTH RESEARCH, PRACTICE, AND POLICY (Select only one response)”. b Abbreviation: CI, confidence interval. a Survey question to assess usual types of alcohol was, “During the past 30 days, what type of liquor did you usually drink? (Select only one response)”. b N bi g d i k d fi d t d t h h d t l t 1 d i k f l h l t l t 1 d d i g th 30 d b f th b t did t t bi g a Survey question to assess usual types of alcohol was, “During the past 30 days, what type of liquor did you usually drink? (Select only one response)”. b Nonbinge drinkers were defined as students who had at least 1 drink of alcohol on at least 1 day during the 30 days before the survey, but did not report binge drinking. c Binge drinkers were defined as students who had 5 or more drinks of alcohol in a row within a couple of hours on at least 1 day during the 30 days before the sur- vey. d Other place was defined as bar or restaurant; while riding or driving in a car; at a park, beach, concert, or sporting event; or on school property. (continued on next page) The opinions expressed by authors contributing to this journal do not necessarily reflect the opinions of the U.S. Department of Health and Human Services, the Public Health Service, the Centers for Disease Control and Prevention, or the authors’ affiliated institutions. 10 Centers for Disease Control and Prevention www cdc gov/pcd/issues/2015/15 0290 htm a Survey question to assess usual types of alcohol was, “During the past 30 days, what type of liquor did you usually drink? (Select only one response)”. b Nonbinge drinkers were defined as students who had at least 1 drink of alcohol on at least 1 day during the 30 days before the survey, but did not report binge drinking Survey question to assess usual types of alcohol was, During the past 30 days, what type of liquor did you usually drink? (Select only one response) . b Nonbinge drinkers were defined as students who had at least 1 drink of alcohol on at least 1 day during the 30 days before the survey, but did not report binge drinking. PREVENTING CHRONIC DISEASE PUBLIC HEALTH RESEARCH, PRACTICE, AND POLICY VOLUME 12, E194 NOVEMBER 2015 Table 4. Distribution of Usual Type of Liquor Consumeda Among Current (Binge and Nonbinge) Drinkers, by Demographic Charac- teristics and Consumption Patterns, Michigan Youth Tobacco Survey, 2011 Characteristic Didn’t Drink Liquor, % (95% CI) Vodka, % (95% CI) Rum, % (95% CI) Whiskey, % (95% CI) Tequila, % (95% CI) No Usual Type, % (95% CI) Other Type, % (95% CI) Overall (n = 1,146) 19.1 (15.6–22.5) 37.3 (33.2–41.1) 11.6 (8.9–14.2) 8.7 (5.6–11.7) 4.6 (3.0–6.3) 10.1 (7.8–12.5) 8.6 (6.0–11.2) Sex Female (n = 577) 16.7 (11.4–22.0) 42.8 (36.4–49.1) 7.8 (5.5–10.1) 4.4 (1.8–7.0) 5.8 (3.6–8.0) 13.0 (8.9–17.0) 9.6 (5.4–13.8) Male (n = 561) 21.4 (18.6–24.3) 31.7 (28.2–35.2) 15.4 (11.5–19.4) 13.0 (9.3–16.8) 3.2 (1.2–5.2) 7.4 (4.1–10.6) 7.8 (4.8–10.8) Grade 9th (n = 384) 21.1 (13.9–28.3) 37.0 (30.9–43.2) 9.7 (5.8–13.6) 9.4 (5.8–13.1) 4.4 (2.4–6.4) 8.0 (4.2–11.9) 10.4 (6.5–14.2) 10th (n = 359) 20.6 (13.4–27.8) 33.5 (26.5–40.4) 9.1 (5.6–12.6) 8.2 (4.3–12.1) 4.1 (2.0–6.3) 11.6 (7.7–15.5) 12.9 (8.0–17.8) 11th (n = 212) 23.6 (16.4–30.8) 36.8 (27.6–45.9) 10.9 (6.5–15.4) 5.7 (3.1–8.4) 3.6 (1.3–5.9) 10.0 (4.7–15.3) 9.4 (3.9–15.0) 12th (n = 190) 13.7 (7.2–20.2) 40.4 (31.1–49.6) 14.8 (10.5–19.1) 10.6 (3.9–17.3) 5.7 (1.8–9.6) 10.6 (5.8–15.5) 4.2 (1.3–7.1) Race/ethnicity White (n = 871) 19.9 (15.6–24.1) 39.4 (34.2–44.5) 12.3 (9.7–15.0) 8.9 (5.5–12.4) 3.7 (1.8–5.6) 9.7 (7.1–12.4) 6.0 (4.4–7.6) Black (n = 141) 16.5 (9.2–23.9) 32.9 (21.4–44.5) 7.3 (1.2–13.3) 5.1 (1.3–8.9) 5.8 (0.5–11.1) 12.6 (3.6–21.5) 19.8 (11.0–28.6) Hispanic (n = 63) 15.2 (7.4–23.0) 29.8 (16.5–43.1) 12.0 (2.5–21.4) 10.5 (1.4–19.7) 18.5 (2.2–34.9) 7.9 (2.0–13.9) 6.1 (0.1–12.0) Drinking behavior Nonbinge drinkersb (n = 491) 26.2 (22.3–30.0) 30.7 (23.5–38.0) 10.0 (5.9–14.2) 6.1 (3.3–8.9) 5.0 (2.3–7.7) 11.5 (7.7–15.3) 10.5 (6.4–14.6) Binge drinkersc (n = 647) 14.1 (10.0–18.2) 42.1 (38.2–46.1) 12.6 (9.3–15.9) 10.5 (6.8–14.3) 4.4 (2.0–6.8) 9.3 (6.5–12.1) 6.9 (4.3–9.5) Usual place of consumption My home (n = 304) 14.2 (8.4–20.0) 30.9 (22.6–39.2) 10.5 (6.8–14.2) 10.9 (5.4–16.4) 7.6 (4.2–11.0) 14.3 (8.4–20.3) 11.6 (5.7–17.5) Someone else’s home (n = 513) 8.3 (5.2–11.5) 49.7 (43.8–55.7) 12.7 (8.8–16.5) 7.9 (4.8–11.0) 3.0 (1.1–4.8) 10.1 (7.0–13.3) 8.2 (4.9–11.6) Other placed (n = 144) 13.2 (8.3–18.0) 30.9 (23.2–38.7) 20.0 (11.6–28.4) 9.5 (1.5–17.5) 8.1 (4.6–11.6) 10.0 (1.9–18.1) 8.3 (4.5–12.2) Abbreviation: CI, confidence interval. iquor Consumeda Among Current (Binge and Nonbinge) Drinkers, by Demographic Charac- chigan Youth Tobacco Survey, 2011 Abbreviation: CI, confidence interval. a Survey question to assess usual types of alcohol was, “During the past 30 days, what type of liquor did you usually drink? Abbreviation: CI, confidence interval. a Survey question to assess usual types of alcohol was, “During the past 30 days, what type of liquor did you usually drink? (Select only one response)”. b Nonbinge drinkers were defined as students who had at least 1 drink of alcohol on at least 1 day during the 30 days before the survey, but did not report binge drinking. c Binge drinkers were defined as students who had 5 or more drinks of alcohol in a row within a couple of hours on at least 1 day during the 30 days before the sur- vey. d Other place was defined as bar or restaurant; while riding or driving in a car; at a park, beach, concert, or sporting event; or on school property. PREVENTING CHRONIC DISEASE PUBLIC HEALTH RESEARCH, PRACTICE, AND POLICY c Binge drinkers were defined as students who had 5 or more drinks of alcohol in a row within a couple of hours on at least 1 day during the 30 days before the sur- vey. d Other place was defined as bar or restaurant; while riding or driving in a car; at a park, beach, concert, or sporting event; or on school property. (continued on next page) The opinions expressed by authors contributing to this journal do not necessarily reflect the opinions of the U.S. Department of Health and Human Services, the Public Health Service, the Centers for Disease Control and Prevention, or the authors’ affiliated institutions. 10 Centers for Disease Control and Prevention • www.cdc.gov/pcd/issues/2015/15_0290.htm 10 Centers for Disease Control and Prevention • www.cdc.gov/pcd/issues/2015/15_0290.htm 10 PREVENTING CHRONIC DISEASE PUBLIC HEALTH RESEARCH, PRACTICE, AND POLICY pes of alcohol was, “During the past 30 days, what type of liquor did you usually drink? (Select only one response)”. Abbreviation: CI, confidence interval. a PREVENTING CHRONIC DISEASE PUBLIC HEALTH RESEARCH, PRACTICE, AND POLICY VOLUME 12, E194 NOVEMBER 2015 Abbreviation: CI, confidence interval. a Survey question to assess usual types of alcohol was, “During the past 30 days, what type of liquor did you usually drink? (Select only one response)”. b Binge drinkers were defined as students who had 5 or more drinks of alcohol in a row within a couple of hours on at least 1 day during the 30 days before the sur vey. d Other place was defined as bar or restaurant; while riding or driving in a car; at a park, beach, concert, or sporting event; or on school property. Abbreviation: CI, confidence interval. (continued) (continued) Table 4. Distribution of Usual Type of Liquor Consumeda Among Current (Binge and Nonbinge) Drinkers, by Demographic Charac- teristics and Consumption Patterns, Michigan Youth Tobacco Survey, 2011 Characteristic Didn’t Drink Liquor, % (95% CI) Vodka, % (95% CI) Rum, % (95% CI) Whiskey, % (95% CI) Tequila, % (95% CI) No Usual Type, % (95% CI) Other Type, % (95% CI) Number of drinking days 1 or 2 (n = 556) 23.7 (19.2–28.2) 34.9 (28.4–41.4) 10.2 (7.0–13.4) 5.0 (1.6–8.4) 4.2 (2.1–6.4) 10.9 (7.5–14.2) 11.1 (7.2–15.0) 3–9 (n = 412) 15.4 (10.2–20.6) 42.2 (36.9–47.6) 13.4 (9.4–17.4) 11.2 (6.4–15.9) 4.4 (1.9–6.8) 8.8 (5.7–12.0) 4.6 (2.1–7.1) 10–30 (n = 178) 14.2 (7.7–20.6) 32.7 (24.5–40.9) 11.3 (5.9–16.8) 13.5 (5.2–21.9) 6.5 (1.2–11.8) 11.1 (4.3–17.9) 10.7 (5.5–15.9) Largest number of drinks on any occasion 1–3 (n = 510) 21.7 (17.4–26.1) 36.6 (30.6–42.6) 9.5 (6.1–13.0) 6.3 (3.1–9.5) 4.3 (1.8–6.7) 10.2 (6.1–14.3) 11.3 (7.2–15.5) 4 or 5 (n = 188) 14.7 (8.0–21.4) 41.9 (33.8–50.0) 10.5 (4.9–16.0) 9.1 (5.2–13.0) 5.9 (2.2–9.6) 14.2 (6.9–21.4) 3.9 (0.7–7.0) ≥6 (n = 366) 12.3 (7.9–16.7) 41.0 (34.6–47.4) 13.9 (8.2–19.6) 10.9 (5.8–16.1) 5.1 (1.8–8.4) 8.9 (4.3–13.4) 7.8 (4.2–11.5) Mixed energy drinks and alcohol (n = 404) 5.9 (2.1–9.7) 47.5 (42.2–52.7) 12.6 (8.2–17.1) 10.8 (6.4–15.1) 6.3 (3.3–9.3) 9.4 (5.4–13.4) 7.5 (4.5–10.5) Table 4. Distribution of Usual Type of Liquor Consumeda Among Current (Binge and Nonbing teristics and Consumption Patterns, Michigan Youth Tobacco Survey, 2011 The opinions expressed by authors contributing to this journal do not necessarily reflect the opinions of the U.S. Department of Health and Human Services, the Public Health Service, the Centers for Disease Control and Prevention, or the authors’ affiliated institutions. g to this journal do not necessarily reflect the opinions of the U.S. Department of Health and Human Services, ce, the Centers for Disease Control and Prevention, or the authors’ affiliated institutions. The opinions expressed by authors contributing to this journal do not necessarily reflect the opinions of the U.S. Department of Health and Human Services, the Public Health Service, the Centers for Disease Control and Prevention, or the authors’ affiliated institutions. www cdc gov/pcd/issues/2015/15 0290 htm • Centers for Disease Control and Prevention 11 www.cdc.gov/pcd/issues/2015/15_0290.htm • Centers for Disease Control and Prevention 11 www.cdc.gov/pcd/issues/2015/15_0290.htm • Centers for Disease Control and Preven
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Propensity for COVID-19 severe epidemic among the populations of the neighborhoods of Fortaleza, Brazil, in 2020
BMC public health
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Open Access Propensity for COVID-19 severe epidemic among the populations of the neighborhoods of Fortaleza, Brazil, in 2020 Jose Ueleres Braga1,2* , Alberto Novaes Ramos Jr3,4, Anderson Fuentes Ferreira4, Victor Macêdo Lacerda5, Renan Monteiro Carioca Freire5 and Bruno Vieira Bertoncini6,7 Abstract Background: The state of Ceará (Northeast Brazil) has shown a high incidence of coronavirus disease (COVID-19), and most of the cases that were diagnosed during the epidemic originated from the capital Fortaleza. Monitoring the dynamics of the COVID-19 epidemic is of strategic importance and requires the use of sensitive tools for epidemiological surveillance, including consistent analyses that allow the recognition of areas with a greater propensity for increased severity throughout the cycle of the epidemic. This study aims to classify neighborhoods in the city of Fortaleza according to their propensity for a severe epidemic of COVID-19 in 2020. Methods: We conducted an ecological study within the geographical area of the 119 neighborhoods located in the city of Fortaleza. To define the main transmission networks (infection chains), we assumed that the spatial diffusion of the COVID-19 epidemic was influenced by population mobility. To measure the propensity for a severe epidemic, we calculated the infectivity burden (ItyB), infection burden (IonB), and population epidemic vulnerability index (PEVI). The propensity score for a severe epidemic in the neighborhoods of the city of Fortaleza was estimated by combining the IonB and PEVI. Results: The neighborhoods with the highest propensity for a severe COVID-19 epidemic were Aldeota, Cais do Porto, Centro, Edson Queiroz, Vicente Pinzon, Jose de Alencar, Presidente Kennedy, Papicu, Vila Velha, Antonio Bezerra, and Cambeba. Importantly, we found that the propensity for a COVID-19 epidemic was high in areas with differing socioeconomic profiles. These areas include a very poor neighborhood situated on the western border of the city (Vila Velha), neighborhoods characterized by a large number of subnormal agglomerates in the Cais do Porto region (Vicente Pinzon), and those located in the oldest central area of the city, where despite the wealth, low-income groups have remained (Aldeota and the adjacent Edson Queiroz). * Correspondence: ueleres@gmail.com * Correspondence: ueleres@gmail.com p g 1Department of Epidemiology, Institute of Social Medicine, State University of Rio de Janeiro, Rua São Francisco Xavier 524 / 7017D, 7° floor, Rio de Janeiro, RJ CEP 20550-013, Brazil 1Department of Epidemiology, Institute of Social Medicine, State University of Rio de Janeiro, Rua São Francisco Xavier 524 / 7017D, 7° floor, Rio de Janeiro, RJ CEP 20550-013, Brazil 2Department of Epidemiology and Quantitative Methods, Sergio Arouca National School of Public Health (ENSP), Oswaldo Cruz Foundation (FIOCRUZ), Rio de Janeiro, RJ, Brazil Full list of author information is available at the end of the article Braga et al. BMC Public Health (2020) 20:1486 https://doi.org/10.1186/s12889-020-09558-9 Braga et al. BMC Public Health (2020) 20:1486 https://doi.org/10.1186/s12889-020-09558-9 (Continued from previous page) (Continued from previous page) Conclusion: Although measures against COVID-19 should be applied to the entire municipality of Fortaleza, the classification of neighborhoods generated through this study can help improve the specificity and efficiency of these measures. Keywords: COVID-19, Public health surveillance, Disease outbreaks Background overall epidemic burden and requires the use of sensitive tools for epidemiological surveillance, including analyses that allow the recognition of areas with a greater pro- pensity for increased severity throughout the epidemic cycle. In addition to their technical-scientific character, these analyses must be strongly integrated with operational aspects of surveillance and control, with cooperation of public managers of the municipality in the health, trans- port, and education sectors, to achieve timely and effective integrated responses like allocation of resources and med- ical personnel depending on determined propensity of severe epidemic. The first case of coronavirus disease (COVID-19) was recorded on December 8, 2019, in Wuhan, Hubei prov- ince, China. Approximately 3 months later, on March 11, 2020, this disease was declared as a pandemic by the World Health Organization (WHO) [1]. On February 3, 2020, the Brazilian Ministry of Health declared COVID-19 as a Public Health Emergency of National Importance (ESPIN), through Ordinance MS n. 188 of 2020, in line with Decree n. 7616, of November 17, 2011. The first confirmed COVID-19 case in Latin America was reported on February 26, 2020, in the city of São Paulo, the most populous city in the Southern Hemisphere (approximately 12 million inhabitants), and involved a person who had travelled to northern Italy (Lombardia region) [2, 3]. Approximately a month later, on March 20, Ordinance MS n. 454 was published in the Official Gazette (Edition: 55-F / Section 1 - Extra), declaring the existence of community transmission of COVID-19 throughout the national territory of Brazil. Since then, Brazil has registered the largest number of confirmed cases in Latin America (n = 20,727, 18:00, April 11, 2020) [4]. The study proposes to increase the understanding of the COVID-19 epidemic in Fortaleza, enabling the rec- ognition of urban spaces with a greater propensity for a severe epidemic based on population mobility in order to promote evidence-based actions by the municipality public authorities. This study aims to classify neighbor- hoods in the city of Fortaleza by their propensity for a severe COVID-19 epidemic in 2020. © The Author(s). 2020 Open Access This article is licensed under a Creative Commons Attribution 4.0 International License, which permits use, sharing, adaptation, distribution and reproduction in any medium or format, as long as you give appropriate credit to the original author(s) and the source, provide a link to the Creative Commons licence, and indicate if changes were made. The images or other third party material in this article are included in the article's Creative Commons licence, unless indicated otherwise in a credit line to the material. If material is not included in the article's Creative Commons licence and your intended use is not permitted by statutory regulation or exceeds the permitted use, you will need to obtain permission directly from the copyright holder. To view a copy of this licence, visit http://creativecommons.org/licenses/by/4.0/. The Creative Commons Public Domain Dedication waiver (http://creativecommons.org/publicdomain/zero/1.0/) applies to the data made available in this article, unless otherwise stated in a credit line to the data. Page 2 of 11 Braga et al. BMC Public Health (2020) 20:1486 Study design Ceará has become one of the states with the highest number of cases of the disease (1374), with only São Paulo and Rio de Janeiro reporting higher numbers (6708 and 1938, respectively). While the average inci- dence rate in Brazil is 7.5 cases per 100,000 inhabitants, the rate in Ceará is double at 14.1 per 100,000 inhabi- tants [5]. The highest number of cases and deaths is concentrated in Fortaleza (1291 confirmed cases and 53 deaths), making it the Brazilian state capital with the highest incidence rate (35/100,000), and a mortality rate of 3.6% among confirmed cases [4]. This is an ecologic study based on the geographical unit of the 119 neighborhoods in the municipality of Forta- leza (Fig. 1). In order to define the main transmission networks (and chains), we hypothesized that the spatial diffusion of the COVID-19 epidemic is influenced by population mobility. We also postulated that the spatial spread of the COVID-19 epidemic follows the hierarch- ical model based on networks and population mobility that plays a key role in the constitution of the viral transmission chains. The rationale of the study assumed that based on the incidence rate of the initial phase of the epidemic (in- fectivity load), the flow of passengers moving between neighborhoods can influence the infection load in each neighborhood of the municipality. If one considers the epidemic vulnerability attributed to populations living in these neighborhoods, combined with the mentioned infection burden, one can estimate the propensity for a serious COVID-19 epidemic. Numerous factors have been considered important to explain the magnitude, intensity, and early viral circula- tion of the epidemic in Fortaleza. Of these, the following two aspects appear to be the most crucial: firstly, the Pinto Martins International Airport in Fortaleza, which became a “flight hub” in 2018, facilitated the influx of a large number of national and international flights and tourists; and secondly, the measures adopted by the state epidemiological surveillance services included testing a relatively large number of suspected cases. To measure the propensity of a severe epidemic, the following events were initially calculated: (i) infectivity burden - ItyB; (ii) infection burden - IonB; and (iii) popu- lation epidemic vulnerability index (PEVI). Then, the (iv) Monitoring the dynamics of the COVID-19 epidemic in Fortaleza is of strategic importance for reducing Braga et al. BMC Public Health (2020) 20:1486 Page 3 of 11 Fig. Infectivity burden Infectivity burden was calculated using the epidemio- logical surveillance records of COVID-19 in Fortaleza, so the number of confirmed cases were denoted as in- fectivity burden. These data were formally obtained from the Municipal Health Department on April 7, 2020. The suspected cases of COVID-19 had been investigated according to the recommendations of the Ministry of Health. All confirmed COVID-19 cases with SARS-CoV-2 PCR positive status reported to the Municipal Health De- partment of Fortaleza until March 12, 2020, residing in the municipality, were included. This period corresponds to the initial phase of the epidemic, when predominantly imported cases (travelers) initiated the transmission; indigenous cases Study design 1 Map of the 119 neighborhoods in the municipality of Fortaleza Fig. 1 Map of the 119 neighborhoods in the municipality of Fortaleza Propensity score for a severe epidemic in the neighbor- hoods of the city of Fortaleza was estimated. would be detected later after being in contact with the initial cases through community transmission [6]. In this phase, the Fortaleza City Council adopted more general measures for surveillance, prevention, and con- trol of COVID-19 [7]. Infection burden The Infection burden was measured by combining the infectivity burden and population mobility between the neighborhoods of Fortaleza. The mobility burden - MtyB, was evaluated through the daily travel flows, by looking at public transport use with work motivation between the two neighborhoods. Notably, the measurement of the flow of people between the neighborhoods of Forta- leza (excluding displacements within the same neighbor- hood) only became possible after a strategic study was conducted by the city of Fortaleza and other institutions Braga et al. BMC Public Health (2020) 20:1486 Page 4 of 11 Page 4 of 11 Page 4 of 11 on this theme in 2019 called Home Origin-Destination Survey (OD survey). on this theme in 2019 called Home Origin-Destination Survey (OD survey). HDUs were designed to generate more homogeneous areas, based on socioeconomic conditions, than the weighted areas of the IBGE. To calculate the proportion of subnor- mal agglomerations, data from the IBGE were used, consid- ering the population living under these conditions by the total population of the neighborhood, thereby obtaining the percentage of people living in subnormal agglomerations per neighborhood. HDUs were designed to generate more homogeneous areas, based on socioeconomic conditions, than the weighted areas of the IBGE. To calculate the proportion of subnor- mal agglomerations, data from the IBGE were used, consid- ering the population living under these conditions by the total population of the neighborhood, thereby obtaining the percentage of people living in subnormal agglomerations per neighborhood. The OD survey comprised a sample survey performed through interviews in households, providing the values of the variables in this analysis with a detailed matrix of the trip patterns and travel choices. Data were collected in a database, which describes the various attributes of the activities and trips of the city’s inhabitants, as well as the respective socioeconomic status and characteristics of individuals and their families, in order to describe a pattern of displacement of people and the chain of their activities throughout a typical business day. The infor- mation from the OD Matrix is of immense importance in the analysis of transport systems, comprising funda- mental elements for planning and decision making, and has therefore been integrated in this study. PEVI The PEVI was constructed according to the Urban Health Index approach recommended by WHO [8], to demonstrate the population attributes that best repre- sent, from a collective point of view, the vulnerability of the population to COVID-19. This index comprises seven (7) sociodemographic indicators, based on the 2010 Brazilian census of the Brazilian Institute of Geog- raphy and Statistics (IBGE). The indicators that make-up the PEVI are: (i) proportion of households with more than two residents per bedroom, (ii) illiteracy proportion, (iii) proportion of the population in extreme poverty, (iv) proportion of households without running water and sani- tation, (v) proportion of unemployment, (vi) Gini of family income, and (vii) proportion of people living in subnormal agglomerations. Since the Ip values are obtained for all indicators and units, the next step was to integrate Ip into a single com- posite index, here called VEPI. VEPI is calculated for each unit, using the geometric mean of the Ip values for each unit. Considering that there are j indicators, the formula used for this calculation was: VEPI ¼ Y j i¼1 Ip i !1 j Where, Ip i is the standardized value of the seven indi- cators for a given neighborhood, and j corresponds to all other neighborhoods. In addition to the point estimate, VEPI variance and standard error were also calculated, with respective confidence interval estimates based on these measures. Considering that the purpose of this index is to identify the “geographical” disparities of the studied phenomenon, a diagnosis of its distribution, evaluating the differences between the highest and lowest values as well as its visual comparison with the homogeneous distribution of these values, was carried out through the “qqnorm” graph (Fig. 2). IBGE classifies subnormal agglomerates groups as consisting of 51 or more housing units, characterized by the absence of ownership titles, and at least one of the following characteristics: irregularity of circulation routes, size and shape of the lots, and lack of essential public services (such as garbage collection, sewage, water, electricity, and public lighting). The data for each of the indicators above were previously obtained and translated into the Human Development Units (HDUs) by the Institute for Applied Economic Research (IPEA), with the exception of the proportion of subnormal agglomerations. The HDUs represent units of analysis with relatively homogeneous socioeconomic characteristics, and the original data were used to produce the Metropolitan Region Human Development Atlas. The Infection burden The infection burden indicator was calculated using the following formula: After the values of the indicators were stored in a database, the accuracy, completeness, and consistency of the data were verified for calculation of the summary measure. There are two main steps in calculating the index: (1) standardization of indicators, and (2) amal- gamation of standardized indicators. Each of these steps can be performed in a mathematically straightforward manner. The standardization of the values of each indicator is performed using the following formula: Ip ¼ I −min Ið Þ max Ið Þ −min Ið Þ IonBi ¼ X 118 j¼1 ItyBMtyB Ip ¼ I −min Ið Þ max Ið Þ −min Ið Þ where Ip is the standardized value of I, max (I) is the highest value of I among all observations, and min (I) is the lowest value of I among all observations. Severe epidemic population propensity The main study outcome is the Severe Epidemic Popula- tion Propensity. The estimated score of the propensity for a severe epidemic amongst the population of the Fortaleza neighborhoods was calculated by combining Braga et al. BMC Public Health (2020) 20:1486 Page 5 of 11 Fig. 2 Flow Diagram for constructing the population epidemic vulnerability index Fig. 2 Flow Diagram for constructing the population epidemic vulnerability index the infection burden with PEVI. The calculation was carried out using the multiplicative approach, involving the multiplication of these two indexes. distribution is, therefore, similar to the distribution of incidence, and compatible with the surveillance strategy adopted by the state and municipal governments for this first stage of the epidemic curve caused by COVID-19. Results The distribution of the infection burden (Fig. 4) was quite heterogeneous, with some areas showing higher levels than those of the central and eastern areas, as this measure reflects the combination of the infectivity bur- den and population mobility between the neighborhoods of Fortaleza. During the initial phase of the epidemic described in this study, from February 27 to March 12, 2020, 54 cases of COVID-19 were detected and reported, and this was the scenario used to study the propensity for a severe epidemic of populations living in the neighborhoods of the municipality of Fortaleza. The VEPI index had a very heterogeneous spatial dis- tribution, similar to the distribution of the seven indica- tors that were combined for its construction. The distribution shown in Fig. 5 indicates low vulnerability in the central and central-eastern regions of the city and increased levels of vulnerability as the neighborhoods move away from this region. However, it can be observed that some areas of the southern periphery, the western zone, and the eastern coastal zone have the highest levels of vulnerability. The spatial distribution of COVID-19 incidence in this period indicates that the epidemic was restricted to spe- cific areas of the city. This distribution reflects important heterogeneity, with a concentration of cases in the central area and in neighborhoods that represent better socioeconomic conditions, and also in areas with more diverse socioeconomic conditions, such as Meireles, Aldeota, Papicu, and Cocó (Fig. 3). During the beginning of the epidemic, the spatial distribution of the infectivity burden by neighborhood reflects heterogeneity, concentrated mainly in central areas and in neighborhoods with the highest economic conditions. This pattern indicates, at this stage, a low burden of COVID-19 in the outskirts of the city. This When looking at the distribution of vulnerability to the COVID-19 epidemic, the combination of the various attributes related to the seven indicators appears to re- veal something different than the individual indicators. An example is the vulnerability of the neighborhoods in Braga et al. BMC Public Health (2020) 20:1486 Page 6 of 11 Fig. 3 Map of the distribution of confirmed cases of COVID-19 in the neighborhoods of the Municipality of Fortaleza, from February 27 to March 12, 2020 Discussion the Cais do Porto region, the eastern coastal zone, and the eastern part of the city, which seemed to have been mainly influenced by the existence of subnormal agglomerations. The neighborhoods with the highest propensity for a severe COVID-19 epidemic were Aldeota, Cais do Porto, Centro, Edson Queiroz, Vicente Pinzon, Jose de Alencar, Presidente Kennedy, Papicu, Vila Velha, Antonio Bezerra, and Cambeba. The main finding of this study indicates higher levels of propensity to the COVID-19 epidemic in areas with a wide spectrum of socioeconomic profiles, including a group of very poor neighborhoods on the western border of the city (Vila Velha), a set of neighborhoods characterized by a large number of subnormal agglomerates in the Cais do Porto region (Vicente Pizon), and neighbor- hoods in the oldest central area of the city, where low-income areas exist despite the overall wealth in the area (Aldeota and the adjacent Edson Queiroz). The distribution of the propensity shown in Fig. 6 indicates that, besides being heterogeneous, the de- tected weaknesses (epidemic vulnerability) combined with the initial situation of the COVID-19 epidemic (period until March 12, 2020) and the population mo- bility lead to the prediction that neighborhoods such as Centro, Aldeota, and Papicu are among those in the top 10% with the highest propensity score. Other neighborhoods are also classified in the same group (western zone, bordered by Caucaia). These results are not as evident when the various aspects that in- fluence the epidemic are evaluated separately. Braga et al. BMC Public Health (2020) 20:1486 Page 7 of 11 Fig. 4 Map of the distribution of the COVID-19 infection burden in the neighborhoods of the Municipality of Fortaleza Fig. 4 Map of the distribution of the COVID-19 infection burden in the neighborhoods of the Municipality of Fortaleza Indeed, the propensity for a severe COVID-19 epi- demic in the neighborhoods of Fortaleza is very hetero- geneous and reflects not only the population’s mobility in the urban space, but also the dynamics of transmis- sion of a disease that is influenced by the living situation of a population in a city. Recently, an article indicating that the heterogeneity of the distribution of the inci- dence of COVID-19 is determined by socio-economic factors was published on the ABC American communi- cation network. Discussion This article states that in New York City, a ‘stark contrast’ in COVID-19 infection rates can be observed, based on education and ethnicity [9]. in neighborhoods with better economic conditions, such as those in the eastern and coastal zones. Almost in a complementary way, the distribution of the proportion of unemployment showed higher rates in neighborhoods located in the outskirts of the municipality. A similar situation was observed for indicators of household agglomeration (more than two people per bedroom) and households without access to water or sanitation. The inequality of these distributions indicates that these phenomena are correlated and probably express the evolution of the urban space occupation process in Fortaleza. Very few studies have assessed the spread of the COVID-19 epidemic, and so far, no articles have been published that appreciat the influence of specific popula- tion factors linked to people’s mobility and to predict the occurrence of severe outbreaks in areas within cities. The seven sociodemographic indicators assessed sep- arately had spatial distributions with relevant heterogen- eity. Inequality expressed by income (data not shown) had a distribution with an expression of less inequality in the periphery of the municipality and greater inequality Braga et al. BMC Public Health (2020) 20:1486 Page 8 of 11 Fig. 5 Map of the distribution of the population epidemic vulnerability to COVID-19 according to neighborhoods in the Municipality of Fortaleza Spatial analysis was used by Kang et al. [10] to understand the epidemic spread of COVID-19. While the authors described the spatiotemporal pattern and evaluated the spatial association of the early stages of the COVID-19 epidemic in mainland China from January 16 to February 6, 2020, they sought only to identify the occurrence of spatial autocorrelation measured by Moran’s I for the vari- ous periods studied. between a given location and the surrounding spatial units by local Moran’s I (LISA). Giuliani et al. [12] studied the spatiotemporal spread of COVID-19 in Italy. They sought to model and predict the number of COVID-19 infections, drawing out the effects of its spatial diffusion. They argue that “forecasts about where and when the disease will occur may be of great usefulness for public decision-makers, as they give the time to intervene on the local public health systems”. However, the authors did not consider the population heterogeneities and their influence in predicting the epi- demic in the studied regions. Fan et al. Discussion [11] studied the epidemiology of the Novel COVID-19 in Gansu Province, China. They concluded that different from findings from Wuhan Province, the spatial distribution pattern analysis indicated hot spots and spatial outliers in Gansu Province. To detect the spatial distribution pattern of COVID-19 cases at county levels during the study periods, they used local indica- tors of spatial association to evaluate the relationship The study that used the methodological approach most similar to our study was conducted by Pluchino et al. [13]. They proposed a data-driven framework for assessing the epidemic risk of a geographical area (in a Braga et al. BMC Public Health (2020) 20:1486 Page 9 of 11 Fig. 6 Map of COVID-19 severe epidemic propensity according to neighborhoods in the city of Fortaleza Fi 6 M f COVID 9 id Fig. 6 Map of COVID-19 severe epidemic propensity according to neighborhoods in the city of Fortaleza Our study aims to contribute to mathematical model- ling studies in a complementary manner, in order to predict the dynamics of the COVID-19 epidemic in Brazil. Complementary methodological approaches are required to broaden the understanding of the epidemic and its possible determinants. Indeed, many mathemat- ical models were used to estimate the epidemic curve of the COVID-19 outbreak in Brazilian cities. Rocha-Filho et al. [16] used a variant of the SEIR (Susceptible, Exposed, Infectious, Recovered) classical model, including hospital- ized variables (SEIHR model) and an age-stratified structure to analyze the expected time evolution during the onset of the epidemic in the metropolitan area of São Paulo. predictive way), and to identify high-risk areas within a country. They constructed a risk index combining three different features: (1) the disease hazard, (2) the infection exposure of the area, and (3) its vulnerability. However, vulnerability was considered based on the local data regarding air pollution, mobility, winter temperature, housing concentration, health care density, population size, and age. g Public transport is presented as a definite spatial trend, with trips mainly concentrated in the central and western regions of the city of Fortaleza, which are directly related to the provision of public transport and radio-concentric bus lines. However, as a part of the assumptions in this approved study, these dimensions should be considered while studying the transmission of SARS-CoV-2, in particular using samples of other respiratory-based infectious diseases [14, 15]. Ethics approval and consent to participate IRB approval is considered unnecessary in accordance with Resolution No. 510, of April 7, 2016 of the National Health Council of Brazil in its first paragraph and according to the Letter of Consent issued by the Municipal Health Secretariat of Fortaleza. Discussion One of the main limitations of this study is that the prediction inherent to the methodological approach does not specify the time at which the severe epidemic will most intensely occur in the neighborhoods of Fortaleza. Page 10 of 11 Page 10 of 11 Braga et al. BMC Public Health (2020) 20:1486 Braga et al. BMC Public Health (2020) 20:1486 The simplicity of the approach used in our study, which does not exhaust all potential factors that influence the epidemic, is also an advantage over other methods. long as the precepts contained in the data transfer rules of Brazilian law are met. long as the precepts contained in the data transfer rules of Brazilian law are met. Author details 1 1Department of Epidemiology, Institute of Social Medicine, State University of Rio de Janeiro, Rua São Francisco Xavier 524 / 7017D, 7° floor, Rio de Janeiro, RJ CEP 20550-013, Brazil. 2Department of Epidemiology and Quantitative Methods, Sergio Arouca National School of Public Health (ENSP), Oswaldo Cruz Foundation (FIOCRUZ), Rio de Janeiro, RJ, Brazil. 3Department of Community Health, School of Medicine, Federal University of Ceará (UFC), Fortaleza, Brazil. 4Postgraduate Program in Public Health, School of Medicine, Federal University of Ceará (UFC), Fortaleza, Brazil. 5Municipal Secretariat of Conservation and Public Services (SCSP), Fortaleza, Brazil. 6Department of Transport Engineering, Technology Centre, Federal University of Ceará (UFC), Fortaleza, Brazil. 7Postgraduate Program in Transport Engineering, h l d l f á ( ) l l No funding was obtained for this study. No funding was obtained for this study. References 1 WHO C ESPIN: Public Health Emergency of National Importance; OD: Origin- Destination; HDU: Human Development Units; IPEA: Institute for Applied Economic Research; IBGE: Brazilian Institute of Geography and Statistics; LISA: Local Moran’s I; PEVI: Population epidemic vulnerability index; WHO: World Health Organization 1. WHO. Coronavirus disease 2019 Situation Report – 51. 2020. https://www. who.int/emergencies/diseases/novel-coronavirus-2019. 2. Faria NR. First cases of coronavirus disease (COVID-19) in Brazil, South America (2 genomes, 3rd march 2020) - novel 2019 coronavirus / genome reports - Virological. 2020. http://virological.org/t/first-cases-of-coronavirus- disease-covid-19-in-brazil-south-america-2-genomes-3rd-march-2020/409. Accessed 13 Apr 2020. 2. Faria NR. First cases of coronavirus disease (COVID-19) in Brazil, South America (2 genomes, 3rd march 2020) - novel 2019 coronavirus / genome reports - Virological. 2020. http://virological.org/t/first-cases-of-coronavirus- disease-covid-19-in-brazil-south-america-2-genomes-3rd-march-2020/409. Accessed 13 Apr 2020. Competing interests The author declares that he has no competing interests. None of the authors of this paper have a financial or personal relationship with other people or organizations that could inappropriately influence or bias the content of the paper. Supplementary information y Supplementary information accompanies this paper at https://doi.org/10. 1186/s12889-020-09558-9. y Supplementary information accompanies this paper at https://doi.org/10. 1186/s12889-020-09558-9. Supplementary information accompanie 1186/s12889-020-09558-9 Additional file 1. Letter of consent from the Municipal Health Secretariat of Fortaleza. Document referring to the consent of the Municipal Health Secretariat of Fortaleza secretariat giving the notification data aggregated by neighborhood of residence, which were published in the epidemiological bulletins and on the Fortaleza city hall website. In this document there is also reference about the need not submit the referred project to the ethics committee of that institution, nor of the other institutions. Received: 27 April 2020 Accepted: 17 September 2020 Received: 27 April 2020 Accepted: 17 September 2020 Acknowledgements The authors would like to thank Edenilo Baltazar Barreira Filho, Francisco Moraes de Oliveira Neto, Geziel dos Santos de Sousa, Jader de Oliveira Santos, Manoel Mendonca de Castro Neto, Verônica Teixeira Franco Castelo Branco, for the valuable suggestions on the analysis and discussion processes. We would like to thank Joana Angélica Paiva Maciel, Fortaleza’s municipal health secretary for the transfer of COVID-19 notification data. We also thank Jorge Ubirajara Pedreira Junior (Polytechnic School of the Federal University of Bahia - EPUFBA) and Márcio Santos da Natividade (Institute of Collective Health - ISC / UFBA) for the initial conception of the study. 3. Rodriguez-Morales AJ, Gallego V, Escalera-Antezana JP, Méndez CA, Zambrano LI, Franco-Paredes C, et al. COVID-19 in Latin America: The implications of the first confirmed case in Brazil. Travel Med Infect Dis. 2020: 101613. https://doi.org/10.1016/j.tmaid.2020.101613. 3. Rodriguez-Morales AJ, Gallego V, Escalera-Antezana JP, Méndez CA, Zambrano LI, Franco-Paredes C, et al. COVID-19 in Latin America: The implications of the first confirmed case in Brazil. Travel Med Infect Dis. 2020: 101613. https://doi.org/10.1016/j.tmaid.2020.101613. 4. Brasil. COVID-19 - Painel Coronavírus. 2020. https://covid.saude.gov.br/. Accessed 13 Apr 2020. 5. Brasil. Boletim Epidemiológico 8 - COE Coronavírus. 2020. https://www. saude.gov.br/images/pdf/2020/April/09/be-covid-08-final-2.pdf. 5. Brasil. Boletim Epidemiológico 8 - COE Coronavírus. 2020. https://www. saude.gov.br/images/pdf/2020/April/09/be-covid-08-final-2.pdf. 6. WHO. Vigilância mundial da COVID-19 causada por infecção humana pelo vírus COVID-19; 2020. p. 1–4. https://apps.who.int/iris/bitstream/handle/1 0665/331506/WHO-2019-nCoV-SurveillanceGuidance-2020.6-por. pdf?sequence=14&isAllowed=y. 6. WHO. Vigilância mundial da COVID-19 causada por infecção humana pelo vírus COVID-19; 2020. p. 1–4. https://apps.who.int/iris/bitstream/handle/1 0665/331506/WHO-2019-nCoV-SurveillanceGuidance-2020.6-por. pdf?sequence=14&isAllowed=y. Authors’ contributions JUB was responsible for statistical modelling, data analysis, result presentation, discussion and writing of the manuscript. ANRJ and BVB were responsible for data analysis, result presentation, discussion, and writing of the manuscript. AFF, VML, and RMCF were responsible for data analysis, result presentation, and discussion. All authors reviewed, revised and approved the manuscript. 7. Fortaleza. Novo Coronavírus - Informações da Prefeitura de Fortaleza. Prefeitura Municipal de Fortaleza. 2020. https://coronavirus.fortaleza.ce.gov. br/acoes.html. Accessed 17 Apr 2020. 8. WHO. The urban health index: a handbook for its calculation and use. Geneva PP - Geneva: World Health Organization. https://apps.who.int/iris/ handle/10665/136839. Conclusion In conclusion, although universal actions should be ap- plied to the entire municipality of Fortaleza, the classifi- cation of neighborhoods generated in this study can help improve specific measures in order to be more efficient. A set of recommendations were made to the municipal government of Fortaleza. The neighborhoods that were identified with a greater propensity for severe epidemics should receive special attention in the adoption of mea- sures to control the epidemic. Funding 9. Schumaker E, In NYC. “stark contrast” in COVID-19 infection rates based on education and race - ABC News. ABC News; 2020. p. 1. https://abcnews.go. com/Health/nyc-stark-contrast-covid-19-infection-rates-based/story?id=6992 0706. Accessed 16 Apr 2020. 9. Schumaker E, In NYC. “stark contrast” in COVID-19 infection rates based on education and race - ABC News. ABC News; 2020. p. 1. https://abcnews.go. com/Health/nyc-stark-contrast-covid-19-infection-rates-based/story?id=6992 0706. Accessed 16 Apr 2020. Availability of data and materials Giving TB wheels: public transportation as a risk factor for tuberculosis transmission. Tuberculosis. 2011;91(SUPPL. 1):S16–23. https://doi.org/10.1016/j.tube.2011.10.005. 15. Feske ML, Teeter LD, Musser JM, Graviss EA. Giving TB wheels: public transportation as a risk factor for tuberculosis transmission. Tuberculosis. 2011;91(SUPPL. 1):S16–23. https://doi.org/10.1016/j.tube.2011.10.005. 16. Filho TMR, Santos FSG d, Gomes VB, TAH R, JHR C, Ramalho WM, et al. Expected impact of COVID-19 outbreak in a major metropolitan area in Brazil. medRxiv. 2020;2020:03.14.20035873. https://doi.org/10.1101/2020.03. 14.20035873. Availability of data and materials 10. Kang D, Choi H, Kim J-H, Choi J. Spatial epidemic dynamics of the COVID-19 outbreak in China. Int J Infect Dis. 2020:1–21. https://doi.org/10.1016/j.ijid. 2020.03.076. I confirm that the map in Figs. 1, 3, 4, 5, and 6. is my own. It was done with GIS software. The notification data for COVID-19 was obtained by request from the municipal health department of Fortaleza and the authors made a commitment to confidential use and confidentiality was guaranteed. Any researcher can request this data to the municipal health department of Fortaleza (https://www.fortaleza.ce.gov.br/institucional/a-secretaria-327) as 10. Kang D, Choi H, Kim J-H, Choi J. Spatial epidemic dynamics of the COVID-19 outbreak in China. Int J Infect Dis. 2020:1–21. https://doi.org/10.1016/j.ijid. 2020.03.076. 11. Fan J, Liu X, Pan W, Douglas MW, Bao S. Epidemiology of 2019 novel coronavirus Disease-19 in Gansu Province, China, 2020. Emerg Infect Dis. 2020;26:1–8. https://doi.org/10.3201/eid2606.200251. 11. Fan J, Liu X, Pan W, Douglas MW, Bao S. Epidemiology of 2019 novel coronavirus Disease-19 in Gansu Province, China, 2020. Emerg Infect Dis. 2020;26:1–8. https://doi.org/10.3201/eid2606.200251. Page 11 of 11 Braga et al. BMC Public Health (2020) 20:1486 Braga et al. BMC Public Health (2020) 20:1486 12. Giuliani D, Dickson MM, Espa G, Santi F. Modelling and Predicting the Spatio-Temporal Spread of Coronavirus Disease 2019 (COVID-19) in Italy. SSRN Electron J. 2020. https://doi.org/10.2139/ssrn.3559569. 12. Giuliani D, Dickson MM, Espa G, Santi F. Modelling and Predicting the Spatio-Temporal Spread of Coronavirus Disease 2019 (COVID-19) in Italy. SSRN Electron J. 2020. https://doi.org/10.2139/ssrn.3559569. 13. Pluchino A, Inturri G, Rapisarda A, Biondo AE, Moli RL, Zappala’ C, et al. A Novel Methodology for Epidemic Risk Assessment: the case of COVID-19 outbreak in Italy. arXiv Prepr arXiv200402739. 2020; http://arxiv.org/abs/2004 02739. Accessed 16 Apr 2020. 13. Pluchino A, Inturri G, Rapisarda A, Biondo AE, Moli RL, Zappala’ C, et al. A Novel Methodology for Epidemic Risk Assessment: the case of COVID-19 outbreak in Italy. arXiv Prepr arXiv200402739. 2020; http://arxiv.org/abs/2004. 02739. Accessed 16 Apr 2020. 14. Troko J, Myles P, Gibson J, Hashim A, Enstone J, Kingdon S, et al. Is public transport a risk factor for acute respiratory infection? BMC Infect Dis. 2011; 11:16. https://doi.org/10.1186/1471-2334-11-16. 14. Troko J, Myles P, Gibson J, Hashim A, Enstone J, Kingdon S, et al. Is public transport a risk factor for acute respiratory infection? BMC Infect Dis. 2011; 11:16. https://doi.org/10.1186/1471-2334-11-16. 15. Feske ML, Teeter LD, Musser JM, Graviss EA. 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Evaluation of Leading Safety Performance of Primary School Buildings in Alexandria, Egypt: Cross-Sectional Study
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Evaluation of Leading Safety Performance of Primary School Buildings in Alexandria, Egypt: Cross- Sectional Study Gehan R. Zaki1 ¥, Kholoud Y. Tayel2, Mayada M. Reda3, 4, Aleya H. Mahmoud3, Engy I. Labib 1 Department of Occupational Health and Air Pollution, High Institute of Public Health, Alexandria University, Egypt 2 Department of Family Health, High Institute of Public Health, Alexandria University, Egypt 3 Department of Environmental Health, High Institute of Public Health, Alexandria University, Egypt 4 Hamdan Bin Mohammed Smart University, Dubai, UAE ublic, by (SP) igher 1.8%) eas." thed m would ety safety; Available on line at: www.jhiph.alexu.edu.eg ¥Correspondence Email: gehanra17@gmail.com Suggested Citations: Zaki GR, Tayel KY, Reda MM, Mahmoud AH, Labib EI. Evaluation of Leading Safety Performance of Primary School Buildings in Alexandria, Egypt: Cross-Sectional Study. JHIPH. 2018;48(2):77-84. Journal of High Institute of Public Health 2018;48(2):77-84. Original Article Background: Unsafe school buildings may adversely affect students, teachers, administrative workers, cleaning workers, and visitors. Objective(s): The aim of the present study was to evaluate the leading safety performance of public, private, and experimental primary school buildings in Alexandria, Egypt. Objective(s): The aim of the present study was to evaluate the leading safety performance of public, private, and experimental primary school buildings in Alexandria, Egypt. Methods: A cross-sectional study was conducted in 30 primary schools in Alexandria selected by stratified cluster sampling. Two observational checklists were designed, validated, and used; including school building safety inspection checklist (SBSIC), and classroom safety inspection checklist (CSIC). The completed checklists were reviewed and coded. The safety performances (SP) for each category and for the overall checklists were then calculated. Results: The SP in private primary schools and classrooms [(66.1±13.0%), (68.7±12.5)] were higher than that of experimental [(59.9±14.0%), (65.1±14.2%)] and public ones [(39.3±7.8%), (46.0±11.8%) respectively]. The most common causes of reduced school SP were "the absence of protective measures against vectors & insects," "the non-daily refuse disposal," "the non-inspected play areas." Other causes included "the irregular fire drills," "the absence of the alarm system," "the non-earthed electrical equipment," "the absence of emergency plan," and "the unmarked tripping/slipping locations." The most frequent unsafe classroom conditions were "the absence of classroom alarm point," "the on-board glare," and "the non-compliant windows to class area ratios." Conclusion: Many safety violations were found to occur in Alexandria primary schools. This would d ti f th f t f d tl l k f f t t S f t than that of experimental [(59.9±14.0%), (65.1±14.2%)] and public ones [(39.3±7.8%), (46.0±11.8%) respectively]. The most common causes of reduced school SP were "the absence of protective measures against vectors & insects," "the non-daily refuse disposal," "the non-inspected play areas." Other causes included "the irregular fire drills," "the absence of the alarm system," "the non-earthed electrical equipment," "the absence of emergency plan," and "the unmarked tripping/slipping locations." The most frequent unsafe classroom conditions were "the absence of classroom alarm point," "the on-board glare," and "the non-compliant windows to class area ratios." Conclusion: Many safety violations were found to occur in Alexandria primary schools. This would cause a reduction of the safety performance and consequently a lack of safety management. Safety performance in private schools was better than that in experimental and public ones. Keywords: Classroom safety; Electrical safety; Emergency preparedness; Fire safety; Ground safety; Housekeeping; Leading safety performance; School safety Abstract Background: Unsafe school buildings may adversely affect students, teachers, administrative workers, cleaning workers, and visitors. INTRODUCTION factors that might lead to future disease or disability.” School safety management is a vital element of the healthy school environment, which is an essential part of the teaching and learning activities. It focuses on controlling the accident promoting factors within the school.(2) According to the proverb "you cannot manage what you cannot measure," it is essential to assess safety performance in order to manage safety. Previously, safety performance was measured by calculation of accident parameters, including frequency rate, severity rate, fatality rate, and frequency severity index (lagging indicators). Nowadays, it is periodically assessed using both factors that might lead to future disease or disability.” School safety management is a vital element of the healthy school environment, which is an essential part of the teaching and learning activities. It focuses on controlling the accident promoting factors within the school.(2) According to the proverb "you cannot manage what you cannot measure," it is essential to assess safety performance in order to manage safety. Previously, safety performance was measured by calculation of accident parameters, including frequency rate, severity rate, fatality rate, and frequency severity index (lagging indicators). Nowadays, it is periodically assessed using both P rimary education stage usually occurs in primary schools, which are occupied by students, teachers, administrative employees, and cleaning workers. The World Health Organization (WHO) defines a health-promoting school as “one that constantly strengthens its capacity as a healthy setting for living, learning and working”.(1) The American Academy of Pediatrics defines a “healthful school environment” as “one that protects students and staff against immediate injury or disease and promotes prevention activities and attitudes against known risk P 77 Journal of High Institute of Public Health 2018;48(2):77-84. 78 classroom safety inspection checklists (SBSIC and CSIC). The two checklists were designed with Yes, No or Not Applicable responses. The Yes response was scored as "one" and the No as "zero." (5-8) The first checklist (SBSIC) consisted of six categories, including housekeeping, maintenance, fire safety, electrical safety, emergency preparedness, in addition to ground safety, which had eight, four, eleven, ten, nine, and ten statements, with score ranges of (0-8), (0-4), (0-11), (0-10), (0-9), and (0-10) respectively added to the school data. INTRODUCTION The second checklist (CSIC) had three main categories, including classroom conditions, safety and emergency preparedness, as well as indoor air quality, which had eight, twenty eight, and six statements in the checklist, with score ranges of (0-8), (0-28), and (0-6) respectively in addition to the classroom data. lagging and leading indicators. Leading safety performance is regularly used by employing the traditional safety checklist with yes, no, or not applicable responses. It aims at finding out the accident promoting factors and immediately executing the suitable corrective actions to prevent it.(3) In Egypt, leading school safety performance is not periodically assessed. Hence, school safety cannot be managed. To bridge this gap it is important to assess the current situation of school safety. So, the aim of the present study was to evaluate the leading safety performance of public, private, and experimental primary school buildings in Alexandria, Egypt. METHODS A cross-sectional study design was conducted in primary schools in Alexandria, Egypt. Multistage stratified cluster sampling from Alexandria public, private, and experimental primary schools was conducted to cover all educational zones, including Eastern, Western, Central, El-Gomrok, El-Montazah, El-Amreya and Borg el Arab. According to the WHO practical manual 1991(4), the minimum sample size was 26 clusters at 95% confidence level and 5% expected error. It was raised to 30 schools. Two classrooms were randomly selected from each school. The sample was selected using a multistage stratified cluster sampling technique. The strata were classified into public, private and experimental primary schools in Alexandria. The seven educational zones in Alexandria Governorate (Eastern, Western, Middle, El-Gomrok, El-Montazah, El-Amreya and Borg El- Arab) were included in the present study. An occupational safety professor in High Institute of Public Health, Alexandria University was consulted to review the two checklists, which were later tested for their validation. This was conducted through completing the checklists by three researchers for the same five schools. Those schools were not included within the study sample. Pearson's correlation coefficients were calculated for the results of the first and second; first and third; as well as for second and third researchers respectively.(9) The data were collected using the validated checklists. The completed checklists were reviewed and coded. The safety performance (percent safety score) for each category and for the overall checklist were then calculated according to equations (1) and (2). It was classified into "poor" (<60%), "moderate" (60-70%), "satisfactory" (70-80%), and "Good" (≥80%).(9) Two observational checklists were designed after extensive literature review, school building and The Number of Yes responses of the school = Number of Yes responses in SBSIC+ Number of Yes responses of CSIC of Class-1+Number of Yes responses of CSIC of Class-2 (1) The Number of Yes responses of the school = Number of Yes responses in SBSIC+ Number of Yes responses of CSIC of Class-1+Number of Yes responses of CSIC of Class-2 (1) (1) Where SP of SBSIC is the safety performance of school building inspection checklist, SP of CSIC of Class-1 is the safety performance of first classroom inspection checklist, and SP of CSIC of Class-2 is the safety performance of second classroom inspection checklist. No. of YES Responses × 100 No. of YES Responses + No. of NO Responses (2) Safety Performance % = No. of YES Responses × 100 No. of YES Responses + No. RESULTS Regarding validation of the checklists, the correlation coefficients of the overall SP in schools and classrooms between the first and second researchers were 0.81, 0.87; the second and third were 0.84, 0.90; Table 1: Safety performance (percent safety scores) of primary schools and classrooms at different school types in Alexandria, Egypt 2014 Variable n % Mean % Safety Score SD Minimum Maximum p- value* School Type Public 16 53.3 39.3 7.8 29.7 52.5 ≤0.05 Private 8 26.7 66.1 13.0 50.0 91.4 Experimental 6 20.0 59.9 14.0 59.8 81.5 Classrooms Public 32 53.3 46.0 11.8 30.9 64.3 ≤0.05 Private 16 26.7 68.7 12.5 50.0 91.2 Experimental 12 20.0 65.1 14.2 53.7 92.9 *One-Way ANOVA Test; it is significant at P-value ≤ 0.0 e (percent safety scores) of primary schools and classrooms at different school mance (percent safety scores) of primary schools and classrooms at different school gypt 2014 performance (percent safety scores) of primary schools and classrooms at different school dria, Egypt 2014 Table 1: Safety performance (percent safety scores) of primary schools and classrooms types in Alexandria, Egypt 2014 *One-Way ANOVA Test; it is significant at P-value ≤ 0.0 The housekeeping SP in private schools was good (84.0%), while that of experimental and public schools were satisfactory (70.0%) and poor (41.0%) respectively (Figure 1). The SPs of the housekeeping category were significantly varied (p≤0.05, at C.I.=95%) among different school types. Further analysis disclosed the significant differences between public and private as well as between public and experimental schools (p≤0.05, at C.I.=95%). The reduction of the housekeeping SP below the 100% was attributed to the safety standards' violations. The major unsafe housekeeping conditions were "the absence of protective measures against vectors & insects" (100.0%, 50.0%, 66.7%), and "the non-daily disposal of Refuse" (100.0%, 12.5%, and 50.0%) in public, private, and experimental schools respectively as illustrated in (Table 2). The poor SPs of the maintenance (<60%) in each of public, private, and experimental schools (Figure 1) were obvious in the non-conformity with the safety benchmarks. One-Way ANOVA Test revealed significant variation in maintenance SP among the three school types (p≤0.05, at C.I.=95%). Meanwhile, significant differences in maintenance SP were noticed between public and private schools as well as between public and experimental ones (p≤0.05, at C.I.=95%). The most frequent unsafe maintenance condition was "the non- inspected play areas" (100.0%, 87.5%, and 100.0%) for public, private, and experimental schools respectively (Table 2). Ethical Considerations The study was approved by the Institutional Review Board and the Ethics Committee of High Institute of Public Health. The study conformed to the principles of Helsinki declaration (2013) and the international ethics guidelines. Confidentiality of collected information was ensured. METHODS of NO Responses (2) No. of YES Responses × 100 Safety Performance % = Safety Performance % = (2) Post-Hoc Multiple Comparison Least Significant Difference (LSD) Tests were used to check the significance of variation for variables of more than two classes. The significance of the results was The data were entered and statistically analyzed using IBM SPSS 21 software package (IBM SPSS Statistics, Somers, NY, USA). The safety performance (% safety score) was checked for normality using One-Sample Kolmogorov-Smirov Test. One-Way ANOVA and the 79 Zaki et al. estimated at 95% confidence interval (C.I). The results were considered significant at P-value ≤ 0.05.(10, 11) and between the first and third were 0.83, 0.89 respectively. The SP showed non-significant Kologorov Smirov Test (p>0.05, at 95% C.I.). Table (1) indicated that the SP of private primary schools and classrooms [(66.1±13.0%), and (68.7±12.5)] were higher than that of experimental [(59.9±14.0%) and (65.1±14.2%)] and public ones [(39.3±7.8%) and (46.0±11.8%) respectively]. Significant differences (p≤0.05, at C.I.=95%) of schools and classrooms' SP were observed among the three school types (public, private, and experimental). Further analysis showed highly significant variation in the schools and classrooms' SP between public and private as well as between public and experimental schools (P≤0.05, at C.I.=95%). Safety performance of schools and classrooms showed non-significant One-Way ANOVA Test among different districts (p>0.05, at C.I.=95%). RESULTS The fire-safety SP in private schools was moderate (65.0%), while that of experimental (38.0%) and public (8.0%) were poor (Figure 1). The fire SP showed highly significant variation (p≤0.05, at C.I.=95%) among different school types (One-Way ANOVA Test). Further analysis showed significant differences between public and private as well as between public and experimental schools (p≤0.05, at C.I.=95%). The most frequent unsafe condition was Journal of High Institute of Public Health 2018;48(2):77-84. 80 "the irregular fire drills" that present 100.0%, 75.0%, and 83.3% of public, private, and experimental schools respectively. "The absence of the alarm system" was found in 100.0% of public, 62.5% of private, and 83.3% of the experimental schools (Table 3). g p and 83.3% of public, private, and experimental schools respectively. "The absence of the alarm system" was found in 100.0% of public, 62.5% of private, and 83.3% of the experimental schools (Table 3). Figure 1: Primary Schools’ Safety Performance (percent safety Scores) of different checklist categories at different school types in Alexandria, Egypt 2014. 41 20 8 28 6 48 46 84 54 65 48 31 70 69 70 50 38 30 30 73 65 0 10 20 30 40 50 60 70 80 90 100 Housekeeping Maintenance Fire safety Electrical safety Emergency Preparedness Ground safety Classroom safety % Safety Score of the School (Safety Performance) Public n=16 Private n=8 Experimental n=6 ≤0.05 ≤0.05 ≤0.05 >0.05 >0.05 ≤0.05 ≤0.05 41 20 8 28 6 48 46 84 54 65 48 31 70 69 70 50 38 30 30 73 65 0 10 20 30 40 50 60 70 80 90 100 Housekeeping Maintenance Fire safety Electrical safety Emergency Preparedness Ground safety Classroom safety % Safety Score of the School (Safety Performance) Public n=16 Private n=8 Experimental n=6 ≤0.05 ≤0.05 ≤0.05 >0.05 >0.05 ≤0.05 ≤0.05 ary Schools’ Safety Performance (percent safety Scores) of different checklist categories at types in Alexandria, Egypt 2014. Figure 1: Primary Schools’ Safety Performance (percent safety Scores) of different checklist categories at different school types in Alexandria, Egypt 2014. Table 2: Distribution of the violated safety standards of housekeeping and maintenance at primary schools in Alexandria, Egypt 2014 Variables Public n=16 Private n=8 Experimental n=6 No. % No. % No. RESULTS % Fire Safety Extinguishers are absent 13 81.3 1 12.5 3 50.0 Extinguishers are not properly mounted 2 12.5 1 12.5 0 0.0 Extinguishers are not accessible 2 12.5 0 0.0 0 0.0 There is not alarm system 16 100.0 5 62.5 5 83.3 Alarm system is not working -- -- 5 62.5 0 0.0 Fire lane is absent 15 93.8 6 75.0 5 83.3 Lane is not clear 1 6.3 6 75.0 0 0.0 Fire drills are not undertaken regularly 16 100.0 6 75.0 5 83.3 Electrical Safety Electrical cords near the foot traffic area 4 25.0 4 50.0 2 33.3 All electrical cords near the foot traffic area are not tied up 0 0.0 0 0.0 0 0.0 Power Strips are not used instead of extension cords 13 81.3 4 50.0 4 66.7 Electrical Panels are not properly labeled 15 93.8 5 62.5 5 83.3 Electrical Panels have open slots 15 93.8 6 75.0 6 100.0 Electrical panels' rooms are not locked 7 43.8 3 37.5 5 83.3 Electrical switch/junction boxes are not covered 16 100.0 6 75.0 4 66.7 There are charred outlet faces 11 68.8 2 25.0 3 50.0 Flammable materials are stored in heater rooms 9 56.3 2 25.0 4 66.7 All electrical equipment are not earthed 16 100.0 8 100.0 6 100.0 on of the studied primary schools according to violated safety standards of Fire and exandria, Egypt 2014 Concerning the SP of the electrical safety, it was poor at the three school types (28.0%, 48.0%, and 30.0% respectively), although the difference was not significant (Figure 1). The main unsafe electrical safety condition was "the non-earthed electrical equipment" at all of the studied schools (Table 3). The safety performance of the emergency preparedness Table 4: Distribution of the studied primary schools according to the violated safety standards of Emergency, and of Ground safety, Alexandria, Egypt 2014 Variable Public n=16 Private n=8 Experimental n=6 No. % No. % No. RESULTS % Emergency preparedness Emergency exits are absent 12 75.0 5 62.5 4 66.7 Emergency Exits are not clear 4 25.0 1 12.5 1 16.7 Emergency evacuation maps are absent 16 100 7 87.5 4 66.7 Emergency Planning Manual is absent 16 100 7 87.5 5 83.4 MSDSs are absent in the school 15 93.8 5 62.5 4 66.7 Ground Safety There are slippery and broken stairs 12 75.0 2 25.0 2 33.3 Yard ground texture is slippery 2 12.5 1 12.5 0 0.0 Ground is not clean and not free from obstacles 2 12.5 0 0.0 0 0.0 Aisles, stairs, and walking surfaces are not clear of obstacles 5 31.3 0 0.0 1 16.7 Floors are not in good conditions with missing tiles 15 93.8 1 12.5 1 16.7 Floors are slippery 0 0.0 1 12.5 0 0.0 Areas with high fall potential (around drinking fountains, entryways, etc.) have not been marked 15 93.8 5 62.5 4 66.7 High-visibility tape or paint are not used to point out hard to see steps, cracks, trip hazard 15 93.8 8 100.0 5 83.3 Table 3: Distribution of the studied primary schools according to violated safety standards of Fire and electrical safety, Alexandria, Egypt 2014 Variables Public n=16 Private n=8 Experimental n=6 No. % No. % No. RESULTS % Housekeeping Presence of hazards in corridors 7 43.8 0 0 1 16.7 Unclean of workers' uniform of kitchen or canteen 5 31.3 3 33.3 4 66.7 Absence of protective measures against vectors & insects 16 100.0 4 50.0 4 66.7 Absence of refuse container in each room 1 6.3 0 0 1 16.7 Refuse disposal does not occur daily 16 100.0 1 12.5 3 50.0 Water basins (sinks) are not clean 11 68.8 1 12.5 0 0.0 The closets are not clean 13 81.3 1 12.5 1 16.7 Maintenance Play areas are not inspected for accidents hazards 16 100.0 7 87.5 6 100.0 Drinking tabs are not maintained 10 62.5 1 12.5 0 0.0 Water basins are not regularly maintained 10 62.5 2 25 2 33.3 Closets are not maintained 15 93.8 4 50 4 66.7 on of the violated safety standards of housekeeping and maintenance at primary schools in 2014 Zaki et al. 81 81 Table 3: Distribution of the studied primary schools according to violated safety standards of Fire and electrical safety, Alexandria, Egypt 2014 Variables Public n=16 Private n=8 Experimental n=6 No. % No. % No. RESULTS % Fire Safety Extinguishers are absent 13 81.3 1 12.5 3 50.0 Extinguishers are not properly mounted 2 12.5 1 12.5 0 0.0 Extinguishers are not accessible 2 12.5 0 0.0 0 0.0 There is not alarm system 16 100.0 5 62.5 5 83.3 Alarm system is not working -- -- 5 62.5 0 0.0 Fire lane is absent 15 93.8 6 75.0 5 83.3 Lane is not clear 1 6.3 6 75.0 0 0.0 Fire drills are not undertaken regularly 16 100.0 6 75.0 5 83.3 Electrical Safety Electrical cords near the foot traffic area 4 25.0 4 50.0 2 33.3 All electrical cords near the foot traffic area are not tied up 0 0.0 0 0.0 0 0.0 Power Strips are not used instead of extension cords 13 81.3 4 50.0 4 66.7 Electrical Panels are not properly labeled 15 93.8 5 62.5 5 83.3 Electrical Panels have open slots 15 93.8 6 75.0 6 100.0 Electrical panels' rooms are not locked 7 43.8 3 37.5 5 83.3 Electrical switch/junction boxes are not covered 16 100.0 6 75.0 4 66.7 There are charred outlet faces 11 68.8 2 25.0 3 50.0 Flammable materials are stored in heater rooms 9 56.3 2 25.0 4 66.7 All electrical equipment are not earthed 16 100.0 8 100.0 6 100.0 Concerning the SP of the electrical safety, it was poor at the three school types (28.0%, 48.0%, and 30.0% respectively), although the difference was not significant (Figure 1). The main unsafe electrical safety condition was "the non-earthed electrical equipment" at all of the studied schools (Table 3). The safety performance of the emergency preparedness was poor in the public, private, and experimental schools (6.0%, 31.0%, and 30.0% respectively), with no significant difference was between the three school types (p>0.05, at C.I.=95%) (Figure 1). The most frequent unsafe emergency condition was "the absence of emergency planning manual" (100.0%, 87.5%, and 83.4% respectively) (Table 4). Table 4: Distribution of the studied primary schools according to the violated safety standards of Emergency, and of Ground safety, Alexandria, Egypt 2014 Variable Public n=16 Private n=8 Experimental n=6 No. % No. % No. RESULTS The most recurrent unsafe classroom situations were "the absence of classroom alarm point" (100.0%, 87.5%, and 83.3%), "the on- board glare" (75.0%, 50.0%, and 75.0%), and "the non-compliant windows to class area ratios" (71.9%, 75.0%, and 58.3%) in public, private, and experimental schools respectively. Table 5: Distribution of the studied primary schools' classrooms according to violated safety standards, Al d i E t 2014 of the studied primary schools' classrooms according to violated safety standards, Table 5: Distribution of the studied primary schools' classrooms according to violated safety standards, Alexandria, Egypt 2014 Safety Standards Public n=32 Private n=16 Experimental n=12 No. % No. % No. RESULTS % Emergency preparedness Emergency exits are absent 12 75.0 5 62.5 4 66.7 Emergency Exits are not clear 4 25.0 1 12.5 1 16.7 Emergency evacuation maps are absent 16 100 7 87.5 4 66.7 Emergency Planning Manual is absent 16 100 7 87.5 5 83.4 MSDSs are absent in the school 15 93.8 5 62.5 4 66.7 Ground Safety There are slippery and broken stairs 12 75.0 2 25.0 2 33.3 Yard ground texture is slippery 2 12.5 1 12.5 0 0.0 Ground is not clean and not free from obstacles 2 12.5 0 0.0 0 0.0 Aisles, stairs, and walking surfaces are not clear of obstacles 5 31.3 0 0.0 1 16.7 Floors are not in good conditions with missing tiles 15 93.8 1 12.5 1 16.7 Floors are slippery 0 0.0 1 12.5 0 0.0 Areas with high fall potential (around drinking fountains, entryways, etc.) have not been marked 15 93.8 5 62.5 4 66.7 High-visibility tape or paint are not used to point out hard to see steps, cracks, trip hazard 15 93.8 8 100.0 5 83.3 ution of the studied primary schools according to the violated safety standards of Emergency afety, Alexandria, Egypt 2014 Journal of High Institute of Public Health 2018;48(2):77-84. 82 Considering the SP of the ground safety, it was the highest for experimental schools (73% Satisfactory), followed by private (70% Satisfactory) and public (48% poor) ones (Figure 1). One-Way ANOVA Test indicated the highly significant variation of SP between the three school types (p≤0.05, at C.I.=95%). Further analysis disclosed statistically significant difference between public and private as well as between public and experimental schools (p≤0.05, at C.I.=95%). As obvious in table (4), the unsafe condition of the highest frequency was "the unmarked tripping/slipping locations" (93.8%, 100.0%, and 83.3%) in public, private, and experimental schools respectively. According to the classrooms' SP, it was the highest at private schools (69% moderate), followed by experimental (65% moderate) and public (46% poor) ones (Figure 1), and it was statistically significant (p≤0.05, at C.I.=95%) at different school types. Additional statistical analysis declared significant differences between public and private as well as between public and experimental schools (p≤0.05, at C.I.=95%). RESULTS % Area available for each student is non-compliant 19 59.4 7 43.8 6 50.0 Classroom Conditions Board is not maintained regularly 14 59.4 1 6.3 1 8.3 Classroom is crowded 19 59.4 7 43.8 6 50.0 Illumination source is not clean 17 53.1 0 0.0 0 0.0 There is glare on board 24 75.0 8 50.0 9 75.0 Windows are not clean 24 75.0 0 0.0 8 66.7 Windows are not maintained periodically 12 37.5 1 6.3 4 33.3 There is broken glass windows 18 56.3 4 25.0 0 0.0 There is odor in the classroom 17 53.1 3 18.8 4 33.3 Classroom Safety and emergency preparedness Absence of emergency exits 32 100.0 10 62.5 10 83.3 Absence of emergency routes 32 100.0 10 62.5 10 83.3 Absence of fire extinguishers in the class 32 100.0 11 68.8 10 83.3 Absence of alarm point within the class 32 100.0 14 87.5 10 83.3 There are Obstacles in the classroom 1 3.1 2 12.5 2 16.7 There are extra cables in the classroom 4 12.5 4 25.0 2 16.7 There are storage area in the classroom 0 0.0 3 18.8 2 16.7 All classroom equipment is not checked on a regular basis 21 65.6 6 37.5 2 16.7 First aid measures is absent 28 87.5 10 62.5 2 16.7 Classroom is not vacuumed regularly 19 59.4 4 25.0 2 16.7 Blackboards/whiteboards are not cleaned properly 8 25.0 0 0.0 2 16.7 Trash is not removed daily 21 67.7 0 0.0 3 9.0 Food is kept in the classroom overnight 21 65.6 10 62.5 4 33.3 Desks and lockers are not cleaned regularly 27 84.4 4 25.0 8 66.7 Indoor air quality On whiteboards, markers that release high levels of volatile organic compounds (VOCs) are used 1 4.8 0 0.0 1 8.3 Windows to class area ratio is not compliant 23 71.9 12 75.0 7 58.3 Windows are not facing each other's 7 21.9 5 31.3 0 0.0 DISCUSSION safety performance (SP) is essential for safety safety performance (SP) is essential for safety management,(3) which is necessary for improving SP.(12) The SP in public schools, and classrooms were poor. This can be explained based on two factors, including safety expenditure, and lack of safety rules' enforcement. The first is too high as compared with DISCUSSION This study assessed the presence and application of the minimal safety standards in school buildings of different types. Ensuring school safety is a very important public health issue worldwide. Evaluation of Zaki et al. 83 the economic conditions in the Egypt. So, the private schools had the highest SP followed by experimental and public and ones. Safety rules in Egypt are enforced by the Ministry of Manpower and Immigration, which grants permits and approvals for any foundation after ensuring compliance with the minimal safety rules. Previously, it granted these permissions routinely for governmental foundations, but now it becomes stricter with both governmental and private institutions. earthquakes respectively, while the unusual crises were deaths of staff and students at school, and terrorism.(22) Unsafe grounds may lead to falling, tripping, or slipping. The ground safety category revealed satisfactory SP for experimental and private schools, and poor for public ones. The most common unsafe situation was "the unmarked tripping/slipping locations." In accordance with these results, the findings revealed from a project conducted in Kenyatta University to assess school safety reported that about 17% of school accidents occur due to ground problems, and more than one-quarter of the students' injuries are on the ground.(23) There is strong relationship between housekeeping and safety performance as stated in two Finnish studies 1989, and 2014.(13, 14) The housekeeping showed good SP in private, satisfactory in experimental, and poor in public schools. The major frequent causes of low SP in the present study were "the absence of protective measures against vectors and insects," and "non-daily refuse disposal." A Saudi study 1998 conducted safety inspection in large and small construction projects. It concluded that the housekeeping safety scores of large projects were good, while that of small projects were poor.(15) The classrooms' SP was moderate for private and experimental schools and poor for public ones. The most recurrent unsafe classroom situations at the classroom conditions, safety and emergency preparedness, and indoor air quality categories were "the on-board glare," "the absence of classroom alarm point," and "the non-compliant windows to class area ratios" respectively. Two studies conducted in Swedish and Arizona revealed the positive impact of classroom safety on the students' achievements and their ability to learn.(24, 25) Designers and managers must consider the fire- safety standards to ensure sustainability.(16) In this study; the fire SP was moderate for private schools, and poor for experimental and public ones. DISCUSSION The most common fire hazards were "the irregular fire drills" as well as "the absence of the alarm system." In compliance with the present study, a Chinese study (1999) developed a fire-safety assessment system for existing buildings, and applied it on residence in Hong Kong. It found that the low safety score was observed for the alarm system.(17) In contrary to the present study, A US study 2009 checked 1052 public schools in Texas and revealed that the most frequent cause of reduced fire safety was the absence of fire sprinkler, and the absence of the alarm system was common in just 5.4% of the public schools.(18) CONCLUSIONS AND RECOMMENDATIONS The findings of the present study can lead us to conclude that there are many safety violations that occur in Alexandria primary schools. This causes reduction of the safety performance and lack of safety management. Safety performances of private schools are better than that in experimental and public ones. Emergency preparedness category is of the least SP, while housekeeping is of the highest in the three school types. It is recommended to activate the licensing and inspection roles of the Ministry of Manpower and Immigration to enable enforcement of safety standards. In addition, the decision makers in the Ministry of Education must train and motivate the health and safety committee for each school to implement the school safety standards. Moreover, they must provide the suitable financial allocations necessary for safety equipment. Also, they had to consider categories, including housekeeping, maintenance, fire safety, electrical safety, emergency preparedness, ground safety, and classroom safety. The electrical safety performances were poor in the three school types. The unsafe electric situation of the highest popularity was "the non-earthed electrical equipment," which is an actual problem of non- industrial buildings in Egypt. In agreement with the present work, the results of a study conducted in Tampere University of Technology 2010 revealed that the most common cause of electrical accidents was the failure to earth the electrical equipment.(19) The SP of the emergency preparedness was poor at the three school types. The most frequent violations were "the absence of emergency planning manual," and "the lack of evacuation maps." Similarly, the main emergency problems in Saudi and Turkish schools were the absence of emergency plan, long response time to incidents, and lack of emergency training.(20, 21) The New Zealand study (2017) revealed that the most common emergencies were due to weather, fires, and REFERENCES 13. Saari J, Näsänen M. The effect of positive feedback on industrial housekeeping and accidents; A long-term study at a shipyard. International Journal of Industrial Ergonomics. 1989;4(3):201-11. 1. Organization WH. School and youth health: Global school health initiative. Geneva: WHO. www. who. int/school youth health/gshi/hps/en; 1995 int/school_youth_health/gshi/hps/en; 1995. int/school_youth_health/gshi/hps/en; 1995. g 14. Leino A, Heinonen R, Kiurula M, editors. Improving safety performance through 5s program. Proc 22nd Ann Conf of the Int’l Group for Lean Construction; 2014. 2. Organization WH. The physical school environment: an essential element of a health-promoting school. 2004. 3. Hinze J, Thurman S, Wehle A. Leading indicators of construction safety performance. Safety science. 2013;51(1):23-8. 15. Jannadi MO, Assaf S. Safety assessment in the built environment of Saudi Arabia. Safety Science. 1998;29(1):15-24. 4. Lwanga SK, Lemeshow S. Sample size determination in health studies: a practical manual. Switzerland: WHO Geneva; 1991. Available from: http://www.who.int/iris/handle/10665/40062. 16. Buchanan AH, Abu AK. Structural design for fire safety: John Wiley & Sons; 2017. 17. Lo SM. A fire safety assessment system for existing buildings. Fire Technology. 1999;35(2):131-52. 5. Health and safety checklist for classrooms [Internet]. [cited February 7, 2018]. Available from: http://www.hse.gov.uk/risk/ classroom-checklist.pdf. 18. Shelton AJ, Owens EW, Song H. An examination of public school safety measures across geographic settings. Journal of school health. 2009;79(1):24-9. 6. Verginia Department of Education. 2016 School Safety Inspection Checklist for Virginia Public School. Virginia Virginia Department of Education 2016. [cited May 23, 2018]. Available from: https://www.dcjs.virginia.gov/sites/dcjs virginia.gov/files/publications/law-enforcement/school-safety- inspection-checklist_0.pdf 19. Tulonen T. Electrical accident risks in electrical work. Tukes- Publication. 2010;2:2010. 20. Alolah T, Stewart RA, Panuwatwanich K, Mohamed S. Determining the causal relationships among balanced scorecard perspectives on school safety performance: Case of Saudi Arabia. Accident Analysis & Prevention. 2014;68:57-74. p p 7. Self-Inspection Safety Checklist for Schools and Educational Facilities [Internet]. Church Mutual Insurance Company. 2011 [cited February 7, 2018]. Available from: https://www.churchmutual.com/media/safetyResources/files/Self _Inspection_Checklist_Schools.pdf. ا Accident Analysis & Prevention. 2014;68:57-74. 21. Tabancalı E, Bektaş T. Student safety in primary schools: A sample of Büyükçekmece county. Procedia - Social and Behavioral Sciences. 2009;1(1):281-4. 22. Tipler K, Tarrant R, Johnston D, Tuffin K. Are you ready? Emergency preparedness in New Zealand schools. International Journal of Disaster Risk Reduction. 2017;25:324-33. _ p _ _ p 8. التعليمية اال. معايير واشتراطات صالحية المواقع والمباني المدرسية بمدارس )التعليم األساسي والثانوي العام (بالمدن والقرى القائمة. In: والدراسات اال, editor. القاهرة مصر: مطبعة الهيئة ا ;لعامة لألبنية التعليمية2011 . 23. Nyakundi ZO. Conflict of interest: None to declare. Conflict of interest: None to declare. Conflict of interest: None to declare. 12. Kin RYC. The Impact of Safety Management Systems on Safety Performance: Commercial Aviation Operations: Griffith University; 2015. Journal of High Institute of Public Health 2018;48(2):77-84. Journal of High Institute of Public Health 2018;48(2):77-84. 84 ACKNOWLEDGEMENTS Great thanks and appreciation are presented to the decision makers in the Ministry of Education in Alexandria for their enlightened minds and permission to conduct the study. Unlimited gratitude is awarded to the school managers, who accepted to make their schools included in this study. REFERENCES Implementation of safety standards and guidelines in public secondary schools in Marani District, Kisii County, Kenya 2012. 9. Mendoza JA, Watson K, Baranowski T, Nicklas TA, Uscanga DK, Hanfling MJ. Validity of instruments to assess students' travel and pedestrian safety. BMC Public Health. 2010;10(1):257-63. 24. Rosén M. On the Importance of a Safe School and Classroom Climate for Student Achievement in Reading Literacy. The 4th IEA International Research Conference; Göteborg, 2009. p. 1-14. 10. Ott RL, Longnecker M. An Introduction to Statistical Methods and Data Analysis. USA: Cengage Learning, Inc.; 2010. 25. Holley LC, Steiner S. Safe space: Student perspectives on classroom environment. Journal of Social Work Education. 2005;41(1):49-64. 11. Rosner B. Fundamentals of biostatistics. USA: Cengage Learning, Inc.; 2011.
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Russia’s Sovereignty and Emergence of Pragmatic Polycentrism
Upravlenie i politika
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2022, Vol. 1, No. 1 Received: February 15, 2022 / Accepted: March 10, 2022 Political Science / Country in Focus 2022, Vol. 1, No. 1 Received: February 15, 2022 / Accepted: March 10, 2022 Political Science / Country in Focus 2022, Vol. 1, No. 1 Received: February 15, 2022 / Accepted: March 10, 2022 Political Science / Country in Focus Governance and Politics UDC: 323, 327, 369.032 1 Ladislav Zemánek – PhDr., Analyst and Researcher, Faculty of Arts, Institute of World History, Charles University, Czech Republic E-mail: zemanek.ml@gmail.com The author declares the absence of conflict of interest Russia’s Sovereignty and Emergence of Pragmatic Polycentrism LADISLAV ZEMÁNEK1 Charles University (Czech Republic) LADISLAV ZEMÁNEK1 Abstract The article researches Russia’s concept of comprehensive sovereignty that is a cornerstone of the official state paradigm. Sovereignty is analyzed in its internal as well as external dimen- sion, both synchronically and diachronically. The concept is interconnected with national secu- rity expanding into different realms. Sovereignty-based securitization tendencies are examined through methods of discursive analysis based upon constructivist assumptions. The present study puts emphasis on conceptual constructions and discursive practices significantly affecting individual perceptions, interpretation and Weltanschauung of political leadership as a whole, thereby shaping behavior, strategies and policies of individual actors concerned. The analy- sis reveals an affinity between concepts and discursive practices of the actors included in the research – Russia, the United States, China, and the European Union. It manifests itself in the concepts of comprehensive sovereignty, America First, dual circulation, and strategic autonomy respectively. These strategies are examined in relation to the transformation of the global order from US-led hegemonism towards polycentrism. The author draws attention to the interactions within the “quadrilateral” consisting of Russia, China, the EU and the US in order to demonstrate the emergence and dynamics of autonomization or regionalization, which is to be regarded as a dialectical moment in the globalization process towards glocalization and pragmatic polycen- trism. The author concludes that the EU’s strategic autonomy is a positive feature, but requires the abandonment of Euro-Atlanticism, which thwarts restructuring the Eurasian macroregion in general, and settling Russia-Europe relations in particular. Key words Russia, United States, China, European Union, sovereignty, national security, polycentrism, Eu- ro-Atlanticism, strategic autonomy, hegemonism For citation Ladislav Zemánek (2022). Russia’s Sovereignty and Emergence of Pragmatic Polycentrism. Gov- ernance and Politics, 1(1), P. 63–99. Key words Russia, United States, China, European Union, sovereignty, national security, polycentrism, Eu- ro-Atlanticism, strategic autonomy, hegemonism 2 Presidential Address to Federal Assembly. Kremlin, 2019, February 20. URL: http://en.kremlin.ru/events/president/ news/59863 (accessed 20.02.2022) For citation For citation Ladislav Zemánek (2022). Russia’s Sovereignty and Emergence of Pragmatic Polycentrism. Gov- ernance and Politics, 1(1), P. 63–99. For citation Ladislav Zemánek (2022). Russia’s Sovereignty and Emergence of Pragmatic Polycentrism. Gov- ernance and Politics, 1(1), P. 63–99. 63 Ladislav Zemánek “Without sovereignty, Russia cannot be a state. Some countries can do this, but not Russia.” These words were spoken by President Vladimir Putin during his address to the Federal Assembly in 2019.2 Sovereignty is a crucial concept of Russia’s hegemonic paradigm. It is of a comprehensive nature, not limited to the realm of security or international policy. This concept is an analogous phe- nomenon to EU’s strategic autonomy, China’s dual circulation or Donald Trump’s America First policy. All these strategies are symptoms of the transformation of the global order towards a polycentric model, thus embodying complex dialec- tics of globalization and deglobalization, integration and regionalization. Global restructuring is of utmost relevance and significance in terms of contemporary socio-economic as well as political processes, also affecting the very concept of governance. The present article examines Russia’s concept of sovereignty from both in- ternal and external perspectives. A detailed discursive analysis is made focusing on basic normative documents (Military Doctrine, National Security Strategy), bilateral diplomatic agreements and declarations (Joint Statement on the Twen- tieth Anniversary of the Treaty of Good Neighborliness and Friendly Cooperation between the Russian Federation and the People’s Republic of China; Joint Statement on the International Relations Entering a New Era and the Global Sustainable Development), as well as articles and statements of the highest-ranking repre- sentatives of the relevant sides (Vladimir Putin, Sergey Lavrov, Dmitry Med- vedev, Donald Trump, Mike Pompeo, Joe Biden and Josep Borrell). The chosen method is based upon a constructivist assumption of the importance of ideas and ideology in social life including politics (Peter Berger and Thomas Luck- man, Alexander Wendt). The discursivity-oriented research can be effectively combined with related analyses of the interstate interactions, international re- lations and practical behavior of individual actors concerned. Both diachron- ic and synchronic methods are employed in order to shed light on the ongo- ing processes under investigation. For citation In the article, a multidisciplinary attitude is adopted, making use of findings from political science and philosophy (Glenn Diesen, Alexander Lukin, Carl Schmitt, Dmitry Trenin), international relations Governance and Politics Governance and Politics 64 UDC: 323, 327, 369.032 Political Science / Country in Focus (Richard Sakwa, Alexander Sergunin, Dmitry Suslov, Ivan Timofeev), history (Richard Pipes, Andrei Tsygankov), and last but not least social anthropology (Chris Hann). The analysis begins with the internal model of Russia’s comprehensive sovereignty and its constitutive elements in different fields, proceeding to the external model. The inquiry is therefore put into the context of relations with the United States, China, and the European Union on the one hand, and of the increasingly strong move towards polycentrism on the other. The article thus presents an in-depth insight into the status quo of Russia’s state paradigm, based upon the concept of sovereignty, contributing to a better understanding of the metamorphoses of global politics and governance. p // / / / ( ) 4 Voennaia doktrina Rossiiskoi Federatsii (Russia’s Military Doctrine). Kremlin. 2014, December 19. http://static.kremlin.ru/ media/events/files/41d527556bec8deb3530.pdf (accessed 20.02.2022) 3 Kontseptsiia vneshnei politiki Rossiiskoi Federatsii (The Concept of Russian Foreign Policy). Kremlin. 2016, November 30. http://www.kremlin.ru/acts/bank/41451 (accessed 20.02.2022) 3 Kontseptsiia vneshnei politiki Rossiiskoi Federatsii (The Concept of Russian Foreign Policy). Kremlin. 2016, November 30. http://www.kremlin.ru/acts/bank/41451 (accessed 20.02.2022) 4 Voennaia doktrina Rossiiskoi Federatsii (Russia’s Military Doctrine). Kremlin. 2014, December 19. http://static.kremlin.ru/ media/events/files/41d527556bec8deb3530 pdf (accessed 20 02 2022) Kontseptsiia vneshnei politiki Rossiiskoi Federatsii (The Concept of Russian Foreign Policy). Kremlin. 2016, November 30. http://www.kremlin.ru/acts/bank/41451 (accessed 20.02.2022) 4 Voennaia doktrina Rossiiskoi Federatsii (Russia’s Military Doctrine). Kremlin. 2014, December 19. http://static.kremlin.ru/ media/events/files/41d527556bec8deb3530.pdf (accessed 20.02.2022) Security dimension of sovereignty The current state of international relations and development of the global order is characterized by growing tensions, conflicts and rivalry following from competition between the US, as the declining hegemon of the unipolar system that emerged after the end of the Cold War, and the non-Western powers, first and foremost China and Russia. The objective decline in Western states’ poten- tial and the concurrent shift of the political and economic center to the East arouse defiance in the West. From the Russian perspective, the existing system of international relations and security is unfair, discriminating against the rival emerging powers.3 Irrespective of the frictions in Russia’s near neighborhood (Ukraine, Belarus, the Caucasus or Central Asia) and a long-term confronta- tional policy from the West including large-scale sanctions, the Russian leader- ship does not expect a full-fledged war against the country.4 The perception of threats plays a serious role in the leadership’s deci- sion-making. These threats are formulated in the Military Doctrine as a sub- stantial part of the state’s strategic planning. The current version was adopted in 2014. The most serious external threat is seen in NATO, its expansion and vest- 2022, Vol. 1, No. 1 65 Ladislav Zemánek ing the organization with global functions regardless of the mandate of the UN.5 The tension between Russia and NATO is a permanent feature of international politics, which especially endangers European countries. While the Russian side is not against further enlargement of the EU in principle, perceiving the Euro- pean integration project as legitimate and analogous to Eurasian integration in the post-Soviet space and even a model for the EAEU in a sense, it has rejected US-led NATO policies and activities for a long period (Sergunin, 2016, p. 154, 162). The origins of the current controversies lie in the Gorbachev era (Gvozdev & Marsh, 2014, p. 78). Boris Yeltsin allowed for expansion only if Russia itself was included (Gvozdev & Marsh, 2014, p. 211), thus anticipating future projects of a single security area comprising North America, Europe and Russia (Dmitry Medvedev) or common military systems (Vladimir Putin). From a comparative perspective, Russia respects the development of the EU while disapproving of NATO. It follows that the main obstacle for security and stability between Rus- sia and Europe lies in Washington’s hegemonic interests discursively justified by a concept of the Euro-Atlantic community. 5 Ibid. 6 Voennaia doktrina Rossiiskoi Federatsii (Russia’s Military Doctrine). Kremlin. 2014, December 19. http://static.kremlin.ru/ media/events/files/41d527556bec8deb3530.pdf (accessed 20.02.2022) 7 Ibid. 5 Ibid. Governance and Politics Security dimension of sovereignty The second major threat is seen in the destabilization of individual coun- tries and whole regions.6 Undoubtedly, the Russian strategists have the practice of color revolutions in mind. This point can also be interpreted as a criticism of the export of liberal democracy which is connected with Western exception- alism and the false self-perception of this model as a universal one that is to be established globally disregarding specific local conditions. Whereas the first defined threat regards the military practice, the second one applies to the po- litical practice carried out by the Western countries. Another risk is seen in the deployment of military contingents on the territories of the adjacent or allied countries as well as in the expansion of missile systems, undermining agree- ments on the non-proliferation of nuclear weapons, development of the global strike concept or militarization of outer space.7 The current Military Doctrine anticipates that nuclear weaponry will safe- guard the role of deterrence and prevention of an outbreak of full-fledged as well Governance and Politics Governance and Politics 66 Political Science / Country in Focus UDC: 323, 327, 369.032 as limited military conflicts. Therefore, the Russian Federation has been inter- ested in the maintenance of the Strategic Arms Reduction Treaty (New START) as the cornerstone of the stability of the international regime of nuclear arms. The Treaty was signed in Prague in 2010 as a sequel of quite a long history of negotiations between Moscow and Washington in the field of non-proliferation, control and reduction of strategic weapons, which started in the 1960s with the multilateral Partial Test Ban Treaty (PTBT), Nuclear Non-Proliferation Treaty (NPT) and the bilateral Interim Agreement on Offensive Arms (SALT I) from 1972. There have been several bilateral agreements since then. The collapse of the USSR did not make Russia-US treaties irrelevant, as the Russian Federation took over a substantial part of the Soviet military arsenal. Even though overall Russia’s role diminished in the newly emerged unipolar world dominated by the US as a global hegemon in the 1990s, the country remained a military great power. As such, Russia has played a crucial role in the balance and stability of international security, endeavoring to preserve the status quo. However, the security regime was put in danger during Donald Trump’s presidency. ( ) 9 Sanger, D. E., & Broad, W. J. U.S. to Tell Russia It Is Leaving Landmark I.N.F. Treaty. New York Times. 2019, October 19. URL: https://www.nytimes.com/2018/10/19/us/politics/russia-nuclear-arms-treaty-trump-administration.html (accessed 20.02.2022) 8 Marcus, J. INF Nuclear Treaty: US Pulls out of Cold War-era Pact with Russia. BBC, 2019, August 02. URL: https://www.bbc. com/news/world-us-canada-49198565 (accessed 20.02.2022) 8 Marcus, J. INF Nuclear Treaty: US Pulls out of Cold War-era Pact with Russia. BBC, 2019, August 02. URL: https://www.bbc. com/news/world-us-canada-49198565 (accessed 20.02.2022) 9 Sanger, D. E., & Broad, W. J. U.S. to Tell Russia It Is Leaving Landmark I.N.F. Treaty. New York Times. 2019, October 19. URL: https://www.nytimes.com/2018/10/19/us/politics/russia-nuclear-arms-treaty-trump-administration.html (accessed 20.02.2022) 2022, Vol. 1, No. 1 8 Marcus, J. INF Nuclear Treaty: US Pulls out of Cold War-era Pact with Russia. BBC, 2019, August 02. URL: htt com/news/world us canada 49198565 (accessed 20 02 2022) Security dimension of sovereignty From the beginning, Trump took a stand against the bilateral agree- ments in the field, similarly as in the case of the multilateral Joint Comprehensive Plan of Action, regarding the Iranian nuclear program. In 2019, the US with- drew from the Intermediate-Range Nuclear Forces Treaty (INF Treaty) signed by Mikhail Gorbachev and Ronald Reagan in 1987. This step provoked concerns about a situation when no arms control limits would be in force. Washington ac- cused Russia of breaching the treaty, which Moscow denied.8 The real reason be- hind the US attack on the INF Treaty was, nevertheless, connected with growing tensions with China, with the allegations against Russia’s “transgressions” being rather a pretext than anything else. As China was not included in the treaty, it was free to develop and deploy intermediate-range nuclear missiles.9 Trump attempted to establish new security regimes instead of the existing ones, which were considered disadvantageous for the US as putting other rele- 2022, Vol. 1, No. 1 67 Ladislav Zemánek vant actors, first and foremost China, aside. It was not only a question of the INF Treaty but also of the New START. The latter was to expire in February 2021. Under the circumstances when the US withdrew from the INF Treaty and added new conditions to agree with the prolongation, including a redefinition of the agreement and engaging China, it seemed very unlikely that the New START would be extended. All the more so that Washington refused Moscow’s sugges- tions, for instance, temporary extension without any preconditions.10 When the current US President Joe Biden assumed the presidency, it was clear that there would be no quick substantial turn in the relationship with Russia. Standing as a candidate, Biden wrote about the need for countering “Putin’s kleptocratic au- thoritarian system”.11 In his first presidential speech dedicated to foreign affairs, Biden accused Russia of aggression including interference in the US election, conducting cyber-attacks or poisoning its citizens12. In spite of the confronta- tional policy, the new administration avoided the imminent threat of the arms race, supporting the prolongation of the New START, thus manifesting the US readiness to find common ground at least as far as most risk issues are con- cerned. The treaty would probably not have been extended until 2026 without Biden becoming President. The Russian leadership has put a strong emphasis on the use of nuclear arms with regard to the deterrence policy. ) 12 Biden, J. R. Remarks by President Biden on America’s Place in the World. White House, 2021, February 04. URL: https:// www.whitehouse.gov/briefing-room/speeches-remarks/2021/02/04/remarks-by-president-biden-on-americas-place-in- the-world/ (accessed 20.02.2022); Crane, M. Biden Agrees Putin Is a Killer, Says He’ll Pay for Meddling. Bloomberg, 2021, March 17. URL https://www.bloomberg.com/news/articles/2021-03-17/biden-agrees-putin-is-killer-says-he-ll-pay-price- for-meddling (accessed 20.02.2022) 13 Osnovy gosudarstvennoi politiki Rossiiskoi Federatsii v oblasti iadernogo sderzhivaniia (Foundations of State Policy of the Russian Federation in the Area of Nuclear Deterrence). Kremlin. 2020, June 02. URL: http://static.kremlin.ru/media/events/i 10 TASS. 2020, October 16. URL: https://tass.ru/politika/9738037 (accessed 20.02.2022) , p // /p / ( ) 11 Biden, J. R. Why America Must Lead Again. Rescuing U.S. Foreign Policy After Trump. Foreign Affairs, 2020, March/ April. URL: https://www.foreignaffairs.com/articles/united-states/2020-01-23/why-america-must-lead-again (accessed 20.02.2022) 10 TASS. 2020, October 16. URL: https://tass.ru/politika/9738037 (accessed 20.02.2022) 11 Biden, J. R. Why America Must Lead Again. Rescuing U.S. Foreign Policy After Trump. Foreign Affairs, 2020, March/ April. URL: https://www.foreignaffairs.com/articles/united-states/2020-01-23/why-america-must-lead-again (accessed 20.02.2022) 12 Biden, J. R. Remarks by President Biden on America’s Place in the World. White House, 2021, February 04. URL: https:// www.whitehouse.gov/briefing-room/speeches-remarks/2021/02/04/remarks-by-president-biden-on-americas-place-in- the-world/ (accessed 20.02.2022); Crane, M. Biden Agrees Putin Is a Killer, Says He’ll Pay for Meddling. Bloomberg, 2021, March 17. URL https://www.bloomberg.com/news/articles/2021-03-17/biden-agrees-putin-is-killer-says-he-ll-pay-price- for-meddling (accessed 20.02.2022) 13 Osnovy gosudarstvennoi politiki Rossiiskoi Federatsii v oblasti iadernogo sderzhivaniia (Foundations of State Policy of the Russian Federation in the Area of Nuclear Deterrence). Kremlin. 2020, June 02. URL: http://static.kremlin.ru/media/events/ files/ru/IluTKhAiabLzOBjIfBSvu4q3bcl7AXd7.pdf (accessed 20.02.2022) g ( ) 13 Osnovy gosudarstvennoi politiki Rossiiskoi Federatsii v oblasti iadernogo sderzhivaniia (Foundations of State Policy of the Russian Federation in the Area of Nuclear Deterrence). Kremlin. 2020, June 02. URL: http://static.kremlin.ru/media/events/ files/ru/IluTKhAiabLzOBjIfBSvu4q3bcl7AXd7.pdf (accessed 20.02.2022) Governance and Politics Security dimension of sovereignty President Putin signed a strategic docu- ment dedicated to this area which was made public for the first time ever in 2020. In the new Foundations of State Policy of the Russian Federation in the Area of Nuclear Deterrence, the nuclear arsenal is considered a crucial element for Russia’s sovereignty.13 The strategy postulates a strictly defensive character of the latter. At the same time it allows the active use of nuclear arms but solely in the case of nuclear attack against the country or its allies, as well as in case Governance and Politics 68 Political Science / Country in Focus UDC: 323, 327, 369.032 of any aggression endangering the existence of the Russian state.14 Similarly, ar- ticle 22 of the Military Doctrine enables employing the Armed Forces in the case of aggression against Russia or allies, in the case of the decision of the UN Security Council or other collective security bodies and, last but not least, in the case of need for protection of Russian citizens abroad.15 Article 31 expands the legitimacy of the external military intervention if this is carried out in line with principles and norms of international law, international agreements and Russian legislation. These provisions might be used to active military actions abroad, making the defensive posture more offensive. Russia’s action readiness was demonstrated especially in Syria and Crimea in the last years. Russia has been endeavoring to strengthen its security, protect the sovereignty and multiply military potential through cooperation with other partners, first and foremost within the Collective Security Treaty Organization (CSTO), the Commonwealth of Independent States (CIS) and the Shanghai Cooperation Organization (SCO). The allies sensu stricto are countries partic- ipating in the CSTO which is a military alliance, unlike two other multilateral institutions. The asset of the SCO lies in the fact that transcends the limited post-Soviet area including a wide array of Eurasian countries and provides the states with an opportunity to interconnect and converge national development strategies as well as grand initiatives as the Belt and Road Initiative or the Eura- sian Economic Union (Kaczmarski, 2017, p. 1036). The significance of the SCO consists in the fact that the member states generate more than 20% of the global GDP, accounting for virtually 42% of the world’s population. 14 Ibid. 15 Voennaia doktrina Rossiiskoi Federatsii (Russia’s Military Doctrine). Kremlin. 2014, December 19. http://static.kremlin.ru/ media/events/files/41d527556bec8deb3530.pdf (accessed 20.02.2022) 14 Ibid. Security dimension of sovereignty It is the most im- portant non-Western regional organization which can contribute considerably to reshaping the global order according to the reformist agenda of the partici- pants (Zemánek, 2020, p. 202). Naturally, the leading role has been played by the engines of Eurasian inte- gration – Russia and China. Moscow found common ground with Beijing as far as the fight against terrorism, separatism and extremism are concerned (Chi- na calls these threats “The Three Evils”). Security and military cooperation be- 2022, Vol. 1, No. 1 69 Ladislav Zemánek tween Russia and China have been deepening, especially after 2014. Both major powers reject the idea of establishment of the alliance, thus clearly demonstrat- ing an innovative attitude towards interstate relations and cooperation, based on openness, overlapping interests and heterogeneous integration model, the basis of which lies in different speed and levels of integration in individual areas, sometimes even going beyond the traditional alliance (Lukin, 2018b, p. 128). Both countries are aware of the Western hegemonic ambitions, export of liberal democracy and destabilizing actions in relation to the countries with different values and political systems. The SCO, therefore, serves to the deterrence of the Western quasi-universalism and gradual transformation of the international order to be based on polycentrism, equality, inclusiveness and the principles of peaceful coexistence, the prerequisite of which is the very existence of different models and a plurality of development as well as modernization patterns. Rus- sia’s sovereignty can be protected in a more effective way through the above- mentioned institutions. Paradoxically, engagement in supranational organiza- tions and integration, thus openness and not introversion, may become the way towards stronger sovereignty and national security for Russia. 16 The 2021 National Security Strategy leaves the term “strategic partnership” of the 2015 NSS out, replacing it with “win-win international cooperation”. Comprehensive sovereignty: an internal perspective The principle of sovereignty is enshrined in strategic documents in a wide array of fields. It applies not only to national security, external relations or econ- omy but also to the energy sector, information security or cultural policy. Its significance, therefore, cannot be overestimated, sovereignty being a quintes- sence of the current Russian state and hegemonic paradigm. Russia’s concept of sovereignty can be interpreted as based on the threefold principle of strate- gic autonomy/independence, strategic stability and strategic partnership.16 These categories define both internal and external dimensions of the existence of the state. The National Security Strategy (hereinafter referred to as NSS) follows the tradition of patrimonialism and etatism (see: Pipes, 1974, p. 24) when defining the need for strengthening the power of the state to accomplish social security. Governance and Politics Governance and Politics 70 Political Science / Country in Focus UDC: 323, 327, 369.032 Such an attitude preserves the crucial role of the power vertical and its respon- sibility for addressing people’s interests. The strong role of the vertical is also to secure protection against external interference which is frequently thematized in different contexts by the NSS. An emphasis is put on the threat posed by foreign intelligence services and other foreign organizations, terrorist, extrem- ist and radical forces undermining the sovereignty, territorial integrity, political and social stability or traditional religious and moral Russian values. Besides, the strong state vertical is to protect citizens against external propaganda as well as the one carried out by “inner enemies”.17 Such a strategy clearly demonstrates that the elites consider the strong state, its apparatus and institutions as a key to maintaining the country’s sovereignty. Economic sovereignty is an inseparable part of the political one. The impor- tant point is a crucial role ascribed to the state – analogous to its role in social development. A strong role of the state in the economic processes is a constant of Russian history (Tsygankov, 2014, p. 7). Neither the free market nor the We- berian protestant ethic has been characteristic of Russian society. On the contra- ry, economy was perceived as a secondary social institution (always subjugated either to religious principles or political and social goals) in the pre-revolution- ary epoch. Even the history of the Russian socialist and communist movement is suffused with the adoration of the state and its role, despite the Marxist theory of the economic base and social superstructure. i 18 Not by coincidence, the first Soviet People’s Commissar for Education Anatolii Lunacharskii propounded a specific theory of bogostroitel’stvo, “God-Building”, emphasizing that socialism is the most religious of all religions. See: Lunacharskii (1908). 17 Strategiia natsional’noi bezopasnosti Rossiiskoi Federatsii (National Security Strategy). Kremlin. 2015, December 31. URL: http://static.kremlin.ru/media/acts/files/0001201512310038.pdf (accessed 20.02.2022)ii Comprehensive sovereignty: an internal perspective The Bolsheviks did not win because of the economic conditions – they win because of their political will, credo and quasi-religious zeal.18 The Soviet era further deepened the embedded Russian mistrust of market mechanisms and entrepreneurship. Russia’s civiliza- tional trajectory confirms the observations presented by social anthropologist Chris Hann, according to whom the Eurasian societies have been characteristic of “inclusive embeddedness”, the economy being subordinated to broader social goals and needs (Hann, 2016, p. 4). Similarly, the Soviet system was nothing absolutely extraneous, but at least partially followed older traditions and devel- opmental tendencies based on the strong role played by the Russian state. 2022, Vol. 1, No. 1 71 Ladislav Zemánek The principles of central planning, the state’s control over the economic ac- tivities, priority of political goals as well as the dominant position of the state in the modernization and reforms were presented in different forms both in the pre-revolutionary Russia and the Soviet Union. One can conclude that the So- viet experiment radicalized older patterns, reinterpreting them in a modernist way. However, a different perspective also existed. Reformists Sergei Witte and Pyotr Stolypin started to pave the way for Russia with the middle class, more inclusive and participatory model with wider space for individual activities and market mechanisms. In post-Soviet Russia, Dmitry Medvedev followed this line. Amidst the economic recession in 2009, he formulated a modernization program in which the Russian President harshly criticized economic backward- ness, commodity dependence, low productivity and innovations or fragility of democratic institutions. Medvedev rejected paternalism and historic forms of Russian modernization connected with Peter I and the Bolsheviks. The new program was to develop Russian democracy and establish a new, post-industri- al economy.19 The liberal ethos of the three mentioned reformists did not deny local peculiarities or conservative moral values arising from the Orthodoxy but represented a more open, more liberal model of Russia’s modernization as well as external behavior. Both at the beginning of the 20th century and in the 2010s, this reformist shift was interrupted due to the external factors – the Great War, which was joined by the Russian Empire with an utter reluctance, and the color revolu- tion in Ukraine in 2014, the subsequent anti-Russian campaign launched by the Western countries and deteriorating relations. Vladimir Putin has accentuated different elements, but the policies throughout his terms have not been incom- patible with the reformist modernization agenda essentially. 19 Medvedev, D. Go Russia! Kremlin. 2009, September 10. URL: http://en.kremlin.ru/events/president/news/5413 (accessed 20.02.2022) 20 According to President Putin, the Internet and media should be regulated not only by the positive legal norms but also by moral, natural law. Putin Calls for Internet Bound by Moral Rules, Criticizes Opposition Rallies. Reuters. 2021, March 04. URL: https://www.reuters.com/article/us-russia-internet-idUSKCN2AW2D4 (accessed 20.02.2022); Priniat zakon o «suver- ennom internete» (Adopting Sovereign Internet Law). Gosudarstvennaia Duma. 2019, April 16. URL: http://duma.gov.ru/ news/44551/ (accessed 20.02.2022)i 22 Russia to Launch Own TikTok Developed With Putin’s Alleged Daughter. AFP. 2020, December 23. URL: https://www. themoscowtimes.com/2020/12/23/russia-to-launch-own-tiktok-developed-with-putins-alleged-daughter-a72467 (ac- cessed 20.02.2022); Rossiiskii analog “Vikipedii” oboidetsia biudzhetu RF pochti v 2 mldr rublei (Russian analogous Wikipe- dia will cost 2 bln rubles). Interfax. 2019, September 26. URL: https://www.interfax.ru/russia/677964 (accessed 20.02.2022) n, Q. China, Russia to Form Closer Partnership on 5G in Defiance of US. Global Times. 2020, August 24. URL: https w.globaltimes.cn/content/1198654.shtml (accessed 20.02.2022) 2022, Vol. 1, No. 1 Comprehensive sovereignty: an internal perspective Putin’s program has also been aimed at integration in the global economy, acceleration of the economic growth, progressive development of the national economy, support for the business environment, and the political system development. The agen- da, nevertheless, gradually became more securitized and the key goal was not “mild” Westernization and building the Greater Europe anymore but protec- /en.kremlin.ru/events/president/news/5413 (accessed Governance and Politics Governance and Politics 72 UDC: 323, 327, 369.032 Political Science / Country in Focus tion of sovereignty and Russian peculiarity, deepening of autonomy in all nec- essary fields and Eurasian integration where possible. Modernization, regional and interregional integration or sovereign opening up was not abandoned but modified under adverse external conditions and growing frictions worldwide (Sakwa, 2021, p. 5-6). Safeguarding of sovereignty was accompanied by stronger emphasis on self-sufficiency and development of own systems independent of the global ones (which creates a comfortable position in the cases of crises or conflicts) in the post-2014 period. It applies to the payment system, interbank financial telecom- munication system, navigation satellite system, 5G systems or the Internet. Pro- tection of the Russian Internet against external threats should be accomplished on the basis of the act passed in 2019 in response to the US National Cyber Strat- egy which declared the ambition to spread US particular interests worldwide.20 Russia’s path towards information and digital sovereignty is not isolated. It has to be perceived in an international context when many actors seek the same self-protection. In general, the assertion of information sovereignty is one of the most important tasks states have in the digital era (Romashkina, 2019). Moscow introduced numerous acts in this direction, including a data localization law or a law regarding import substitution of foreign IT products. Foreign encryp- tion protocols of the Russian Internet should be supplemented by the domestic ones in the near future and the 5G networks are to be based on Russian or Chi- nese technologies.21 One of Russia’s leading media holding Gazprom-Media has started to prepare a Russian version of TikTok while a national alternative of Wikipedia is also under preparation.22 Similarly, the US, the EU and China seek digital sovereignty, trying to minimize mutual interdependence and vulnerabil- ity. Washington banned TikTok and WeChat, preventing Huawei and ZTE from 2022, Vol. 1, No. 1 73 Ladislav Zemánek doing business in the country. 23 The Clean Network. U.S. Department of State. 2020. URL: https://2017-2021.state.gov/the-clean-network/index.html (ac- cessed 20.02.2022) 24 Csernatoni, R. The EU’s Rise as a Defense Technological Power: From Strategic Autonomy to Technological Sovereignty. Carnegie Europe, 2021, August 12. URL: https://carnegieeurope.eu/2021/08/12/eu-s-rise-as-defense-technological-pow- er-from-strategic-autonomy-to-technological-sovereignty-pub-85134 (accessed 20.02.2022) 25 Epifanova, A. Digital Sovereignty on Paper: Russia’s Ambitious Laws Conflict with Its Tech Dependence. Wilson Center. 2020, October 23. URL:.https://www.wilsoncenter.org/blog-post/digital-sovereignty-paper-russias-ambitious-laws-con- flict-its-tech-dependence (accessed 20.02.2022) 23 The Clean Network. U.S. Department of State. 2020. URL: https://2017-2021.state.gov/the-clean-network/index.html (ac- cessed 20.02.2022) g y g g y p 25 Epifanova, A. Digital Sovereignty on Paper: Russia’s Ambitious Laws Conflict with Its Tech Dependence. Wilson Center. 2020, October 23. URL:.https://www.wilsoncenter.org/blog-post/digital-sovereignty-paper-russias-ambitious-laws-con- flict-its-tech-dependence (accessed 20.02.2022) Governance and Politics 24 Csernatoni, R. The EU’s Rise as a Defense Technological Power: From Strategic Autonomy to Technological So Carnegie Europe, 2021, August 12. URL: https://carnegieeurope.eu/2021/08/12/eu-s-rise-as-defense-technolo er-from-strategic-autonomy-to-technological-sovereignty-pub-85134 (accessed 20.02.2022)l 23 The Clean Network. U.S. Department of State. 2020. URL: https://2017-2021.state.gov/the-clean-network/index.html (ac- cessed 20.02.2022) 24 Csernatoni, R. The EU’s Rise as a Defense Technological Power: From Strategic Autonomy to Technological Sovereignty. Carnegie Europe, 2021, August 12. URL: https://carnegieeurope.eu/2021/08/12/eu-s-rise-as-defense-technological-pow- f t t i t t t h l i l i t b 85134 ( d 20 02 2022) Comprehensive sovereignty: an internal perspective Trump’s administration introduced the Clean Network program aimed at the protection of data, privacy, security and rights against “aggressive intrusions by malign actors”.23 The EU promotes the policy of strategic autonomy within which cooperation with the pioneering Chinese companies is jeopardized.24 However, Russia lags behind the other major pow- ers in many respects, and the overall competitiveness, innovative potential and technological development are not comparable to the US or China’s. Therefore Russia is likely to deepen cooperation with Chinese partners to offset this con- siderable disadvantage.25 The National Security Strategy is a cornerstone of Russia’s official state para- digm. Unlike other strategic documents (for instance, Military Doctrine, Foreign Policy Concept, Economic Security Strategy, Energetic Security Doctrine, Infor- mation Security Doctrine or Foundations of State Cultural Policy), it deals with all these areas, synthesizing them into a comprehensive whole. Dmitry Trenin considers the 2021 NSS “the most important Kremlin strategy statement” and “a manifesto” for the present era characteristic of a deep confrontation with the Western world (Trenin, 2021). Indeed, the two last versions of the NSS were formulated under different circumstances. The preceding one was issued in re- sponse to the Ukrainian crisis, sudden deterioration of relations with the West, the pivot to the East and strong uncertainty about the impacts of the pres- sure and sanctions on the Russian economy and society as such. The last years showed that the country was able to withstand, transforming economic pro- cesses, diversifying external ties and deepening integration within the Greater Eurasia (Lukin & Novikov, 2021, p. 52-53). The current NSS reflects the further deterioration of relations with Europe and the US, missing them out completely. Similarly, prospects of cooperation with NATO are omitted, thus signaling that Moscow does not expect any substantial progress in the relations with the West in the upcoming years. 74 UDC: 323, 327, 369.032 Political Science / Country in Focus In this regard, the 2021 Strategy is much more skeptical and explicit as far as the confrontational actions of the US, its allies as well as transnational cor- porations are concerned. 26 Strategiia natsional’noi bezopasnosti Rossiiskoi Federatsii. Kremlin (National Security Strategy 2021). 2021, July 02. URL: http://static.kremlin.ru/media/events/files/ru/QZw6hSk5z9gWq0plD1ZzmR5cER0g5tZC.pdf (accessed 20.02.2022) 27 U.S. – Russia Presidential Joint Statement on Strategic Stability. Kremlin 2021, June 16. URL: http://en.kremlin.ru/supple- ment/5658 (accessed 20.02.2022) 28 Strategiia natsional’noi bezopasnosti Rossiiskoi Federatsii (National Security Strategy). Kremlin. 2015, December 31. URL: http://static.kremlin.ru/media/acts/files/0001201512310038.pdf (accessed 20.02.2022) 29 Strategiia natsional’noi bezopasnosti Rossiiskoi Federatsii. Kremlin (National Security Strategy 2021). 2021, July 02. URL: http://static.kremlin.ru/media/events/files/ru/QZw6hSk5z9gWq0plD1ZzmR5cER0g5tZC.pdf (accessed 20.02.2022) 2022, Vol. 1, No. 1 / ( ) tegiia natsional’noi bezopasnosti Rossiiskoi Federatsii (National Security Strategy). Kremlin. 2015, December 31. U ://static.kremlin.ru/media/acts/files/0001201512310038.pdf (accessed 20.02.2022) 26 Strategiia natsional’noi bezopasnosti Rossiiskoi Federatsii. Kremlin (National Security Strategy 2021). 2021, July 02. URL: http://static.kremlin.ru/media/events/files/ru/QZw6hSk5z9gWq0plD1ZzmR5cER0g5tZC.pdf (accessed 20.02.2022) 27 i id i l i i bili li h k li l i p ( ) tegiia natsional’noi bezopasnosti Rossiiskoi Federatsii. Kremlin (National Security Strategy 2021). 2021, July 02. U ://static.kremlin.ru/media/events/files/ru/QZw6hSk5z9gWq0plD1ZzmR5cER0g5tZC.pdf (accessed 20.02.2022) / / /i / / g q p g p ( ) ussia Presidential Joint Statement on Strategic Stability. Kremlin 2021, June 16. URL: http://en.kremlin.ru/supple- 658 (accessed 20.02.2022) tegiia natsional’noi bezopasnosti Rossiiskoi Federatsii. Kremlin (National Security Strategy 2021). 2021, July 02. U // i k li / di / /fil / /QZ 6hSk5 9 W 0 lD1Z R5 ER0 5 ZC df ( d 20 02 2022) Strategiia natsional noi bezopasnosti Rossiiskoi Federatsii (National Security Strategy). Kremlin. 2015, December 31. URL: http://static.kremlin.ru/media/acts/files/0001201512310038.pdf (accessed 20.02.2022) 29 Strategiia natsional’noi bezopasnosti Rossiiskoi Federatsii. Kremlin (National Security Strategy 2021). 2021, July 02. URL: http://static.kremlin.ru/media/events/files/ru/QZw6hSk5z9gWq0plD1ZzmR5cER0g5tZC.pdf (accessed 20.02.2022) Comprehensive sovereignty: an internal perspective The illiberal wording becomes obvious when the cul- tural Westernization is called a fatal threat for Russia and the absolutization of freedom of individuals, egoism and immorality is criticized as proof of the West’s decline while emphasizing the significance of the traditional values and norms as well as religious foundations of societies.26 Such rhetoric corresponds with Putin’s assessment of today’s Western liberalism. In an interview given to the Financial Times in 2019, he refused the hegemonic ambitions of the liberal elites, castigating them for their contempt for the rights of the majority, assaults on the traditional values and silencing opponents. Russian President expressed his belief that biblical values are more universal than liberal ones.27 The 2021 NSS thus embodies the conservative worldview of the Russian leadership, ar- ticulating and shaping the hegemonic discourse, which will be reflected in the adjustments of the partial doctrines in the following months and years. The conservative character of the ruling paradigm (Diesen, 2021, p. 205) is confirmed by the definition of the traditional Russian values, put forward by the 2015 NSS, with the priority of the spiritual over the material at the top and followed by the protection of human life, rights and freedoms, family, creative labor, service to the motherland, the norms of morality, humanism, charity, fair- ness, mutual assistance, collectivism, unity of Russia’s nations and the continuity of the Russian history. This conservative discourse is complemented by a strict refusal of external interference undermining cultural sovereignty. The Russian state stipulates the right to protect citizens against external expansion in terms of ideology and values, as well as to control the information sphere.28 The 2021 Strategy rearranges the enumeration of the Russian values only minimally.29 The establishment and reinforcement of sovereignty in different spheres of the life of the state and society brings multiple risks, especially in terms of 2022, Vol. 1, No. 1 75 Ladislav Zemánek individual rights and freedoms. The dominant conservative paradigm under Vladimir Putin is nevertheless moderate. Potential radicalization is dependent mainly on the objective external pressure and subjective sense of danger. This combination is risky if taking the Russian tradition of patrimonialism, strong state, and collectivism into consideration. It could result in a substantial closure of the country, the building of a fortress and isolationism. In that case, Rus- sia might become a real international threat – which is not absolutely at this moment. Comprehensive sovereignty: an internal perspective On the contrary, it is the anti-Russian narrative together with actual political steps aimed against Russia which forces the country to the adoption of defensive, partially hostile stance. Governance and Politics Comprehensive sovereignty: an external perspective Russia’s concept of sovereignty includes the principles of autonomy and independence, strategic stability and partnership. Whereas the autonomy and independence reflect predominantly the internal dimension, the stability and partnership concern the external one – the external relations as well as a vision of and practical steps towards reform of the global system in line with the gen- eral normative outlook on how the system should be designated and structured in the 21st century. In this chapter, we will turn to the external dimension, going through the strategic framework defined by the National Security Strategies and subsequently focusing on the relations with the US and China. It is worth noticing that both the 2015 and 2021 NSS do not position the relations with the West (the US and Europe) on the top of priorities. It has a symbolic significance connected with the deep disillusionment with the West- ern policies in the last decades, turn to the East and renunciation of Westerni- zation. It shows that the West is not in the spotlight anymore, thus underlining the objective reality of multipolarization and the rise of the East. Taking this development into account, the secondary position of the US and Europe within the NSS exceeds the genuinely symbolic meaning and expresses the rational and pragmatic choice made in response to the transformation of the global order, economy and politics. Not surprisingly, therefore, the 2015 NSS emphasized the multilateral cooperation within BRICS, SCO, APEC and G20, highlighting the relationship with China and India. Besides, stress was laid on the near neigh- Governance and Politics 76 Political Science / Country in Focus UDC: 323, 327, 369.032 borhood, the CIS and integration processes in Eurasia, first and foremost, the Eurasian Economic Union (EAEU). As far as Europe is concerned, the NSS mentioned the support for win-win cooperation with the European states and the EU, harmonization of integration processes in Europe and on the post-So- viet territory (EAEU) as well as the establishment of joint collective security architecture in the Euro-Atlantic region.30 These were long-term goals pursued by the Russian establishment. Their inclusion in the 2015 NSS can be perceived as proof of continuity and transparency of Russia’s foreign policy if we become aware of the context of the sharp deterioration in the relations with the West. Irrespective of obvious Washington’s involvement in the coup d’état in Ukraine, Moscow reaffirmed its readiness to develop the bilateral partnership. 30 Strategiia natsional’noi bezopasnosti Rossiiskoi Federatsii (National Security Strategy). Kremlin. 2015, December 31. URL: http://static.kremlin.ru/media/acts/files/0001201512310038.pdf (accessed 20.02.2022) 31 Ibid Ibid. 32 Strategiia natsional’noi bezopasnosti Rossiiskoi Federatsii. Kremlin (National Security Strategy 2021). 2021, July 02. URL: http://static.kremlin.ru/media/events/files/ru/QZw6hSk5z9gWq0plD1ZzmR5cER0g5tZC.pdf (accessed 20.02.2022) 30 Strategiia natsional’noi bezopasnosti Rossiiskoi Federatsii (National Security Strategy). Kremlin. 2015, December 31. URL: http://static.kremlin.ru/media/acts/files/0001201512310038.pdf (accessed 20.02.2022) 31 Ibid. 32 Strategiia natsional’noi bezopasnosti Rossiiskoi Federatsii. Kremlin (National Security Strategy 2021). 2021, July 02. URL: http://static.kremlin.ru/media/events/files/ru/QZw6hSk5z9gWq0plD1ZzmR5cER0g5tZC.pdf (accessed 20.02.2022) Comprehensive sovereignty: an external perspective Moreover, it admitted the development of relations with NATO on the condition that the latter respects international law and Russia’s interests.31 The wording of the 2021 NSS is different. It contains the keystones of the long-term Russian strategy such as maintenance of the central coordination role of the UN and the UN Security Council, deepening cooperation within CIS, CSTO, SCO, BRICS, EAEU or ASEAN. However, no explicit mentions of Europe, the US and NATO are made. It is especially the part dedicated to ex- ternal affairs which distinguishes the present strategy from the former. And it is sharper in criticism. The world is characterized as a place where contradictions and conflicts have been intensifying and where the declining powers of the West assault on others, violate principles and norms of the international law as well as international treaties, breach sovereignty, undermine trust among individual actors and stability of the system, impose sanctions and attempt to revise the ex- isting rules. Obviously, Russia endeavors to present itself as a crucial stabilizing element and protector of the status quo. In this regard, the NSS addresses the need for reinforcement of coherence and resilience of the international system and its legal base, strengthening of international peace and collective security, prevention of military conflicts and, last but not least, – strategic stability.32 2022, Vol. 1, No. 1 77 Ladislav Zemánek Russia’s self-perception as a protector of the status quo and concurrent accu- sation against the declining Western actors of revisionism is not new, since it has appeared in recent articles published by the highest representatives, especially Minister of Foreign Affairs Sergei Lavrov. In the article The World at a Cross- roads and a System of International Relations for the Future from 2019, Lavrov subjected the West and its liberal democratic ideology to criticism. Lambasting the West for endeavors to usurp the fruits of globalization to the detriment of the “rest”, he describes the Western countries as hypocrites, for the narrative based on freedom, democracy and human rights entails inequality, injustice, selfish- ness and exceptionalism (Lavrov, 2019, p. 10). More or less explicitly, Minister follows the concepts of multiple modernities, multipolarity and multilateralism which fit the reality of the transforming global order. From the Russian perspec- tive, the West invented the concept of a rules-based order which is revisionist in its essence. 33 Ibid, p. 11. 34 Sanger, D. E. Biden Defines His Underlying Challenge with China: ‘Prove Democracy Works’. New York Times. 2021, March 26. URL: https://www.nytimes.com/2021/03/26/us/politics/biden-china-democracy.html (accessed 20.02.2022) 35 President Biden to Convene Leaders’ Summit for Democracy. The White House. 2021, August 11. URL: https://www.white- house.gov/briefing-room/statements-releases/2021/08/11/president-biden-to-convene-leaders-summit-for-democra- cy/ (accessed 20.02.2022) Comprehensive sovereignty: an external perspective Lavrov characterizes it as a calculated, selective combination of rules, unilaterally employed with the aim to circumvent multilateral, collective decision-making and international legal instruments and processes based on the UN Charter as a core of the post-war order.33 The rules-based order introduces a division between liberal democracies and “authoritarian powers”, “autocracies”, between allies and systemic rivals, friends and enemies. Inevitably, it leads to confrontation, conflicts and insta- bility of the international system. The Western concept is opposite to pragma- tism, openness and pluralism, being another sequel of Western universalism. Moreover, it is utterly moralistic, introducing the ethical categories of good and evil into international politics, making it very ideologized. The US President is trying to establish an “alliance of democracies” to confront “autocracies”. At his first news conference after the election, Biden spoke about a “battle” between these two artificially invented camps.34 Later on, the US highest representative announced the plan to organize a summit of the “democratic states” to counter global authoritarianism.35 The Biden administration develops continuously the Governance and Politics 78 Political Science / Country in Focus UDC: 323, 327, 369.032 confrontational narrative created during Donald Trump’s presidency, albeit in a more sophisticated, ostensibly more diplomatic and moderate way. Biden´s “alliance of democracies” is a reformulation of Trump’s and Pompeo’s antinomy between the “free world” and “new tyranny”, i.e. China and its kind.36 Continu- ity in external policy between these two seemingly contradictory Presidents is indisputable, even though Biden pursues a more multilateral, more collective, more inclusive attitude. This attitude is, nevertheless, conditioned by adherence to the “democratic” camp, to the universalist and hegemonic Western paradigm. The others are excluded. That is why the UN-based order ceases being con- venient for the West since the objective power decline results in an inability to control the system. And that is why the Western actors are seeking to replace the UN-based order with the rules-based one. The West’s attempts to marginalize the UN and weaken collective deci- sion-making irrespective of socioeconomic, political, ideological or cultural differences are obvious, so the Russian accusations of revisionism are justifiable. On the other hand, however, the post-1991 world was dominated by the West with the US on the top, being greatly unipolar, and these dominating actors – be they states, transnational corporations or other non-state subjects – are interest- ed in the preservation of the model and their hegemony. g / / ( ) 37 In this article, I put aside the difference between reformism and revisionism as it is not substantial for the reasoning (com- pare with: Sergunin, 2016, p. 25, 27, 32-35). Revisionism is simply meant as the effort to change the status quo. 36 Pompeo, M. R. Communist China and the Free World’s Future. U.S. Department of State 2020, July 23. URL: https://2017-2021. state.gov/communist-china-and-the-free-worlds-future-2/index.html (accessed 20.02.2022)f Comprehensive sovereignty: an external perspective The reality is therefore more complex and the role of the West is ambiguous, being typical of contra- dictory tendencies towards the maintenance of the status quo and revisionism.37 The revisionist tendencies can be seen in the US withdrawal from the An- ti-Ballistic Missile Treaty (ABM Treaty) in 2002, Joint Comprehensive Plan of Action (JCPOA), the UN Human Rights Council and UNESCO in 2018, In- termediate-Range Nuclear Forces Treaty (INF Treaty) in 2019, Paris Agreement and Treaty on Open Skies in 2020. The US position towards the Comprehensive Nuclear Test Ban Treaty (CTBT) is also questionable as Washington is reluctant to ratify it. Similarly, the destiny of the Treaty on Measures for the Further Re- duction and Limitation of Strategic Offensive Arms (New START) was extreme- ly uncertain for a long period. Many of these steps disrupted the foundations 2022, Vol. 1, No. 1 79 Ladislav Zemánek of the system, gradually forming after World War II amidst the systemic con- frontation between and coexistence of the two camps. The US withdrawals thus undermined relative stability and transparency brought about by the bilateral agreements between the US and the USSR. The Russian Federation assumed ob- ligations and inherited armaments from the Soviet period and as such, it contin- ues to be one of the pillars of international security architecture with the UN Se- curity Council as its core. In this regard, it is Washington that has attempted to reverse the long-term status quo. The US have ignored the UN Security Council resolutions (for instance regarding the settlement of the Israeli-Palestinian con- flict), launched unilateral military operations without authorization by this UN body and carried out multiple measures aimed against other countries – extra- territorial use of the US legislation, spying on their closest allies, abuse of the status of the USD as the main means of payment, introduction of protectionist barriers and, last but not least, imposition of sanctions. These practices have become an integral part of the US-led rules-based order. The imposition of sanctions is an increasingly frequent practice used by lib- eral democracies against others. Given the intensifying frictions and transfor- mation of the global order, continuation and deepening of this tendency are to be expected in the following period. Governance and Politics Comprehensive sovereignty: an external perspective It is worth noting that in spite of the strong pressure, the impacts on the Russian economy have been relatively low com- pared to other external factors such as the drop in oil prices, global pandemic and related economic slump. The data published by the International Monetary Fund follows that the role of sanctions accounted only for -0.2% in terms of GDP growth. At the same time, low macroeconomic influence is accompanied by high risks for individual businesses and projects (Timofeev, 2021, p. 4). In his report Sanctions Against Russia: A Look into 2021, Ivan Timofeev, therefore, comes to the conclusion that the best long-term answer for Russia is to lower dependency on the Western structures through concurrent development of the national payment system and a single payment system within the EAEU and the SCO, de-dollarization of the Russian economy as well as the development of international/regional mechanisms of counter-sanctions (Timofeev, 2021, p. 19). The significance of the latter is highlighted by the fact that it is Chi- na and Russia together with Iran that is the most frequent targets. Adaptation on the Western restrictive regimes thus could be made more effective through Governance and Politics 80 UDC: 323, 327, 369.032 Political Science / Country in Focus the SCO, especially after the Islamic Republic becomes a full member of the organization.38 Not only sanctions show that the discourse dividing countries into “democ- racies” and “autocracies” is dangerous. Instead, the division into those who ad- vocate unipolarity or multipolarity is relevant. We can hardly avoid delimita- tion, as the world is full of antagonism, contradictions, divergent interests and conflicts. As long the sphere of the political exists, dividing lines are inevitable. The difference between friends and foes, allies and challengers remains. As Carl Schmitt (2015) put it, the political world is pluriverse in its essence, since the existence of genuine universality requires complete depoliticization, hence the elimination of the state as a social institution. At this stage of development, the elimination of the state or withering away of the state – if we use the Marxist term coined by Friedrich Engels in his Anti-Dühring – is not only unfeasible but also undesirable. The opponents of Western hegemonism and unipolarity need to have a strong state to break those structures that hamper the development to- wards a more democratic, fairer, more equal, more inclusive model. 38 SCO Opposes External Meddling, Builds Shield against Outside Forces. Global Times. 2021, September 17. URL: https:// www.globaltimes.cn/page/202109/1234595.shtml (accessed 20.02.2022) Comprehensive sovereignty: an external perspective Naturally, it raises resistance from the declining powers which have resorted to the narrative of democracies versus autocracies, rules-based order versus a “law-of-the-jun- gle-based” order. Such a picture is false, serving merely the particular interests of the weakening hegemons. Joe Biden gave up the isolationist America First imperative, replacing it with “Democracies First”. Of course, the US only has liberal democracies in mind. The internationalist agenda, however, remains particularist, albeit presented as universalist and objectively valid. The US President has revived ties with the allies, making some initial concessions (Nord Stream 2), but continues estab- lishing a “global democratic phalanx”, as an American scholar called one of the pillars of the Biden doctrine (Brands, 2021). Biden makes use of the existing, frequently overwhelming structures like Group of Seven, the Quad (Quadrilat- eral Security Dialogue), the Indo-Pacific Strategy (“A free and open Indo-Pacif- ic”), América Crece, T-12 (League of Digital Democracies), D-10 (Democracies 10) and the Clean Network, or is developing new ones such as B3W initiative 2022, Vol. 1, No. 1 81 Ladislav Zemánek (Build Back Better World), the Summit for Democracy, AUKUS or Trade and Technology Council (TTC) to advance hegemonic interests and contain “au- tocracies”. The first leader-level summit of the Quad was held in March 2021.39 The Biden administration tries to give a new impetus to this grouping with the aim to incorporate it into the “global phalanx” and employ it against China. The G7 summit in Carbis Bay in June 2021 presented the B3W initiative to provide an alternative to the Belt and Road Initiative (BRI) in relation to the “strategic competition” with Beijing. The ambitious infrastructure project is focused on low- and middle-income countries from the Americas to Africa, Indo-Pacif- ic and beyond. Representatives of the largest liberal democracies agreed on an investment worth at least 40 trillion USD. Both the ideologic background and financial sources come predominantly from the US. The idea can be interpreted as an expanded and updated version of the New Silk Road Initiative announced by the Secretary of State Hillary Clinton in 2011. It also builds on the Blue Dot Network (BDN) launched in 2019. 39 Quad Leaders’ Joint Statement: “The Spirit of the Quad”. The White House. 2021, March 12. URL: https://www.whitehouse. gov/briefing-room/statements-releases/2021/03/12/quad-leaders-joint-statement-the-spirit-of-the-quad/ (accessed 20.02.2022) 39 Quad Leaders’ Joint Statement: “The Spirit of the Quad”. The White House. 2021, March 12. URL: https://www.whitehouse. gov/briefing-room/statements-releases/2021/03/12/quad-leaders-joint-statement-the-spirit-of-the-quad/ (accessed 20.02.2022) 40 President Biden and G7 Leaders Launch Build Back Better World (B3W) Partnership. The White House. 2021c, June 12. URL: https://www.whitehouse.gov/briefing-room/statements-releases/2021/06/12/fact-sheet-president-biden-and-g7- leaders-launch-build-back-better-world-b3w-partnership/ (accessed 20.02.2022) 41 The Czech Republic expressed an interest in the mediation of the meeting in Prague, but the plan was thwarted as a result of an unprecedented anti-Russian campaign in the country based on the allegation of Russia’s “terrorist act” on Czech territory in 2014. For greater detail see Zemánek (2021a; 2021b). p p 41 The Czech Republic expressed an interest in the mediation of the meeting in Prague, but the plan was thwarted as a result of an unprecedented anti-Russian campaign in the country based on the allegation of Russia’s “terrorist act” on Czech territory in 2014. For greater detail see Zemánek (2021a; 2021b). Governance and Politics ) 40 President Biden and G7 Leaders Launch Build Back Better World (B3W) Partnership. The White House. 2021 URL: https://www.whitehouse.gov/briefing-room/statements-releases/2021/06/12/fact-sheet-president-bide leaders-launch-build-back-better-world-b3w-partnership/ (accessed 20.02.2022) ) 40 President Biden and G7 Leaders Launch Build Back Better World (B3W) Partnership. The White House. 2021c, June 12. URL: https://www.whitehouse.gov/briefing-room/statements-releases/2021/06/12/fact-sheet-president-biden-and-g7- leaders-launch-build-back-better-world-b3w-partnership/ (accessed 20.02.2022) 41 The Czech Republic expressed an interest in the mediation of the meeting in Prague, but the plan was thwarted as a result ) 40 President Biden and G7 Leaders Launch Build Back Better World (B3W) Partnership. The White House. 2021c, June 12. URL: https://www.whitehouse.gov/briefing-room/statements-releases/2021/06/12/fact-sheet-president-biden-and-g7- leaders-launch-build-back-better-world-b3w-partnership/ (accessed 20.02.2022) 41 The Czech Republic expressed an interest in the mediation of the meeting in Prague, but the plan was thwarted as a result of an unprecedented anti-Russian campaign in the country based on the allegation of Russia’s “terrorist act” on Czech territory in 2014. For greater detail see Zemánek (2021a; 2021b). Comprehensive sovereignty: an external perspective Washington as a leading force of the B3W plans to engage the Development Finance Corporation, the Agency for Inter- national Development (USAID), the Export-Import Bank (EXIM), the Millen- nium Challenge Corporation, the US Trade and Development Agency or the Transaction Advisory Fund.40 Generally, the US-led global initiative is in line with the Strategic Competition Act of 2021, one of the priorities of which is to counter the “malign influence of the Communist Party of China” worldwide, thus following the anti-Chinese discourse and policies of Trump’s era with its zero-sum-game logic. What, then, is Russia’s position amid the “strategic com- petition” between Washington and Beijing declared by the US political class? R i US Chi t i l Th fi t it f Bid d P ti i G Russia-US-China triangle. The first summit of Biden and Putin in Geneva, following the public accusations made by the US President41, could be interpret- ed as a step towards stabilization of the volatile and deteriorating relations. The 82 Political Science / Country in Focus UDC: 323, 327, 369.032 goal was not a reset of the relationship, and created a more realistic context. As Dmitry Suslov (2021) aptly expressed it, the realistic aim was to lay foundations for a “managed confrontation”. Moscow welcomed this initiative irrespective of the recent US actions perceived as hostile – sanctions, the expulsion of diplo- mats, hesitation in prolongation of the New START, forming a front against “autocracies”, engagement in Belarus, military provocations, labeling Russia’s President as a “killer”, accusations of attacks against other countries (the Czech Republic in 2014 in particular) and so on and so forth. The reasons behind Bid- en’s efforts are likely to be connected with China, as the latter has become the cardinal rival for Washington, replacing Russia.42 Therefore, Biden is motivated to seek sustainability in their relations with Moscow, notwithstanding deeply divergent interests and values. It seems to be a sequel to Trump’s vision to revive cooperation with Russia against China which failed primarily due to historical experience, and the mental and ideological inertia of the US establishment. In both cases, nevertheless, it is still the same zero-sum-game perspective, calculated cooperation with one country against another. Whereas Washington took side with China several times in the last century, such a symbiosis never existed with Russia – with the exception of hints in the 1990s. 42 Annual Threat Assessment of the US Intelligence Community. Office of the Director of National Intelligence. 2021, April 09. URL: https://www.dni.gov/files/ODNI/documents/assessments/ATA-2021-Unclassified-Report.pdf (accessed 20.02.2022) Threat Assessment of the US Intelligence Community. Office of the Director of National Intelligence. 2021, April 09. tps://www.dni.gov/files/ODNI/documents/assessments/ATA-2021-Unclassified-Report.pdf (accessed 20.02.2022) Comprehensive sovereignty: an external perspective The US support- ed Japan financially against Russia in the Russo-Japanese War at the beginning of the 20th century. They refused to recognize the USSR until 1933 as the last of the major powers, even though the rule of the Soviet regime had been a fait accompli long before this. The first bilateral agreement was concluded no earlier than 1964. In spite of the balance of power based on nuclear deterrence, both hegemons waged a wide array of proxy wars against each other. The US also imposed sanctions on the Soviet Union, for example in response to the Soviet invasion of Afghanistan. The unique possibility to incorporate the new Russia into the Euro-Atlantic community after 1991 miscarried predominantly due to the West’s arrogant belief of superiority and hegemonism (NATO expansion eastwards, support for separatism in Russia, disrespect for Russia’s specific con- ditions, interests and needs). Given these experiences, it is hard to expect that any kind of “Russiamerica” (akin to “Chinamerica”) will emerge in the future 2022, Vol. 1, No. 1 83 Ladislav Zemánek and oppose anyone, unless other emerging non-Western actors adopt a radi- cal anti-Russian attitude. The Biden-Putin summit was merely aimed to find an elementary level of understanding and communication which had been lost because of the extreme politicization of the Russian question in the US under Donald Trump. Russia became one of the main factors in the domestic political struggle. It made a constructive policy towards Moscow impossible. Unlike the preceding President, Joe Biden is not accused of having ties with the Kremlin in the public discourse, which paradoxically may facilitate mutual interactions (Kortunov, 2021, p. 7-10). Regardless of discrepancies, the Geneva summit can be interpreted in terms of starting talks about “strategic stability”.43 The US side has made a constructive step when agreeing with the extension of the New START which remains in force until 2026. Both major powers could also find a way of cooperation in the field of cybersecurity, especially cybercrime, and at least define rules of cyberwarfare. Another area of possible cooperation concerns the nuclear programs of Iran and North Korea, as well as climate change and green transition (Suslov, 2021). The latter became easier after the US rejoined the Paris Agreement. The war on terrorism was an exception where Russia and the US managed to intensify co- operation during Trump’s presidency (Shakirov, 2020, p. 8). This tendency could continue under Biden. 43 U.S. – Russia Presidential Joint Statement on Strategic Stability. Kremlin 2021, June 16. URL: http://en.kremlin.ru/supple- ment/5658 (accessed 20.02.2022) Comprehensive sovereignty: an external perspective Similarly, both powers will be confronted with a move towards militarization and exploitation of outer space and new regions such as the Arctic, which will require the establishment of rules and a legal framework (Shakirov, 2020, p. 10). The dynamic development, disruptions and innovations in many fields (cyberspace, the Arctic, outer space) will push both Moscow and Washington into interacting and seeking pragmatic solutions. However, the confrontation will be a harsh reality. Russia belongs to the “autocratic camp” and the unprecedented level of cooperation and understanding between Moscow and Beijing is already raising concerns among the liberal democracies. Some experts, therefore, warn about the West’s possible attempts to undermine the Sino-Russian strategic partnership, taking the side of either the Russians or the Chinese (Kortunov, 2021, p. 27). Others even call upon the Western political Governance and Politics Governance and Politics 84 UDC: 323, 327, 369.032 Political Science / Country in Focus elites to rupture the “axis of the autocratic powers” (Kendall-Taylor & Shullman, 2021). Still, such a scenario is merely wishful thinking. Although the 2021 NSS is very concise as far as the relationship with China is concerned44, the real state of affairs is much more vivid. Possibly, the lacon- ism of the NSS is intended to weaken the assertions, according to which the Sino-Russian “axis” is a de facto alliance, and to emphasize Russia’s multi-vec- tor external orientation, excluding superiority of partnership with one coun- try. Whatever the reasons, the fact is that Sino-Russian relations have reached the highest level in history. The legal framework of the bilateral interactions is defined by the Treaty of Good Neighborliness and Friendly Cooperation con- cluded in 2001. On the occasion of the 20th anniversary of the signing of this fundamental document, both sides issued a Joint Statement45 that reflects the unprecedented development of the comprehensive partnership, demonstrating its specific, innovative nature, being an example of a new type of interstate rela- tions in our era. The document characterizes bilateral ties as a “comprehensive partnership”, “strategic interaction”, “international relations of a new type” and a “model of harmonious coexistence” based on the “comprehensive consideration of the partner´s interests and non-interference in each other´s internal affairs”. p // / / /i / /Q g q p g p ( ) 45 Joint Statement of the Russian Federation and the People’s Republic of China on the Twentieth Anniversary of the Treaty of Good Neighborliness and Friendly Cooperation between the Russian Federation and the People’s Republic of China. Kremlin. 2021, June 28. URL: http://static.kremlin.ru/media/events/files/en/Bo3RF3JzGDvMAPjHBQAuSemVPWTEvb3c.pdf (accessed 20.02.2022) 46 Ibid 44 Strategiia natsional’noi bezopasnosti Rossiiskoi Federatsii. Kremlin (National Security Strategy 2021). 2021, July 02. URL: http://static kremlin ru/media/events/files/ru/QZw6hSk5z9gWq0plD1ZzmR5cER0g5tZC pdf (accessed 20 02 2022) ( 46 Ibid. 2022, Vol. 1, No. 1 44 Strategiia natsional’noi bezopasnosti Rossiiskoi Federatsii. Kremlin (National Security Strategy 2021). 2021 Comprehensive sovereignty: an external perspective Refus- ing the concept of a military or political alliance of the past, both actors stress that the partnership is not directed against third sides.46 Undoubtedly, we are witnesses of a peculiar phenomenon that can anticipate new forms of interac- tions in the reformed, polycentric, regions-based order. After all, China is a pi- oneer of such a shift, if we take into consideration the nature and specific forms of the Belt and Road Initiative, 16+1 (Central and Eastern Europe + China) and other analogic formats in different regions. Unlike these, the Sino-Russian relationship is bilateral and its crucial significance is connected with objective factors, as well as the fact that both major powers are the engines of the Eurasian integration and pillars of the emerging new order. 2022, Vol. 1, No. 1 85 Ladislav Zemánek The June Joint Statement47 defines the overall direction and priorities of the Sino-Russian partnership for the ongoing period. (1) The major emphasis is put on Putin-Xi diplomacy as the cornerstone and guarantee of successful develop- ment. Mutual meetings at leaders´ level are very frequent and regular. The same applies to the lower levels (PMs, ministers or state agencies). (2) Military and military technology cooperation are in second place. It regards not only joint exercises, military exchanges or purchases of armaments but also the reduction of armed forces in the border areas. Both China and Russia have expanded the military cooperation and potential through multilateral platforms, particular- ly the SCO. (3) The economic and trade ties are seen as a substantial element of the social development and improvement of the people´s living standards. The goals are set as follows: (i) to increase the volume of bilateral trade; (ii) to strengthen energy cooperation; (iii) to deepen financial interaction, support the expansion of mutual payments in national currencies in trade; (iv) to strengthen cooperation in the field of industry, information and communication technol- ogies, space and aviation; (v) to enhance cooperation in the scientific and tech- nological innovations; (vi) to ensure protection and enforcement of intellectual property rights; (vii) to increase cooperation in agro-industry; (viii) to deepen interactions in the field of transport; (ix) to strengthen cooperation within the Northern Sea Route (or the so-called Polar Silk Road), promoting sustainable development of the Arctic; (x) to enhance interregional ties. 47 Ibid. Comprehensive sovereignty: an external perspective (4) The expansion of interregional ties is of the utmost importance given the obvious tendency towards regionalization as an accompanying phenomenon of globalization at the current stage of development that is typical of the ambivalent logic of inte- gration and autonomatization, openness and introversion. The Eurasian mac- ro-region is the most dynamic space in this regard as it includes several grand integration processes, starting with the Belt and Road Initiative, the Shanghai Cooperation Organization and the Eurasian Economic Union subsumed into the overarching vision of the Greater Eurasian Partnership (GEP), and ending with the European Union in the Western part of Eurasia. The abovementioned projects (with the exception of the EU) are crucial to redefining the international order embodying the new paradigm as a perspec The abovementioned projects (with the exception of the EU) are crucial to redefining the international order, embodying the new paradigm as a perspec- 47 Ibid. Governance and Politics 86 Political Science / Country in Focus UDC: 323, 327, 369.032 tive of the emancipating and rising non-Western world, seeking full-fledged recognition, equity and participation (Zemánek, 2020, p. 200). Not by coinci- dence, the GEP is mentioned in the recent Russian NSS for the first time ever.48 Agreement on the basic values and principles of the international order between Russia and China is crucial as it multiplies the relevance, attraction and influence of the new paradigm. The Joint Statement replicates the wording of the Russian strategic documents. The mutual understanding can be demonstrated in Russia’s explicit recognition of the China-proposed concept of building a community of shared future for humanity and China’s concurrent recognition of the concept of multipolar global order pursued by Russia for a long period.49 Both major powers have the identical perception of the main risks in terms of the interna- tional stability and security – unilateralism and undermining of the UN-based legal framework (the withdrawal from several arms control agreements made by the US), development of the US missile defense system, building high-precision non-nuclear weapons, militarization and the arms race in outer space, or the so- called Three Evils of terrorism, separatism and extremism. http://static.kremlin.ru/media/events/files/ru/QZw6hSk5z9gWq0plD1ZzmR5cER0g5tZC.pdf (accessed 20.02.2022) 49 Joint Statement of the Russian Federation and the People’s Republic of China on the Twentieth Anniversary of the Treaty of Good Neighborliness and Friendly Cooperation between the Russian Federation and the People’s Republic of China. Kremlin. 2021, June 28. URL: http://static.kremlin.ru/media/events/files/en/Bo3RF3JzGDvMAPjHBQAuSemVPWTEvb3c.pdf (accessed 20.02.2022) 50 Ibid Ibid. 51 Joint Statement by the Foreign Ministers of China and Russia on Certain Aspects of Global Governance in Modern Condi- tions. The Ministry of Foreign Affairs of the Russian Federation. 2021, March 23. URL: https://www.mid.ru/en/foreign_pol- icy/news/-/asset_publisher/cKNonkJE02Bw/content/id/4647776 (accessed 20.02.2022) 48 Strategiia natsional’noi bezopasnosti Rossiiskoi Federatsii. Kremlin (National Security Strategy 2021). 2021, July 02. URL: http://static kremlin ru/media/events/files/ru/QZw6hSk5z9gWq0plD1ZzmR5cER0g5tZC pdf (accessed 20 02 2022) ( 50 Ibid. Comprehensive sovereignty: an external perspective Moscow and Beijing make a commitment to pursue multilateralism and keep global stability through developing regional partnerships and integration as a step towards the commu- nity with a shared future for mankind, protecting security through the establish- ment of a global information security system (China’s Global Initiative on Data Security as an alternative to the US-led Clean Network) with the sovereign states’ right to regulate the national segment of the Internet, as well as promoting a multilateral and open trading system with the WTO as its core.50 Despite being labelled “autocratic”, both Russia and China adhere to democ- racy and human rights. The cause of controversies between these countries and the West rests in the simple fact that Russia and China admit the existence of multiple democracies and conceptions of human rights.51 They are more open, 2022, Vol. 1, No. 1 87 Ladislav Zemánek plural and tolerant than the West with its monism and moralism, hampering pragmatic cooperation to the benefit of all members of the global community. The gradual building of the Greater Eurasian Partnership comprising manifold integration processes, development patterns as well as political models, and based on both common interests and shared principles, creates a new norma- tive framework, significantly influenced by Russian perspective as enshrined in the basic national strategic documents. The rising Eurasian community poses a great challenge to the European Union that has been caught in a trap of Eu- ro-Atlanticism. It inevitably complicates relations with Russia and China.h The EU and Russia: seeking a new modus vivendi. The EU elites decided to follow the path of strategic autonomy. While commenced in the field of the de- fense industry, the concept has gradually spread to other fields including econ- omy, technology, politics, research and development or education. The concept became a part of the Common Security and Defense Policy (CSDP) in 2013, before the split with Moscow and in the times of the record mutual trade exchange and deepening relations (Shkolyar, 2021). After the crisis around Ukraine erupted, the strategic autonomy expanded into the external policies, being present in the 2016 EU Global Strategy. It is a similar process compared to that in Russia where the concept of sovereignty started to be employed in different fields including economy, power industry, information or culture, thus not limiting itself to the foreign policy or military. 52 Borrell, J. B. Why European Strategic Autonomy Matters. European External Action Service. 2020, December 03. URL: https://eeas.europa.eu/headquarters/headquarters-homepage/89865/why-european-strategic-autonomy-matters_en (accessed 20.02.2022) Comprehensive sovereignty: an external perspective Securitization of the individual fields is accompanied by the introduction of the concept of sovereignty into new contexts. The same applies to strategic autonomy in the EU. Josep Borrell, the High Representa- tive of the European Union for Foreign Affairs and Security Policy, published an article Why European Strategic Autonomy Matters in which he explains the reasons for this strategy. Borrell explicitly admits that strategic autonomy is a “process of political survival” given the objective tendency of decline in the Eu- ropean significance and influence in the world.52 It is therefore connected with the protection of the “European way of life” (irrespective of the controversial and obscure nature of this term), development of the independent position and Governance and Politics Governance and Politics 88 UDC: 323, 327, 369.032 Political Science / Country in Focus distinctive “mission” within the global community and, last but not least, with the economic base. Face to face with Trump’s protectionism, unilateralism, growing interna- tional instability and rivalry, as well as the coronavirus pandemic, the EU elites noticed the Union’s vulnerability and weak points of interdependence. From this perspective, strategic autonomy can be interpreted as a positive effort to make the EU one of the poles in the multipolar world, globalized and regionalized at the same time. Donald Trump’s America First policy, China’s dual circula- tion and Russia’s comprehensive sovereignty are of a similar nature even though the individual characteristics vary. Within the EU’s strategic autonomy, Brus- sels endeavors to boost the international role of the euro, regulate and control foreign investment (through screening mechanisms) or critical infrastructure (including 5G, energy sector or health service) as well as increase the military capabilities. It has launched the European Development Fund (EDF), Perma- nent Structured Cooperation (PESCO), European Battery Alliance (EBA), Eu- ropean Raw Material Alliance (ERMA), Future Combat Air System (FCAS) and many other projects in different fields. The EU aspires to deepen self-sufficien- cy in some regards, particularly those having strategic significance, to increase domestic production and decrease the role of import where the latter collides with the Union’s interests. 53 A Globally Connected Europe: Council Approves Conclusions. European Council. 2021, July 12. URL: https://www.con- silium.europa.eu/en/press/press-releases/2021/07/12/a-globally-connected-europe-council-approves-conclusions/ (ac- cessed 20.02.2022) 55 Emmott, R., & Siebold, S. After G7 Pledge, EU Seeks to Rival China’s ‘Belt and Road’ with Own Infrastructure Plan. Reuters, 2021, July 12. URL: https://www.reuters.com/world/china/after-g7-pledge-eu-seeks-rival-chinas-belt-road-with-own-in- frastructure-plan-2021-07-12/ (accessed 20.02.2022) 54 Global Gateway: up to €300 billion for the European Union’s strategy to boost sustainable links around the world. Europe- an Commission. 2021, December 01. URL: https://ec.europa.eu/commission/presscorner/detail/en/ip_21_6433 (accessed 20.02.2022) 53 A Globally Connected Europe: Council Approves Conclusions. European Council. 2021, July 12. URL: https://www.con- silium.europa.eu/en/press/press-releases/2021/07/12/a-globally-connected-europe-council-approves-conclusions/ (ac- cessed 20.02.2022) 54 Global Gateway: up to €300 billion for the European Union’s strategy to boost sustainable links around the world. Europe- an Commission. 2021, December 01. URL: https://ec.europa.eu/commission/presscorner/detail/en/ip_21_6433 (accessed 20.02.2022) 55 Emmott, R., & Siebold, S. After G7 Pledge, EU Seeks to Rival China’s ‘Belt and Road’ with Own Infrastructure Plan. Reuters, 2021, July 12. URL: https://www.reuters.com/world/china/after-g7-pledge-eu-seeks-rival-chinas-belt-road-with-own-in- frastructure-plan-2021-07-12/ (accessed 20.02.2022) Comprehensive sovereignty: an external perspective In response to the results of the G7 summit in Carbis Bay and following the European Commission’s Asian strategy called Connecting Europe and Asia – Building Blocks for an EU Strategy announced in 2018, the Council of the EU approved the basic framework for the initiative A Globally Connected Europe, a rival project of the BRI.53 It subsequently materialized in the project of the Global Gateway that explicitly declares the goal to expand the “EU’s democratic values”.54 The new global infrastructure plan is, however, prepared in close cooperation with Washington, as German Foreign Minister Heiko Maas avowed, and builds on partnerships with India and Japan.55 It will 2022, Vol. 1, No. 1 89 Ladislav Zemánek therefore be predisposed to become misused for the US-led confrontation with “autocracies”. The gradual integration of national military capability and formation of the EU army can also be subsumed into the strategic autonomy. Integration in this field poses serious risks in terms of the individual member states, at the same time, however, it can result in emancipation from Washington and NATO or a substantial transformation of the North Atlantic Alliance. Obviously, Borrell understands the importance of the strategic autonomy and its potential, never- theless, being still unable to overcome the Euro-Atlantic paradigm as demon- strated by the following words: “[N]o one disputes the vital character of the transatlantic relationship and no one advocates the development of a fully au- tonomous European force outside NATO, which remains the only viable frame- work to ensure the territorial defense of Europe”.56 In fact, the opposite is true. If the EU wants to position itself as an independent center of power, if it aspires to a sovereign policy, if the highest representatives are serious about the need for the EU to assume responsibility for itself, the Euro-Atlantic concept must be abandoned. The EU will not play an independent role in the world if remains subordinated to the US. Until the Union does not emancipate from Washing- ton, integration of the Eurasian macro-region, a common space from Lisbon to Vladivostok and Jakarta free of confrontation and dividing lines will be a mere vision. Notwithstanding Russia’s pivot to the East, Moscow still reiterates its read- iness to deepen cooperation with the EU. Formal ties began in 1997 when the Partnership and Cooperation Agreement (PCA) was concluded, establishing a general framework of the EU-Russian political and economic relations. 56 Borrell, J. B. Why European Strategic Autonomy Matters. European External Action Service. 2020, December 03. URL: https://eeas.europa.eu/headquarters/headquarters-homepage/89865/why-european-strategic-autonomy-matters_en (accessed 20.02.2022) 57 EU and Russia Launch New Partnership for Modernization. European Commission. 2010, June 01. URL: https://ec.europa. eu/commission/presscorner/detail/en/IP_10_649 (accessed 20.02.2022) eu/commission/presscorner/detail/en/IP_10_649 (accessed 20.02.2022) 58 Lavrov, S. Foreign Minister Sergey Lavrov’s Remarks at a Conference on Relations between Russia and the European Un- ion. The Ministry of Foreign Affairs of the Russian Federation. 2021a, May 31. URL: https://www.mid.ru/en/foreign_policy/ news/-/asset_publisher/cKNonkJE02Bw/content/id/4759042 (accessed 20.02.2022) 59 EU-Russia Relations: Commission and High Representative Propose the Way forward. European Commission. 2021, June 16. URL: https://ec.europa.eu/commission/presscorner/detail/en/ip_21_3010 (accessed 20.02.2022) 60 Joint Communication to the European Parliament, the European Council and the Council on EU-Russia relations – Push Back, Constrain and Engage. European Commission. 2021, June 16. URL: https://ec.europa.eu/info/sites/default/files/ joint-communication-eu-russia-relations.pdf (accessed 20.02.2022) 2022, Vol. 1, No. 1 57 EU and Russia Launch New Partnership for Modernization. European Commission. 2010, June 01. URL: https://ec.europa. eu/commission/presscorner/detail/en/IP_10_649 (accessed 20.02.2022) 58 Lavrov, S. Foreign Minister Sergey Lavrov’s Remarks at a Conference on Relations between Russia and the European Un- i Th Mi i t f F i Aff i f th R i F d ti 2021 M 31 URL htt // id / /f i li / news/-/asset_publisher/cKNonkJE02Bw/content/id/4759042 (accessed 20.02.2022) 59 EU-Russia Relations: Commission and High Representative Propose the Way forward. European Commission. 2021, June 16. URL: https://ec.europa.eu/commission/presscorner/detail/en/ip_21_3010 (accessed 20.02.2022) 60 Joint Communication to the European Parliament, the European Council and the Council on EU-Russia relations – Push Back, Constrain and Engage. European Commission. 2021, June 16. URL: https://ec.europa.eu/info/sites/default/files/ joint-communication-eu-russia-relations.pdf (accessed 20.02.2022) 58 Lavrov, S. Foreign Minister Sergey Lavrov’s Remarks at a Conference on Relations between Russia and the Eur ion. The Ministry of Foreign Affairs of the Russian Federation. 2021a, May 31. URL: https://www.mid.ru/en/forei news/-/asset_publisher/cKNonkJE02Bw/content/id/4759042 (accessed 20.02.2022) Ibid. 62 Lavrov, S. Foreign Minister Sergey Lavrov’s Remarks at a Conference on Relations between Russia and the European Un- ion. The Ministry of Foreign Affairs of the Russian Federation. 2021a, May 31. URL: https://www.mid.ru/en/foreign_policy/ news/-/asset_publisher/cKNonkJE02Bw/content/id/4759042 (accessed 20.02.2022) 63 Putin, V. Stat’ia Vladimira Putina «Byt’ otkrytymi, nesmotria na proshloe» (Putin: Be Open, Regardless of the Past). Kremlin. 2021, June 22. URL: http://kremlin.ru/events/president/news/65899 (accessed 20.02.2022) 64 Lavrov, S. Foreign Minister Sergey Lavrov’s Remarks at a Conference on Relations between Russia and the European Un- ion. The Ministry of Foreign Affairs of the Russian Federation. 2021a, May 31. URL: https://www.mid.ru/en/foreign_policy/ news/-/asset_publisher/cKNonkJE02Bw/content/id/4759042 (accessed 20.02.2022) Comprehensive sovereignty: an external perspective In 2003, both sides agreed on four “common spaces”, strengthening the strategic partner- ship. It included the creation of common economic space, cooperation in the field of both internal and external security and justice, research, education and culture. In 2010, Moscow and Brussels launched a new Partnership for Modern- ization on the 25th EU-Russian summit in Rostov-on-Don, addressing common Governance and Politics Governance and Politics 90 Political Science / Country in Focus UDC: 323, 327, 369.032 challenges and problems, for instance, those emerging from the global econom- ic crisis.57 Recently, Foreign Minister Lavrov reminded the progress made by both actors prior to 2014 as well as Russia’s proactiveness, when putting forward the European Security Treaty to establish a common security area, the idea of a common energy complex, the joint committee on foreign policy and secu- rity, cooperation in crisis management or visa-free regime, the latter being a step from realization. Mutual interaction was increasingly intensive at different levels, including the political one. Permanent Partnership Council as the main working body of the EU-Russia cooperation was active, joint summits were held every six months, the Russian Government and the European Commission held a joint session once a year.58 The gradual building of Greater Europe was a reality. However, this auspicious development failed in 2014. The EU suspended mutual initiatives, imposed sanctions and joined the US confrontational policy. Since 2016, the EU’s approach to Russia is guided by five principles: (1) full imple- mentation of the Minsk agreements; (2) strengthening the Eastern Partnership, involving other countries from the region; (3) building Union’s resilience in the field of energy security, hybrid threats or strategic communication; (4) selec- tive engagement with Moscow in chosen areas; (5) promoting people-to-people contacts and supporting Russian “civil society”.59 The official discourse vis-à-vis Russia remains hostile. The European Council arranged the first strategic debate on Russia in May 2021. The result was not a plan how to improve the relation- ship but a simple condemnation of the “illegal, provocative and disruptive Rus- sian activities against the EU, its Member States and beyond”.60 The political confrontation does not correspond with other ways of inter- action whatsoever. Irrespective of the restrictive measures, Russia is the EU’s fifth-largest trade partner while the EU remains Russia’s biggest trade partner. The EU also accounts for up to 75% of all foreign direct investment in the coun- 2022, Vol. 1, No. Governance and Politics 61 Ibid. Comprehensive sovereignty: an external perspective 1 91 Ladislav Zemánek try. Similar is the situation in the energy sector. Almost two thirds of Russia’s oil exports, two thirds of the gas exports and approximately half of its coal exports go to the EU. At the same time, up to 26% of EU oil imports and 40% of gas imports come from Russia.61 The economic interdependence has thus remained deep. The mutual interactions and exchange would even intensify in case of po- litical normalization. The Russian side has also repeatedly raised the question of linking the EAEU with the EU, but Brussels conditions the debate by normali- zation.62 The European (and Western) vector is absent in the 2021 NSS, but the Rus- sian leadership have sent messages that Moscow is interested in stable ties and revival of the strategic partnership. Vladimir Putin published an article in the German newspaper Die Zeit in June 2021. The Russian President speaks out in favor of “constructive interdependence”, “comprehensive partnership”, “com- mon space of equal cooperation, security and prosperity from the Atlantic to the Pacific”, thus reaffirming the continuity of the basic principles and goals of Russia’s European policy regardless of the shift after 2014. Putin emphasizes Russia’s cultural and historical affinity with Europe, expressing his long-term belief that the partnership is to be grounded in the close relationship between Moscow and Berlin.63 Sergei Lavrov presented a somewhat more skeptical atti- tude in December 2020, asserting that even though the EU could play a role of an independent pole in the multipolar global system, it has allegedly given up this ambition as shown by German and French recent policies following the concept of “rules-based multilateralism”.64 Multilateralism as such is positive (as opposed to Trump’s unilateralism) but the Western notion of it is exclusive and permeated with pseudo-universalism, moralism and superiority. In a sense, it is an expanded unilateralism as it does not accept “the others” on equal terms. The EU’s multilateralism is conditioned with adherence to the currently proclaimed Governance and Politics 92 Political Science / Country in Focus UDC: 323, 327, 369.032 European (Western) values and rules, to liberal democracy.65 The development of the rules-based order (RBO) can undermine the UN-based international sys- tem that is multilateral and inclusive. The RBO is liberal in its essence and de- nies the equal existence of different patterns and models, whether they are po- litical, economic or cultural. ( ) 70 News Conference Following Russian-Hungarian Talks. Kremlin. 2022, February 01. URL: http://en.kremlin.ru/events/presi- dent/news/67690 (accessed 20.02.2022) 65 Joint Communication to the European Parliament, the European Council and the Council on Strengthening the EU´s Con- tribution to Rules-Based Multilateralism. European Commission. (2021, February 17. URL: https://eeas.europa.eu/sites/de- fault/files/en_strategy_on_strengthening_the_eus_contribution_to_rules-based_multilateralism.pdf (accessed 20.02.2022) 66 Lavrov, S. (2021). O prave, pravakh i pravilakh (On Law, Rights and Rules). Global Affairs, June 28. URL: https://globalaffairs. ru/articles/o-prave-pravah-i-pravilah/ (accessed 20.02.2022) g g 69 Siebold, S., & Emmott, R. France and Germany Drop Russia Summit Plan after EU’s East Objects. Reuters. 2021, June 25. URL https://www.reuters.com/world/europe/france-germany-drop-plans-russia-summit-after-eu-outcry-2021-06-25/ (accessed 20.02.2022) / / p p p / ( ) 67 Rettman, A. Macron: EU Sanctions on Russia Do not Work. EU Observer. 2021, May 26. URL: https://euobse world/151946 (accessed 20.02.2022) 68 Merkel, Macron: ‘Russia Is a Big Challenge for Us’. Deutsche Welle. 2021, June 18. URL: https://www.dw.com/ macron-russia-is-a-big-challenge-for-us/a-57960118 (accessed 20.02.2022) 65 Joint Communication to the European Parliament, the European Council and the Council on Strengthening the EU´s Con- tribution to Rules-Based Multilateralism. European Commission. (2021, February 17. URL: https://eeas.europa.eu/sites/de- fault/files/en_strategy_on_strengthening_the_eus_contribution_to_rules-based_multilateralism.pdf (accessed 20.02.2022) 66 Lavrov, S. (2021). O prave, pravakh i pravilakh (On Law, Rights and Rules). Global Affairs, June 28. URL: https://globalaffairs. ru/articles/o-prave-pravah-i-pravilah/ (accessed 20.02.2022) 67 Rettman, A. Macron: EU Sanctions on Russia Do not Work. EU Observer. 2021, May 26. URL: https://euobserver.com/ world/151946 (accessed 20.02.2022) 68 Merkel, Macron: ‘Russia Is a Big Challenge for Us’. Deutsche Welle. 2021, June 18. URL: https://www.dw.com/en/merkel- macron-russia-is-a-big-challenge-for-us/a-57960118 (accessed 20.02.2022) 69 Siebold, S., & Emmott, R. France and Germany Drop Russia Summit Plan after EU’s East Objects. Reuters. 2021, June 25. URL https://www.reuters.com/world/europe/france-germany-drop-plans-russia-summit-after-eu-outcry-2021-06-25/ (accessed 20.02.2022) 70 News Conference Following Russian-Hungarian Talks. Kremlin. 2022, February 01. URL: http://en.kremlin.ru/events/presi- dent/news/67690 (accessed 20.02.2022) Comprehensive sovereignty: an external perspective These reasons lie behind Lavrov’s critical remarks. In another article, the Foreign Minister relates the EU’s reinterpretation of mul- tilateralism to the need for an ideological justification in maintaining the West´s declining power in the transforming world order.66 Russia wants the EU to be a pole in a multipolar system. Not a few political actors in Europe are interested in equal and win-win cooperation with its biggest neighbor, but the decision-making processes continue to be dominated by ad- herents of Euro-Atlanticism who obstruct normalization and a restart of mutual relations. Signs of a more constructive, rational and pragmatic policy can, nev- ertheless, be observed throughout the continent. French President Emmanuel Macron stated in May 2021 that anti-Russian sanctions were not working, call- ing upon the European Commission to rethink its confrontational position.67 Shortly after, Macron and Chancellor Angela Merkel declared that maintaining dialogue with Russia was crucial in terms of security and stability.68 Both leaders subsequently put forward the idea to convene the EU-Russia summit. However, the initiative was foiled by the Baltic states and Poland.69 Hungary, Greece, Italy and Portugal belong to countries that are in favor of normalization, or that are even developing relations with Moscow. Amidst tensions at the beginning of February 2022, Viktor Orbán made an official visit to Russia, showing an ex- ample of a constructive, win-win relationship despite Hungary’s membership in NATO and the EU.70 Viktor Orbán’s “illiberal democracy” with multi-vector 2022, Vol. 1, No. 1 93 Ladislav Zemánek external orientation and concurrent affiliation to Western structures is an in- spiring model. Furthermore, the prospective successful fulfilment of the Nord Stream 2 gas pipeline would be a symbol of win-win bilateral cooperation with Russia despite US sanctions. It seems that – under the current circumstances – bilateral interactions represent a more viable path compared to the EU-Russia level, which is extremely politicized. Increasing bilateral relations could subse- quently affect the collective decisions and actions. f Neither the EU nor Russia will be able to overcome the essential discrep- ancies in the years to come. The relationship will likely become a “cold partner- ship” (Fischer & Timofeev, 2020). It corresponds with the EU’s selective engage- ment policy presented by the Joint Communication on the EU’s relations with Russia in June 2021 in accordance with the five principles guiding the Union’s Russian strategy. Comprehensive sovereignty: an external perspective Josep Borrell summarized it as follows: “Our ambition should be to explore paths that could help change the current dynamics gradually into a more predictable and stable relationship. The EU will simultaneously push back, constrain and engage with Russia, based on a strong common understanding of Russia’s aims and an approach of principled pragmatism”.71 The dialectics of confrontation, containment, deterrence, sanctioning on one hand and moder- ate selective cooperation on the other seem to be the reality of the EU-Russia interactions of the coming years, whether this is called a “cold partnership” or “managed confrontation”. 71 EU-Russia Relations: Commission and High Representative Propose the Way forward. European Commission. 2021, June 16. URL: https://ec.europa.eu/commission/presscorner/detail/en/ip_21_3010 (accessed 20.02.2022) Conclusion Interactions between Russia and “the rest” can be characterized in terms of binary alternation of openness (and endeavor to follow and adopt external developmental patterns: political, economic, social or cultural) and introversion. Prior to 2014, Russia succeeded in integrating into the international structures in many respects but rather as an independent center with its own interests, goals and pretenses, not as a minor, marginal partner of the dominating West- ern powers. It gradually sobered up from the initial enthusiasm for the West 94 Governance and Politics Governance and Politics Political Science / Country in Focus UDC: 323, 327, 369.032 and the temptation of liberal democracy. Face to face with the NATO expan- sion eastwards, Western support for Chechen Islamists, separatist forces within Russia, anti-Russian forces in the post-Soviet countries, experiences with wars in Afghanistan, Iraq and Yugoslavia, the role of the Western countries in the color revolutions, weakening of the central role of the United Nations, stagna- tion of the West after the 2008 economic crisis and – last but not least –cultur- al wars and progressivist tendencies in the West, Russia revised its conceptual framework, put an end to Westernization, turned to the East and rejected the Western developmental pattern as an example to follow (Bratersky, 2014, p. 54- 55, 60-61). The final reversal happened in 2014 in connection with the crisis in Ukraine. The vision of Russia’s integration in Euro-Atlantic security systems including NATO did not take place either. If Yeltsin, Putin or Medvedev (and Gorbachev before them) hoped for the West to recognize Russia as an equal, full-fledged, important and sovereign member of a single Western community, they must have been deeply disappointed (Kotkin, 2001). Russia opened un- precedentedly to the West but the latter remained closed. What is Russia like today, then? The new, still emergent, post-Soviet Rus- sia is lacking a comprehensive ideology. It avoids extremes, rethinking its posi- tion and role in the international order. It rejected the radical openness of the 1990s, refusing to be an isolated fortress. It can rather be characterized as find- ing a middle path. The absence of a strong, high-profiled ideology need not be a shortcoming for the time being. On the contrary, pragmatism enables flexibility and synthesis of heterogeneous elements. The new Russia has brought an end to dogmatism, messianism and seeking utopia, be it religious (Holy Rus’, the Third Rome) or quasi-religious (Communism) (Lukin, 2018a, p. 2022, Vol. 1, No. 1 Conclusion 150). The recent state is close to the Chinese principle of seeking truth from facts. The new Russia wants to revive or safeguard positive traditions and social patterns while adopt- ing external elements where useful or necessary. The ruling elites have realized that building a strong state is an objective need and a rational choice for their country. Historical experiences teach them that Russia cannot get along without this social institute, as otherwise the society falls into chaos, as happened in the smutnoe vremia (Time of Troubles) of the early 17th century, after the fall of the monarchy in 1917 or in the 1990s. Andrei Tsygankov (2014, p. viii-ix) observes that a strong state has been advocated by socialists, liberals, conservatives as 95 Ladislav Zemánek well as Eurasianists, and so the question is not whether a strong state will be established in Russia or not – the only question is what the Russian strong state will be like. While combining traditional etatism or patrimonialism, social-lib- eral economic policies with conservatism in terms of values, Russia develops a “managed” or “illiberal” democracy internally. From the external point of view, Russia cultivates pragmatic relations with different countries, regions and organ- izations, putting an emphasis on the principles of peaceful coexistence. It wants to be engaged in global processes and institutions, pursuing integration but at the same time insists on independence, autonomy, non-interference. Learning lessons from both the past experiences and the present objective developmental tendencies, the Russian leadership has arrived at comprehensive sovereignty and stability which is the leading principle of official policies in post-2014 Russia. fi In search of a new model of the global order, maintaining some fundamen- tals of the present one, particularly the central role of the UN, while rejecting others with the unipolar US-based hegemonism at the top, Russia collides and clashes with revisionist attempts of the Western actors who oppose the idea of global polycentric architecture, being imbued with cold-war mentality, confron- tational notion of a permanent struggle against “autocrats of the world”, and superiority of liberal democratic model as well as Western values. These char- acteristics are embodied in the concept of the “rules-based international order” that substitutes the international law and principles emerging from the UN for particularist moral categories and haphazardly introduced rules. Such pressure inevitably provokes resistance among the “rest”. 72 Antonov, A., & Qin, G. Russian and Chinese Ambassadors: Respecting People’s Democratic Rights. National Interest. 2021, November 26. URL: https://nationalinterest.org/feature/russian-and-chinese-ambassadors-respecting-people’s-demo- cratic-rights-197165 (accessed 20.02.2022) Conclusion The concurrent containment of Russia and China has also resulted in an unprecedented level of mutual un- derstanding and cooperation between the Eurasian major powers. Both coun- tries’ ambassadors in Washington called for a “harmonious coexistence between countries with different social systems, ideologies, histories, cultures, and devel- opment levels” as a basis of what China calls the “community with a shared fu- ture for mankind”72 in November 2021. Against the Western hegemonism, these Eurasian major powers uphold the legitimacy of pluriversum and diversity, in- Governance and Politics Governance and Politics 96 Political Science / Country in Focus UDC: 323, 327, 369.032 cluding multiple modernities or democracies. This lower-level declaration was reaffirmed by the joint statement of President Xi Jinping and Vladimir Putin “on international relations entering a new era and global sustainable development”, made on the occasion of the 24th Olympic Winter Games in Beijing in Febru- ary 2022. The document can be perceived as a manifesto against hegemonism and militarism as seen in the ongoing NATO expansion or the establishment of AUKUS, thus promoting cooperation and solidarity instead of confrontation and divisions.73 Euro-Atlanticism will be a source of frictions and one of the major obstacles to a peaceful move forward, global restructuring, and the establishment of a common security area including both Europe and Russia. Unless Washington gives up its claims to control the European subcontinent, a sustainable solution of conflicts between Russia and Europe will not be reached. And genuine nego- tiations about Moscow’s security concerns and national interests are also unlike- ly to be held, as was shown by the intensified interactions and crisis bargaining in the first weeks of 2022, in which the US and Joe Biden’s himself played an inauspicious role.74 Russia’s security requirements aimed at a multilateral, com- prehensive security settlement in Europe were denied.75 A new impetus to Euro-Atlanticism was given by Biden’s electoral victo- ry. The US President managed to revive the mutual partnership and Western multilateralism based on liberal principles and embodied by the rules-based order. Washington made necessary concessions to overcome the mistrust that emerged under Donald Trump and amid scandals concerning US spying on their European allies.76 The EU elites, however, cannot forget the experience with Biden’s predecessor who exposed the allies to serious uncertainty. Trump’s unilateralism and “America First” policy contributed to the development and legitimacy of the strategic autonomy in the EU. Paradoxically, Trumpism played 2022, Vol. 76 Henley, J. Denmark Helped US Spy on Angela Merkel and European Allies – Report. The Guardian, 2021, May 31. URL: https://www.theguardian.com/world/2021/may/31/denmark-helped-us-spy-on-angela-merkel-and-european-allies-re- port (accessed 20.02.2022) 73 Joint Statement of the Russian Federation and the People’s Republic of China on the International Relations Entering a New Era and the Global Sustainable Development. Kremlin. 2022, February 04. URL: http://en.kremlin.ru/supplement/5770 74 Tsygankov, A. P. Liberal Messianism and the Ukraine Crisis Have Turned Joe Biden into a Russia Hawk. Canadian Dimen- sion. 2022, February 17. https://canadiandimension.com/articles/view/liberal-messianism-and-the-ukraine-crisis-have- turned-joe-biden-into-a-russia-hawk (accessed 20.02.2022) 73 Joint Statement of the Russian Federation and the People’s Republic of China on the International Relations Entering a New Era and the Global Sustainable Development. Kremlin. 2022, February 04. URL: http://en.kremlin.ru/supplement/5770 74 Tsygankov, A. P. Liberal Messianism and the Ukraine Crisis Have Turned Joe Biden into a Russia Hawk. Canadian Dimen- sion. 2022, February 17. https://canadiandimension.com/articles/view/liberal-messianism-and-the-ukraine-crisis-have- turned-joe-biden-into-a-russia-hawk (accessed 20.02.2022) 75 Obrashchenie Prezidenta Rossiiskoi Federatsii (Presidential Address). Kremlin. 2022, February 21. URL: http://kremlin.ru/ events/president/news/67828 (accessed 20.02.2022) 76 Henley, J. Denmark Helped US Spy on Angela Merkel and European Allies – Report. The Guardian, 2021, May 31. URL: https://www.theguardian.com/world/2021/may/31/denmark-helped-us-spy-on-angela-merkel-and-european-allies-re- port (accessed 20.02.2022) 73 Joint Statement of the Russian Federation and the People’s Republic of China on the International Relations Entering a New Era and the Global Sustainable Development. Kremlin. 2022, February 04. URL: http://en.kremlin.ru/supplement/5770 73 Joint Statement of the Russian Federation and the People’s Republic of China on the International Relations Entering a New Era and the Global Sustainable Development. Kremlin. 2022, February 04. URL: http://en.kremlin.ru/supplement/5770 74 Tsygankov, A. P. Liberal Messianism and the Ukraine Crisis Have Turned Joe Biden into a Russia Hawk. Canadian Dimen- sion 2022 February 17 https://canadiandimension com/articles/view/liberal messianism and the ukraine crisis have chenie Prezidenta Rossiiskoi Federatsii (Presidential Address). Kremlin. 2022, February 21. URL: http://kremlin.ru/ president/news/67828 (accessed 20.02.2022) Conclusion 1, No. 1 97 Ladislav Zemánek a progressive role, and made the “autonomization” of individual countries or regions (the US, the EU, China, Russia) inevitable. All these actors have set out a path of development for mobilizing and focusing on domestic sources and po- tential, and deepening integration with their close partners, thus reaffirming the tendency towards regionalization within globalization. The EU’s strategic au- tonomy, Russia’s comprehensive concept of sovereignty and China’s dual circu- lation can be interpreted as a manifestation of the transformation of the global order towards polycentrism. As such, it may avert a slide into a new, China-US bipolarity, even though bipolarity coincides with the West’s predisposition to a dualist or Manichaean way of thinking (Zhao & Kortunov, 2020). This shift entails the risk of deglobalization, particularism and conflicts, but I expect that given the existing long-term tendencies and trajectories the scenario of glocali- zation and pragmatic polycentrism will prevail eventually. References Brands, H. (2021). The Emerging Biden Doctrine: Democracy, Autocracy, and the Defining Clash of Our Time. Foreign Affairs, June 29. URL: https://www.foreignaffairs.com/articles/unit- ed-states/2021-06-29/emerging-biden-doctrine (accessed: 14.01.2022)f Bratersky, M. (2014). Transformation of Russia’s Foreign Policy. Russia in Global Affairs, 12(2), 54– 62. Diesen, G. (2021). Russian Conservatism: Managing Change under Permanent Revolution. Rowman & Littlefield. Fischer, S., & Timofeev, I. (2020). Alternative futures of EU-Russia relations in 2030. EUREN, No- vember. URL: https://eu-russia-expertnetwork.eu/en/sabine-fischer-ivan-timofeev-alternative-fu- tures-of-eu-russia-relations-in-2030/ (accessed: 14.01.2022) Gvozdev, N. K., & Marsh, C. (2014). Russian Foreign Policy: Interests, Vectors, and Sectors. CQ Press. Hann, C. (2016). A Concept of Eurasia. Current Anthropology, 57(1), 1–27. DOI: 10.1086/684625l Kaczmarski, M. (2017). Two Ways of Influence-building: The Eurasian Economic Union and the One Belt, One Road Initiative. Europe-Asia Studies, 69 (7), 1027-1046. DOI: 10.1080/09668136.2017.1373270 Kendall-Taylor, A., & Shullman, O. (2021). China and Russia’s Dangerous Convergence. How to Counter an Emerging Partnership. Foreign Affairs, May 03. URL: https://www.foreignaffairs.com/arti- cles/china/2021-05-03/china-and-russias-dangerous-convergence (accessed: 14.01.2022) Kortunov, A. (2021). Administratsiia Dzho Baidena i Rossiia (Administration of Joe Biden and Rus- sia). Russian International Affairs Council. URL: https://russiancouncil.ru/papers/Biden-Russia-Paper60. pdf (in Russian) (accessed: 14.01.2022) Kotkin, S. (2001). Armageddon Averted: The Soviet Collapse, 1970-2000. Oxford University Press. Lavrov, S. V. (2019). The World at a Crossroads and a System of International Relations for the Future. Russia in Global Affairs, 17(4), 8–18. Lukin, A. (2018a). Putin’s Political Regime and Its Alternatives. Strategic Analysis, 42(2), 134-153. DOI: 10.1080/09700161.2018.1439331 98 Governance and Politics Governance and Politics UDC: 323, 327, 369.032 Political Science / Country in Focus Lukin, A. (2018b). China and Russia. The New Rapprochement. Polity Press. Lukin, A., & Novikov, D. (2021). Greater Eurasia: From Geopolitical Pole to International Society? In G. Diesen, & A. Lukin (Eds.), The Return of Eurasia. Continuity and Change. Palgrave Macmillan, 33–78. Lunacharskii, A. V. (1908). Religiia i sotsializm. 2 V. Shipovnik. Pipes, R. (1974). Russia under the Old Regime. Charles Scribner’s Sons. Romashkina, N. (2019). Informatsionnyi suverenitet ili pochemu Rossii nuzhna strategiia infor- matsionnoi bezopasnosti (Information Sovereignty or Why Russia Needs an Information Security Strategy). 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URL: https://carnegiemoscow.org/commentary/84893 (accessed: 14.01.2022) Tsygankov, A. P. (2014). The Strong State in Russia: Development and Crisis. Oxford University Press. Zemánek, L. (2020). Belt & Road Initiative and Russia: From Mistrust Towards Cooperation. Human Affairs, 30(2), 199-211. DOI: 10.1515/humaff-2020-0019 Zemánek, L. (2021). Czech RussiaGate: Contemporary McCarthyism in Practice. China-CEE Insti- tute, June 15. URL: https://china-cee.eu/2021/06/15/czech-republic-political-briefing-czech-rus- siagate-contemporary-mccarthyism-in-practice/ (accessed: 14.01.2022) Zemánek, L. (2021). Chastening Experience: Cooperation with Russia Fatal. China-CEE Institute, June 03. URL: https://china-cee.eu/2021/06/03/czech-republic-external-relations-briefing-chastening-ex- perience-cooperation-with-russia-fatal/ (accessed: 14.01.2022) Zhao, H., & Kortunov, A. (2020). The Coming Bipolarity and Its Implications: Views from China and Russia. Russian International Affairs Council, November 23. URL: https://russiancouncil.ru/en/analyt- ics-and-comments/analytics/the-coming-bipolarity-and-its-implications-views-from-china-and-rus- sia/ (accessed: 14.01.2022) 2022, Vol. 1, No. 1 99 99
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Microbiological Oropharyngeal Patterns in Patients with Different Phenotypes of Chronic Obstructive Pulmonary Disease
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Microbiological Oropharyngeal Patterns in Patients with Different Phenotypes of Chronic Obstructive Pulmonary Disease DOI: 10.17691/stm2018.10.2.11 Received April 27, 2017 DOI: 10.17691/stm2018.10.2.11 Received April 27, 2017 M.A. Karnaushkina, MD, PhD, Associate Professor, Department of Hospital Therapy No.21; M.A. Karnaushkina, MD, PhD, Associate Professor, Department of Hospital Therapy No.21; S.V. Fedosenko, MD, Assistant, Department of General Practice and Polyclinic Therapy2; A.E. Sazonov, PhD, Senior Researcher, Central Research Laboratory2; V.A. Petrov, Junior Researcher, Central Research Laboratory2; D.Yu. Ovsyannikov, MD, DSc, Head of the Department of Pediatrics3; L.M. Ogorodova, MD, DSc, Professor, Corresponding Member of the Russian Academy of Sciences, Head of the Department of Pediatrics with the Course of Childhood Diseases, Faculty of Medicine2 1I.M. Sechenov First Moscow State Medical University, 8/2 Trubetskaya St., Moscow, 119991, Russia; 2Siberian State Medical University, 2 Moskovsky trakt, Tomsk, 634050; 3Peoples’ Friendship University of Russia, 6 Miklukho-Maklaya St., Moscow, 117198, Russia p p S.V. Fedosenko, MD, Assistant, Department of General Practice and Polyclin A.E. Sazonov, PhD, Senior Researcher, Central Research Labo V.A. Petrov, Junior Researcher, Central Research Laboratory2; , , y ; D.Yu. Ovsyannikov, MD, DSc, Head of the Department of Pediatrics3; y p L.M. Ogorodova, MD, DSc, Professor, Corresponding Member of the Russian Academy of Sciences, Head of the Department of Pediatrics with the Course of Childhood Diseases, Faculty of Medicine2 . Sechenov First Moscow State Medical University, 8/2 Trubetskaya St., Moscow, 119991, Russia; berian State Medical University, 2 Moskovsky trakt, Tomsk, 634050; 1I.M. Sechenov First Moscow State Medical University, 8/2 Trubetskaya St., Moscow, 119991, Russia; 2Sib i St t M di l U i it 2 M k k t kt T k 634050 y, y , , ; 3Peoples’ Friendship University of Russia, 6 Miklukho-Maklaya St., Moscow, 117198, Russia Persistent bronchial inflammation in chronic obstructive pulmonary disease (COPD) is considered the cause of ventilation disorders and related contamination with conditionally pathogenic microorganisms; the latter can proceed and transform into a full infection, which can aggravate and exacerbate COPD. The aim of the study was to evaluate the relations between the oropharyngeal microbiota in patients with COPD functional, and prognostic parameters of the disease. Materials and Methods. 64 patients with COPD were included in the study; the participants were scheduled to visit our clinic on two occasions. In the first visit, their medical history was studied in detail and the major examination procedures were conducted. сlinical medicine Microbiological Oropharyngeal Patterns in Patients with Different Phenotypes of Chronic Obstructive Pulmonary Disease DOI: 10.17691/stm2018.10.2.11 Received April 27, 2017 M.A. Karnaushkina, MD, PhD, Associate Professor, Department of Hospital Therapy No.21; S.V. Fedosenko, MD, Assistant, Department of General Practice and Polyclinic Therapy2; A.E. Sazonov, PhD, Senior Researcher, Central Research Laboratory2; V.A. Petrov, Junior Researcher, Central Research Laboratory2; D.Yu. Ovsyannikov, MD, DSc, Head of the Department of Pediatrics3; L.M. Ogorodova, MD, DSc, Professor, Corresponding Member of the Russian Academy of Sciences, Head of the Department of Pediatrics with the Course of Childhood Diseases, Faculty of Medicine2 1I.M. Sechenov First Moscow State Medical University, 8/2 Trubetskaya St., Moscow, 119991, Russia; 2Siberian State Medical University, 2 Moskovsky trakt, Tomsk, 634050; 3Peoples’ Friendship University of Russia, 6 Miklukho-Maklaya St., Moscow, 117198, Russia Persistent bronchial inflammation in chronic obstructive pulmonary disease (COPD) is considered the cause of ventilation disorders сlinical medicine Microbiological Oropharyngeal Patterns in Patients with Different Phenotypes of Chronic Obstructive Pulmonary Disease DOI: 10.17691/stm2018.10.2.11 Received April 27, 2017 M.A. Karnaushkina, MD, PhD, Associate Professor, Department of Hospital Therapy No.21; S.V. Fedosenko, MD, Assistant, Department of General Practice and Polyclinic Therapy2; A.E. Sazonov, PhD, Senior Researcher, Central Research Laboratory2; V.A. Petrov, Junior Researcher, Central Research Laboratory2; D.Yu. Ovsyannikov, MD, DSc, Head of the Department of Pediatrics3; L.M. Ogorodova, MD, DSc, Professor, Corresponding Member of the Russian Academy of Sciences, Head of the Department of Pediatrics with the Course of Childhood Diseases, Faculty of Medicine2 1I.M. Sechenov First Moscow State Medical University, 8/2 Trubetskaya St., Moscow, 119991, Russia; 2Siberian State Medical University, 2 Moskovsky trakt, Tomsk, 634050; 3Peoples’ Friendship University of Russia, 6 Miklukho-Maklaya St., Moscow, 117198, Russia Persistent bronchial inflammation in chronic obstructive pulmonary disease (COPD) is considered the cause of ventilation disorders сlinical medicine Microbiological Oropharyngeal Patterns in Patients with Different Phenotypes of Chronic Obstructive Pulmonary Disease DOI: 10.17691/stm2018.10.2.11 Received April 27, 2017 M.A. Karnaushkina, MD, PhD, Associate Professor, Department of Hospital Therapy No.21; S.V. Fedosenko, MD, Assistant, Department of General Practice and Polyclinic Therapy2; A.E. Sazonov, PhD, Senior Researcher, Central Research Laboratory2; V.A. Petrov, Junior Researcher, Central Research Laboratory2; D.Yu. Ovsyannikov, MD, DSc, Head of the Department of Pediatrics3; L.M. Ogorodova, MD, DSc, Professor, Corresponding Member of the Russian Academy of Sciences, Head of the Department of Pediatrics with the Course of Childhood Diseases, Faculty of Medicine2 1I.M. Sechenov First Moscow State Medical University, 8/2 Trubetskaya St., Moscow, 119991, Russia; 2Siberian State Medical University, 2 Moskovsky trakt, Tomsk, 634050; 3Peoples’ Friendship University of Russia, 6 Miklukho-Maklaya St., Moscow, 117198, Russia P i t t b hi l i fl ti i h i b t ti l di (COPD) i id d th f til ti di d сlinical medicine сlinical medicine Microbiological Oropharyngeal Patterns in Patients with Different Phenotypes of Chronic Obstructive Pulmonary Disease Those included an assessment of the respiratory function, the 6-minute walk test, the degree of dyspnea by the Medical Research Council scale, body plethysmography, the diffusion capacity of the lungs, and a chest CT scan. The second visit took place 12 months after the first one to assess the changes in the course of the disease. The result was considered negative if, in the second examination, the patient‘s condition was found more severe. Oropharyngeal samples of all patients were sequenced to identify the V3–V4 variable sites of the 16S rRNA gene. Results. It is found that the microbiological oropharyngeal patterns in COPD patients depend on the source of micro-aspiration. In addition, the changes in the oropharyngeal microbiota correlate with the severity and prognosis of the disease, as well as the patient phenotype. Based on the data obtained by sequencing parts of the 16S rRNA gene, the role of oropharyngeal microbiota in determining the course and prognosis of COPD has been elucidated. Conclusion. The presented clinical and functional characteristics associated with oropharyngeal microbiota indicate that micro- aspirations from other body compartments not only affect the composition of oropharyngeal microbiota in patients with COPD but also have an important prognostic significance. Key words: COPD; oropharyngeal microbiota; sequencing; COPD phenotypes; severity. Key words: COPD; oropharyngeal microbiota; sequencing; COPD phenotypes; severity. Corresponding author: Maria A. Karnaushkina, e-mail: kar3745@yandex.ru Introduction The authors suggested their research would shed light on the causes of chronic pulmonary disorders. However, none of the published studies analyzed the respiratory microbiota in respect to the disease progression and its clinical/functional parameters [8–11]. This may be due to the fact that, firstly, COPD is a clinically heterogeneous disease, and secondly, determination of the microbiota composition using classical microbiological methods does not provide a complete picture of its composition.i bronchodilation test and the pulmonary diffusion index (body plethysmography). The study was performed using a MasterScreen Body instrumentation (Erich Jaeger, Germany). The obtained data were compared to the reference values calculated by the generally accepted formulas recommended by the European Respiratory Society and American Thoracic Society. All patients underwent computed tomography scan of the chest (chest CT) with a Somatom Sensation 40 spiral CT scanner (Siemens, Germany). Densitometry of the lung tissue and bronchial structure was performed using the Pulmo CT program (Siemens, Germany). During the first visit, oropharyngeal swabs were taken from all patients. Based on the clinical and functional parameters obtained at the examination, the patients were divided into groups according to three phenotypes of COPD: “chronic bronchitis” (n=30), “emphysema” (n=14), and “mixed” (n=20). This stratification was based on the emphysema and bronchitis phenotypes as proposed by Burrows (1966) and Dornhorst (1955) and modified by us. In this study, we used the phenotypic classification of COPD based on clinical and functional differences between the patients. As compared with the determination of the forced expiratory volume (FEV1) alone, the proposed parameter better reflects the severity and prognosis of the disease. Here, the composition of microbiota was determined using the methods of molecular-genetic identification of microorganisms [1, 4], which significantly changed our knowledge about the oropharyngeal microbiome. The obtained results enabled us to propose a prognostic model to predict the severity of COPD based on the taxonomic composition of the oropharyngeal microbiota. The phenotype of “chronic bronchitis” describes patients whose major clinical symptom (aside from exacerbation periods) was cough with mucopurulent sputum. At auscultation, harsh breathing sounds and rales were detected; the chest CT showed signs of bronchitis, the diffusion lung capacity was normal. The “emphysema” phenotype refers to patients whose major clinical symptom (aside from exacerbation periods) was shortness of breath; cough was mild and unproductive. Introduction At auscultation, a weakened vesicular breath with wheezing episodes was heard; the characteristic signs and a decreased diffusion capacity were present in the CT scans. The aim of the study was to evaluate the relations between the oropharyngeal microbiota in patients with COPD and the clinical, functional, and prognostic parameters of the disease. Patients, who showed no obvious signs of bronchitis or emphysema during non-exacerbation periods, were included in the “mixed” phenotype group. Introduction contamination of the bronchial tree in COPD has been implicated in the increasing risk of COPD transformation into an infectious process with its exacerbations and disease progression [3, 4]. However, the involvement of the respiratory microbiota associated with the COPD phenotype in this process is still debatable. The development of ventilation disorders in chronic obstructive pulmonary disease (COPD) is manifested by a progressive decrease in airflow associated with an abnormal inflammatory response of the lungs to harmful particles [1, 2], including microorganisms [1]. At the same time, chronic bronchopulmonary inflammation and a decreased airflow create conditions for contamination of the respiratory tract by opportunistic and pathogenic flora that change the composition of the respiratory microbiota [1, 3]. In recent publications, the bacterial Thus, Chambers et al. [5] demonstrated that the microbiota in the respiratory tract of patients with predominantly severe COPD significantly differed from the bacterial composition of the airways in healthy individuals. In the COPD patients, there were less Bacteroides spp. and an increased representation Corresponding author: Maria A. Karnaushkina, e-mail: kar3745@yandex.ru СТМ ∫ 2018 — vol. 10, No.2 101 СТМ ∫ 2018 — vol. 10, No.2 101 Microbiological Oropharyngeal Patterns in Patients with Different Phenotypes of COPD сlinical medicine of Firmicutes and Proteobacteria. Similar results were obtained by Charlson et al. [6]. It has been also suggested that the abnormal changes in microbiota could be initiated at the perinatal period. Thus, Górecka  D. and Puścińska E. [7] analyzed the composition of the respiratory tract microbiota in a newborn and its correlation with baby’s nutrition, and the use of antibiotics by the mother. The authors suggested their research would shed light on the causes of chronic pulmonary disorders. However, none of the published studies analyzed the respiratory microbiota in respect to the disease progression and its clinical/functional parameters [8–11]. This may be due to the fact that, firstly, COPD is a clinically heterogeneous disease, and secondly, determination of the microbiota composition using classical microbiological methods does not provide a complete picture of its composition.i of Firmicutes and Proteobacteria. Similar results were obtained by Charlson et al. [6]. It has been also suggested that the abnormal changes in microbiota could be initiated at the perinatal period. Thus, Górecka  D. and Puścińska E. [7] analyzed the composition of the respiratory tract microbiota in a newborn and its correlation with baby’s nutrition, and the use of antibiotics by the mother. M.A. Karnaushkina, S.V. Fedosenko, A.E. Sazonov, V.A. Petrov, D.Yu. Ovsyannikov, L.M. Ogorodova Materials and Methods this du wit pat CO and mo res dat Comparative characte with different phenoty (Me [Q1; Q3]) Parameters Exacerbations for 12 months Existence of comorbid disorders (n/%) “Triple” therapy, per year (n/%) The number of antibacterial courses, per year BODE index (scores) were tested for the presence of the and the V3–V4 sites sequences 16S Metagenomic Sequencing Lib protocol recommended by Illumina (U sequencer. A taxonomic classificat out according to the sequence data rRNA gene of Greengenes v. 13.5 wi Bayesian classifier and the QIIME (Qu into Microbial Ecology) software. T differences between the phenotype- specified groups of samples at the level of the operating taxonomic unit (OTU), a method based on linear regression analysis and adjusted to the metagenome data by the metagenomeSeq package was used [12]. To detect clusters of microorganisms, the CoNet plugin version 1.1.beta [13] of the Cytoscape software was utilized. Correlations between the amounts of individual microorganisms were determined according to the Spearman method, only the correlations with a coefficient of 0.05 or higher were considered. To quantify the impact of the oropharyngeal microbiota on COPD progression, a prognostic model was developed using the caret package, where the recursive elimination of variables with a 10-fold cross- validation was applied. The composition of oropharyngeal microbiota in patients with different clinical and functional types of COPD was dominated by the Streptococcus, Veillonella, and Prevotella genera of microorganisms (10% and more) in all phenotypes of the disease (Figure 1). Our results on these “highly represented taxa” agree with the data reported by others [14–17]. Comparative characteristics of patients (the extended examination group) with different phenotypes of chronic obstructive pulmonary disease (Me [Q1; Q3]) Parameters COPD phenotypes (n=64) p1–2 p1–3 p2–3 1. “Chronic bronchitis” (n=30) 2. “Emphysema” (n=14) 3. Materials and Methods During the second visit, which took place 12 months after the first one, all patients underwent a clinical and functional examination, an assessment of the exacerbation rate, an evaluation of dyspnea by the MRC scale, a determination of exercise tolerance and a test of external respiration using a bronchodilator probe. We analyzed the dynamics of COPD during the past 12 months of observation, taking into account the severity of the symptoms, the post-bronchodilation value of FEV1 and the risk of exacerbations (GOLD 2015). The dynamics of the disease was considered negative if, at the second examination, the patient presented with a more severe course of COPD than he did on the first visit. The oropharyngeal samples were not taken during the second visit. This prospective non-interventional study included 64 patients with COPD. The average age was 57 (50–63) years. All patients had confirmed COPD according to the GOLD 2015 criteria [1, 2]; the diagnosis was made at least 12 months before the enrollment, and their smoking index was higher than 10 packs/year. For 4 weeks prior to the enrollment, they received no antibiotic therapy. The study was conducted in accordance with the Helsinki Declaration (2013) and approved by the  local Ethics Committee of the Siberian State Medical University (protocol No.1927 of 28.03.2011). An informed consent has been obtained from each patient. The study included two patient visits. During the first visit, all patients were clinically examined and their relevant medical history was carefully studied (below we will refer to these patients as the extended examination group). Tolerance to exercise was determined using a 6-minute walk test. The degree of dyspnea was assessed by the MRC (Medical Research Council) scores; the external respiration was evaluated using the The Statistica 10.0 software package was used for statistical processing of the medical history data and the clinical/functional indices. When comparing the qualitative characteristics, the criterion χ2 was applied. To calculate the difference of the means between non- paired samples, the Mann–Whitney U test was used. The qualitative data are presented as absolute or 102 СТМ ∫ 2018 — vol. 10, No.2 M.A. Karnaushkina, S.V. Fedosenko, A.E. Sazonov, V.A. Petrov, D.Yu. Ovsyannikov, L.M. Ogorodova сlinical medicine relative (%) values. Materials and Methods “Mixed” (n=20) Exacerbations for 12 months 2.0 [1.0; 2.0] 2.0 [1.6; 2.0] 5.0 [3.8; 5.0] p1–2>0.05 p1–3<0.05 p2–3<0.0001 Existence of comorbid disorders (n/%) 7.0/23.3 3.0/21.4 16.0/80.0 p1–2>0.05 p1–3<0.001 p2–3<0.001 “Triple” therapy, per year (n/%) 1.0/3.3 1.0/7.1 9.0/45.0 p1–2<0.05 p1–3<0.0001 p2–3<0.0001 The number of antibacterial courses, per year 2.0 [1.0; 3.0] 1.0 [1.0; 1.8] 3.0 [1.8; 5.0] p1–2<0.05 p1–3<0.05 p2–3<0.0001 BODE index (scores) 2.0 [1.0; 2.0] 2.0 [1.0; 2.8] 5.0 [4.0; 6.0] p1–2>0.05 p1–3<0.05 p2–3<0.05 MRC (scores) 2.0 [1.0; 2.0] 2.0 [1.0; 2.8] 3.0 [2.0; 4.0] p1–2>0.05 p1–3<0.0001 p2–3=0.015 6-minute walk test (m) 500 [426; 518] 380 [348; 398] 308 [224; 376] p1–2<0.05 p1–3<0.05 p2–3<0.0001 FEV1 after bronchodilation (% of reference value) 68.5 [59.0; 71.0] 68.0 [58.0; 75.0] 48.0 [45.0; 49.0] p1–2>0.05 p1–3<0.05 p2–3<0.05 SpO2 after 6-minute walk (%) 98.0 [97.0; 99.0] 96.0 [95.0; 99.0] 94.0 [93.0; 95.0] p1–2>0.05 p1–3<0.05 p2–3<0.05 Comparative characteristics of patients (the extended examination group) with different phenotypes of chronic obstructive pulmonary disease (Me [Q1; Q3]) Materials and Methods The quantitative data are presented as follows: those with a non-normal distribution — as Me [Q25; Q75]; and those with the normal distribution  — as M±SD, where M is the arithmetic mean, SD is the standard deviation. The normality of distributions of was tested with the χ2 criterion. The difference of the means was considered statistically significant at p<0.05. The Spearman coefficient was used for the correlation analysis. The strength of the correlation was denoted as strong at ±0.7 to ±1; average at ±0.3 to ±0.699; and weak at 0 to ±0.299. with the two other phenotypes, “chronic bronchitis” and “emphysema”, patients with the “mixed” phenotype had more exacerbations per year and a higher value of the BODE index. The FEV1 values for the “mixed” phenotype were significantly lower than those for the two other phenotypes (see the Table). The analyzed data indicated that patients with the “mixed” COPD phenotype were treated with inhalations recommended for patients with severe and extremely severe COPD: long-acting anticholinergic drugs + long-acting β2-agonists + inhaled glucocorticoids. In this group, the number of antibacterial therapy courses during the year was significantly higher as compared with the two other groups. Samples of oropharyngeal swabs of all patients were tested for the presence of the 16S rRNA gene and the V3–V4 sites sequences according to the 16S Metagenomic Sequencing Library Preparation protocol recommended by Illumina (USA) for the MiSeq sequencer. A taxonomic classification was carried out according to the sequence database of the 16S rRNA gene of Greengenes v. 13.5 with the help of the Bayesian classifier and the QIIME (Quantitative Insights into Microbial Ecology) software. To find taxonomic differences between the phenotype- specified groups of samples at the level of the operating taxonomic unit (OTU), a method based on linear regression analysis and adjusted to the metagenome data by the metagenomeSeq package was used [12]. To detect clusters of microorganisms, the CoNet plugin version 1.1.beta [13] of the Cytoscape software was utilized. Correlations between the amounts of individual microorganisms were determined according to the Spearman method, only the correlations with a coefficient of 0.05 or higher were considered. To quantify the impact of the oropharyngeal microbiota on COPD progression, a prognostic model was developed using the caret package, where the recursive elimination of variables with a 10-fold cross- validation was applied. Results and Discussion In this study, a comparative intergroup analysis of the clinical and functional parameters of patients with different COPD phenotypes was performed. As a result, clinically important data were obtained to suggest that patients with the “mixed” phenotype of COPD had the most severe manifestations of the disease. Thus, in comparison СТМ ∫ 2018 — vol. 10, No.2 103 Microbiological Oropharyngeal Patterns in Patients with Different Phenotypes of COPD СТМ ∫ 2018 — vol. 10, No.2 103 Microbiological Oropharyngeal Patterns in Patients with Different Phenotypes of COPD сlinical medicine The metagenomeSeq-assisted analysis of low- represented taxa (less than 10% of the identified microbiome) revealed a predominance of bacterial commensals in patients with the phenotype of emphysema. Among those, the Firmicutes bacteria (Acidaminococcus LogFC 1.865; Anaerovibrio LogFC 2.653; Leuconostoc LogFC 1.727; Oceanobacillus LogFC 1.107; Phascolarctobacterium LogFC 2.915; Tetragenococcus LogFC 1.513), the Actinobacteria Actinobacteria, Proteobacteria, Bacteroidetes, Firmicutes, Bacteroides, mainly due to a higher content of opportunistic bacteria of Haemophilus (LogFC 8.662), Actinomyces (LogFC 9.813), Lactobacillus (LogFC 4.758), Leptotrichia (LogFC 10.556), Porphyromonas (LogFC 5.896), Bacillus (LogFC 3.161), Gemella (LogFC 8.303) (p<0.001). Also in these patients, a considerable amount of opportunistic bacteria in the oropharynx was found: Prevotella melaninogenica species (LogFC The “chronic bronchitis” phenotype The “emphysema” phenotype Streptococcus 29% Granulicatella 2% Rothia 2% Actinomyces 3% Porphyromonas 3% Fusobacterium 4% Haemophilus 4% Leptotrichia 4% Veillonella 19% Veillonella 19% other 4% Prevotella 19% Prevotella 19% Streptococcus 22% other 5% Haemophilus 3% Porphyromonas 3% Fusobacterium 5% campylobacter 2% Actinomyces 4% Neisseria 4% Leptotrichia 6% The “mixed” phenotype other 3% Rothia 3% Streptococcus 31% Porphyromonas 2% Granulicatella 2% Fusobacterium 2% Leptotrichia 3% Neisseria 3% Actinomyces 3% Haemophilus 4% Veillonella 17% Prevotella 21% Figure 1. Prevalent genera of microorganisms, comprising 95% of the metagenome of the oropharyngeal microbiota in patients with chronic obstructive pulmonary disease (the extended examination group) The “emphysema” phenotype Veillonella 19% Prevotella 19% Streptococcus 22% other 5% Haemophilus 3% Porphyromonas 3% Fusobacterium 5% campylobacter 2% Actinomyces 4% Neisseria 4% Leptotrichia 6% The “chronic bronchitis” phenotype Streptococcus 29% Granulicatella 2% Rothia 2% Actinomyces 3% Porphyromonas 3% Fusobacterium 4% Haemophilus 4% Leptotrichia 4% Veillonella 19% other 4% Prevotella 19% Prevotella 19% Veillonella 19% The “mixed” phenotype other 3% Rothia 3% Streptococcus 31% Porphyromonas 2% Granulicatella 2% Fusobacterium 2% Leptotrichia 3% Neisseria 3% Actinomyces 3% Haemophilus 4% Veillonella 17% Prevotella 21% Figure 1. M.A. Karnaushkina, S.V. Fedosenko, A.E. Sazonov, V.A. Petrov, D.Yu. Ovsyannikov, L.M. Ogorodova Results and Discussion To identify the respiratory microbiota patterns associated with specific clinical and functional characteristics of the COPD patients, the Spearman’s correlation analysis was performed. In the “chronic bronchitis” phenotype patients, a cluster of conditionally pathogenic bacteria was found; within the cluster, a strong positive correlation between the different microbial taxa was identified: Fusobacterium   +   Prevotella + Mycoplasma + Bulleidia (r=0.701; p<0.05). In the COPD patients with the “emphysema” phenotype, a cluster of normal oropharyngeal microbiota of the Firmicutes type was found. In addition, an Actinobacteria cluster of opportunistic bacteria of the Enterococcus gastrointestinal type and strong positive intra-cluster correlations was also present (r>0.08, p<0.001 and r>0.08, p<0.05, respectively). In the “mixed” phenotype, two clusters of opportunistic oropharyngeal bacteria with a strong positive correlation were found: Porphyromonas + Fusobacterium + Prevotella (r=0.832, p<0.05) and Streptococcus + Rothia (r=0.761, p<0.05). High levels of bacteria typical for the gastrointestinal microbiota were found in oropharyngeal swabs from patients with the “emphysema” phenotype; that included a cluster of normal oropharyngeal microflora (such as Firmicutes) and Actinobacteria + Enterococcus (opportunistic gastrointestinal microbiota). Published [20, 21] studies have demonstrated that gastroesophageal reflux disease (GERD) is associated with chronic bronchopulmonary diseases, including COPD, thus aggravating the course of the main disease. In our study, GERD was found in 11 patients Thus, the patients with COPD showed three patterns of oropharyngeal microbiota composition. In patients with the “chronic bronchitis” phenotype, opportunistic bacteria were mostly represented. In patients with the “emphysema” phenotype, the oropharyngeal normobiota and intestinal microbiota (including conditionally- pathogenic bacteria) were dominant. In patients with the “mixed” phenotype, a pattern with a low species diversity and a predominance of gastrointestinal opportunistic microbiota (Firmicutes), in combination with the opportunistic bacteria cluster (Porphyromonas + Prevotella + Fusobacterium) was found. Figure 2. Multiple correlations between different components of the oropharyngeal microbiota in patients with different prognoses of chronic obstructive pulmonary disease The data includes variables with a Spearman correlation value of ≥0.05 at p<0.05 after introducing the Benjamin–Hochberg correction for multiple comparisons. The blue diamond on the chart shows the optimal sub-selection of variables, which allows to classification of the patients with an accuracy of 76% In this study, it was attempted, for the first time, to develop a prognostic model based on the clinical and functional phenotypes of COPD and their respective microbiological patterns. Results and Discussion Prevalent genera of microorganisms, comprising 95% of the metagenome of the oropharyngeal microbiota in patients with chronic obstructive pulmonary disease (the extended examination group) Prevotella 21% Actinobacteria, Proteobacteria, Bacteroidetes, Firmicutes, Bacteroides, mainly due to a higher content of opportunistic bacteria of Haemophilus (LogFC 8.662), Actinomyces (LogFC 9.813), Lactobacillus (LogFC 4.758), Leptotrichia (LogFC 10.556), Porphyromonas (LogFC 5.896), Bacillus (LogFC 3.161), Gemella (LogFC 8.303) (p<0.001). Also in these patients, a considerable amount of opportunistic bacteria in the oropharynx was found: Prevotella melaninogenica species (LogFC 11.738), Rothia mucilaginosa (LogFC 9.257), and Veillonella parvula (LogFC 10.631) (p<0.001). The metagenomeSeq-assisted analysis of low- represented taxa (less than 10% of the identified microbiome) revealed a predominance of bacterial commensals in patients with the phenotype of emphysema. Among those, the Firmicutes bacteria (Acidaminococcus LogFC 1.865; Anaerovibrio LogFC 2.653; Leuconostoc LogFC 1.727; Oceanobacillus LogFC 1.107; Phascolarctobacterium LogFC 2.915; Tetragenococcus LogFC 1.513), the Actinobacteria bacteria (Micrococcus LogFC 1.002; Dermacoccus LogFC 2.191), and the Proteobacteria bacteria (Cardiobacterium LogFC 1.002; Novosphingobium LogFC 1.561) (p<0.001). Samples of oropharyngeal swabs from patients with COPD with the “mixed” phenotype showed the prevailing representation of the Firmicutes and Bacteroides bacteria (p<0.001) with a less diverse variety of species in comparison with the two other phenotypes. Analysis of the low-represented taxa in patients with the “chronic bronchitis” phenotype revealed a significant predominance of bacteria of Fusobacterium, 104 СТМ ∫ 2018 — vol. 10, No.2 M.A. Karnaushkina, S.V. Fedosenko, A.E. Sazonov, V.A. Petrov, D.Yu. Ovsyannikov, L.M. Ogorodova сlinical medicine oropharyngeal microbiota depends on the source of micro-aspiration (gastrointestinal tract, nasopharynx, oral cavity). Numerous studies [15, 18, 19] demonstrated the role of opportunistic oropharyngeal bacteria in the development of pneumonia and exacerbations of chronic bronchopulmonary diseases. These bacteria enter the lower respiratory tract as a result of micro- aspiration from the upper respiratory tract. According to our study, 40% of patients with the “chronic bronchitis” phenotype and 70% with the “mixed” phenotype suffered from tonsillitis, sinusitis or periodontitis. In these patients, we detected the formation of clusters of oropharyngeal opportunistic bacteria: Fusobacterium + Prevotella + Mycoplasma + Bulleidia, Porphyromonas + Prevotella + Fusobacterium and Porphyromonas + Prevotella + Streptococcus minor. This finding supports the hypothesis that the aspirated oropharyngeal microflora transforms the composition of the airways microbiome in patients with the “mixed” and “chronic bronchitis” phenotypes of COPD. References 1. Chuchalin A.G., Avdeev  S.N., Aysanov Z.R., Belevskiy  A.S., Leshchenko I.V., Meshcheryakova N.N., Ovcharenko S.I., Shmelev E.I. Russian respiratory society. Federal guidelines on diagnosis and treatment of chronic obstructive pulmonary disease. Russian Pulmonology 2014; 3: 15–36, https://doi.org/10.18093/0869-0189-2014-0-3-15-54. 2. Global Initiative for Chronic Obstructive Lung Disease. Global strategy for diagnosis, management, and prevention of COPD. Updated 2015. 3. Fedosenko S.V., Ogorodova L.M., Popenko A.S., Petrov  V.A., Tyakht A.V., Saltykova I.V., Kamaltynova  E.M., Deev I.A., Kulikov E.S., Kirillova N.A., Govorun V.M., Kostryukova  E.S., Karnaushkina M.A. The qualitative and quantitative characteristics of content of oro-pharyngeal microbiota of patients with chronic obstructive disease of lungs. Lechenie i profilaktika 2015; 2(14): 92–99. The presented clinical and functional characteristics of COPD patients and their correlations with the composition of the oropharyngeal microbiota allow us to propose that micro-aspiration from different body compartments affects not only the oropharyngeal microbiota but also contributes to the phenotypic characterization and clinical prognosis of COPD. At present, it is difficult to ascertain, what are the primary factors determining the clinical and functional phenotype of COPD. They can be either the phenotypic traits of the disease (e.g., the impact of GERD) or a disorder of the upper respiratory tract or the composition of the oropharyngeal microbiota. Reasonably, a combination of the above factors should be considered. 4. Avdeev S.N. Symptoms and quality of life in patients with chronic obstructive pulmonary disease: are there patient- defined outcomes or predictive factors? Russian Pulmonology 2016; 26(2): 231–237, https://doi.org/10.18093/0869-0189- 2016-26-2-231-237. 5. Chambers D.C., Gellatly S.L., Hugenholtz P., Hansbro P.M. JTD special edition ‘Hot Topics in COPD’ — the microbiome in COPD. J Thorac Dis 2014; 6(11): 1525–1531, https://doi.org/10.3978/j.issn.2072-1439.2014.11.08. 6. Charlson E.S., Bittinger K., Haas A.R., Fitzgerald A.S., Frank I., Yadav A., Bushman F.D., Collman R.G. Topographical continuity of bacterial populations in the healthy human respiratory tract. Am J Respir Crit Care Med 2011; 184: 957– 963, https://doi.org/10.1164/rccm.201104-0655oc. As shown in the present study, the low species diversity of the oropharyngeal microbiota in patients with COPD of the “mixed” phenotype may favor progression and aggravation of the disease. 7. Górecka D., Puścińska E. Microbiome of the lung. Pneumonol Alergol Pol 2014; 82(6): 481–485, https://doi. org/10.5603/PiAP.2014.0063. g 8. Park H., Shin J.W., Park S.G., Kim W. Microbial communities in the upper respiratory tract of patients with asthma and chronic obstructive pulmonary disease. PLoS One 2014; 9(10): e109710, https://doi.org/10.1371/journal. pone.0109710. Results and Discussion Our analysis indicates that an increase in the oropharyngeal Moraxellaceae, Peptococcaceae, Eubacteriaceae, Enterococcaceae, Staphylococcaceae, and Actinomycetaceae bacterial families is an unfavorable prognostic factor in terms of the disease progression. In the validation test, this model showed a sensitivity of 0.67 and specificity of 1, a prognosis accuracy of 75% with an area under the ROS curve of 0.72 (Figure 2). Figure 2. Multiple correlations between different components of the oropharyngeal microbiota in patients with different prognoses of chronic obstructive pulmonary disease Thus, it can be argued that certain changes in the composition of oropharyngeal microbiota are significant for the progression of COPD. The data includes variables with a Spearman correlation value of ≥0.05 at p<0.05 after introducing the Benjamin–Hochberg correction for multiple comparisons. The blue diamond on the chart shows the optimal sub-selection of variables, which allows to classification of the patients with an accuracy of 76% To interpret the obtained results, we assumed that the composition of the СТМ ∫ 2018 — vol. 10, No.2 105 Microbiological Oropharyngeal Patterns in Patients with Different Phenotypes of COPD сlinical medicine (78.6%) with the phenotype of “emphysema”, in 15 patients (75%) with the “mixed” phenotype, and only 6 patients (20%) with the phenotype of “chronic bronchitis”. It is known [14–17] that the presence of the Porphyromonas and Prevotellgenera pathogenic bacteria detected in patients with the “mixed” phenotype is clinically significant because this is associated with purulent inflammatory processes. Likewise, the impact of the respiratory tract opportunistic microflora on the pulmonary tissue is enhanced by the gastrointestinal microbiota that enters the airways. The low diversity of bacterial species in this COPD phenotype decreases their resistance to pathogens [17] and can severely complicate the course of COPD in patients with the “mixed” phenotype. (78.6%) with the phenotype of “emphysema”, in 15 patients (75%) with the “mixed” phenotype, and only 6 patients (20%) with the phenotype of “chronic bronchitis”. It is known [14–17] that the presence of the Porphyromonas and Prevotellgenera pathogenic bacteria detected in patients with the “mixed” phenotype is clinically significant because this is associated with purulent inflammatory processes. Likewise, the impact of the respiratory tract opportunistic microflora on the pulmonary tissue is enhanced by the gastrointestinal microbiota that enters the airways. The low diversity of bacterial species in this COPD phenotype decreases their resistance to pathogens [17] and can severely complicate the course of COPD in patients with the “mixed” phenotype. Conclusion Each phenotype of chronic obstructive pulmonary disease with its specific clinical and functional features is characterized by a specific pattern of oropharyngeal microbiota, which determines the severity and prognosis of the disease. The bacterial composition of these patterns depends on the source of micro-aspiration (upper gastrointestinal tract or nasopharynx). In addition, the composition of the oropharyngeal microbiota is determined by the severity of the disease and by its phenotype. This phenotype determines the course of the disease in a patient with chronic obstructive pulmonary disease. 9. Molyneaux P.L., Mallia P., Cox M.J., Footitt J., Willis-Owen S.A., Homola D., Trujillo-Torralbo M.B., Elkin  S., Kon  O.M., Cookson W.O., Moffatt M.F., Johnston S.L. Outgrowth of the bacterial airway microbiome following rhinovirus exacerbation of chronic obstructive pulmonary disease. Am J Respir Crit Care Med 2013; 188(10): 1224– 1231, https://doi.org/10.1164/rccm.201302-0341oc. 10. Patel A.R., Hurst J.R. Extrapulmonary comorbidities in chronic obstructive pulmonary disease: state of the art. Expert Rev Respir Med 2011; 5(5): 647–662, https://doi.org/10.1586/ ers.11.62. 11. Hurst J.R. Upper airway. 3: Sinonasal involvement in chronic obstructive pulmonary disease. Thorax 2010; 65(1): 85–90, https://doi.org/10.1136/thx.2008.112888. Financial support. The study was supported by a grant from the Federal target program “Research and development in priority areas for the development of the scientific and technological complex in Russia for 2014–2020” (GC No.14.604.21.0075, unique identifier RFMEFI60414X0075).li 12. Paulson J.N., Olson N.D., Wagner J., Talukder H., Pop M., Bravo H.C. Statistical analysis for sparse high- throughput sequencing, https://doi.org/10.18129/B9.bioc. metagenomeSeq. 13. Faust K., Sathirapongsasuti J.F., Izard J., Segata N., Gevers D., Raes J., Huttenhower C. Microbial co-occurrence relationships in the human microbiome. PLoS Comput Biol 2012; 8(7): e1002606, https://doi.org/10.1371/journal.pcbi.1002606. Conflict of interest. The authors confirm the absence of financial and other conflicting interests that could influence their work. 14. Pragman A.A., Kim H.B., Reilly C.S., Wendt C., 106 СТМ ∫ 2018 — vol. 10, No.2 M.A. Karnaushkina, S.V. Fedosenko, A.E. Sazonov, V.A. Petrov, D.Yu. Ovsyannikov, L.M. Ogorodova сlinical medicine Isaacson R.E. The lung microbiome in moderate and severe chronic obstructive pulmonary disease. PloS One 2012; 7(10): e47305, https://doi.org/10.1371/journal.pone.0047305. Sikaroodi M., Naqvi A., Gillevet P.M. Characterization of the oral fungal microbiome (mycobiome) in healthy individuals. PLoS Pathog 2010; 6(1): e1000713, https://doi.org/10.1371/ journal.ppat.1000713. 15. Sze M.A., Dimitriu P.A., Hayashi S., Elliott W.M., McDonough J.E., Gosselink J.V., Cooper J., Sin D.D., Mohn  W.W., Hogg J.C. The lung tissue microbiome in chronic obstructive pulmonary disease. Am J Respir Crit Care Med 2012; 185(10): 1073–1080, https://doi.org/10.1164/ rccm.201111-2075oc. СТМ ∫ 2018 — vol. 10, No.2 107 18. Ghannoum M.A., Jurevic R.J., Mukherjee P.K., Cui F., Conclusion 19. Hsiao W.W., Li K.L., Liu Z., Jones C., Fraser- Liggett C.M., Fouad A.F. Microbial transformation from normal oral microbiota to acute endodontic infections. BMC Genomics 2012; 13: 345, https://doi.org/10.1186/1471-2164-13-345. g 20. Pei Z., Yang L., Peek R.M., Jr Levine S.M., Pride D.T., Blaser M.J. Bacterial biota in reflux esophagitis and Barrett’s esophagus. World J Gastroenterol 2005; 11(46): 7277–7283, https://doi.org/10.3748/wjg.v11.i46.7277. 16. Sin D., Sze M., Hogg J. Bacterial microbiome of lungs in COPD. Int J Chron Obstruct Pulmon Dis 2014; 9: 229–238, https://doi.org/10.2147/copd.s38932. 21. Sakae T.M., Pizzichini M.M., Teixeira P.J., Silva R.M., Trevisol D.J., Pizzichini E. Exacerbations of COPD and symptoms of gastroesophageal reflux: a systematic review and meta-analysis. J Bras Pneumol 2013; 39(3): 259–271, https:// doi.org/10.1590/s1806-37132013000300002. 17. Huang Y.J., Sethi S., Murphy T., Nariya S., Boushey H.A., Lynch S.V. Airway microbiome dynamics in exacerbations of chronic obstructive pulmonary disease. J  Clin Microbiol 2014; 52(8): 2813–2823, https://doi.org/10.1128/jcm.00035-14. 18. Ghannoum M.A., Jurevic R.J., Mukherjee P.K., Cui F., СТМ ∫ 2018 — vol. 10, No.2 107 Microbiological Oropharyngeal Patterns in Patients with Different Phenotypes of COPD СТМ ∫ 2018 — vol. 10, No.2 107 Microbiological Oropharyngeal Patterns in Patients with Different Phenotypes of COPD
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How Quickly Do Proteins Fold and Unfold, and What Structural Parameters Correlate with These Values?
Biomolecules
2,020
cc-by
12,319
Received: 27 December 2019; Accepted: 26 January 2020; Published: 29 January 2020 Abstract: The correlations between the logarithm of the unfolding rate of 108 proteins and their structural parameters were calculated. We showed that there is a good correlation between the logarithm of folding and unfolding rates (0.79) and protein stability and unfolding rate (0.79). Thus, the faster the protein folds, the faster it unfolds. Folding and unfolding rates are higher for the proteins with two-state kinetics, in comparison with the proteins with multi-state kinetics. At the same time, two-state bacterial proteins folds and unfolds two orders of magnitude faster than two-state eukaryotic proteins, and multi-state bacterial proteins folds and unfolds slower than multi-state eukaryotic proteins. Despite the fact that the folding rates of thermophilic and mesophilic proteins are close, the unfolding rates of thermophilic proteins is about two orders of magnitude lower than for mesophilic proteins. The correlation between unfolding rate and stability of thermophilic proteins is high (0.90). We also found that the unfolding rate correlates with such structural parameters as: size of the protein, radius of the cross-section, logarithm of absolute contact order, and radius of gyration. This information will be useful for engineering and designing new proteins with desired properties. Keywords: protein unfolding; unfolding rates; bacterial proteins; eukaryotic proteins; thermophile; mesophile Anna V. Glyakina 1,2 and Oxana V. Galzitskaya 1,3,* 1 Institute of Protein Research, Russian Academy of Sciences, Pushchino, Moscow Region 142290, Russia; glyakina@rambler.ru 2 Institute of Mathematical Problems of Biology, Russian Academy of Sciences, Keldysh Institute of Applied Mathematics, Russian Academy of Sciences, Pushchino, Moscow Region 142290, Russia 3 Institute of Theoretical and Experimental Biophysics, Russian Academy of Sciences, Pushchino, Moscow Region 142290, Russia * Correspondence: ogalzit@vega.protres.ru; Tel.: +7-903-675-0156 Institute of Protein Research, Russian Academy of Sciences, Pushchino, Moscow Region 142290, Russia; 1 Institute of Protein Research, Russian Academy of Sciences, Pushchino, Moscow Region 142290, Russia glyakina@rambler.ru 1 Institute of Protein Research, Russian Academy of Sciences, Pushchino, Moscow Region 142290, Russia; glyakina@rambler.ru 2 Institute of Mathematical Problems of Biology, Russian Academy of Sciences, Keldysh Institute of Applied Mathematics, Russian Academy of Sciences, Pushchino, Moscow Region 142290, Russia 3 Institute of Theoretical and Experimental Biophysics, Russian Academy of Sciences, Pushchino, Moscow Region 142290, Russia * Correspondence: ogalzit@vega.protres.ru; Tel.: +7-903-675-0156 * Correspondence: ogalzit@vega.protres.ru; Tel.: +7-903-675-0156 Received: 27 December 2019; Accepted: 26 January 2020; Published: 29 January 2020 Biomolecules 2020, 10, 197; doi:10.3390/biom10020197 How Quickly Do Proteins Fold and Unfold, and What Structural Parameters Correlate with These Values? Anna V. Glyakina 1,2 and Oxana V. Galzitskaya 1,3,* biomolecules biomolecules biomolecules biomolecules 1. Introduction The problem of predicting folding rates (kf) for proteins with two-state and multi-state kinetics is still important and extensively studied [1–20]. Many articles are devoted to the study of protein folding rates and their correlation with various structural parameters [2,6–8,16]. In 1998, a relative contact order (rCO) parameter was suggested, which is the average distance along the sequence between all pairs of contacting residues, normalized to the size of the protein (number of amino acid residues, further protein length). This parameter reflects the topological complexity of the protein chain. It was shown that the rCO correlates well (correlation coefficient is 0.81) with the logarithm of the folding rate for 12 two-state proteins [2]. Subsequent studies have shown that there is no correlation between rCO and logarithm of the folding rate of proteins [6,7,16]. It turned out that only absolute contact order (AbsCO, contact order multiplied by protein length) correlated with the logarithm of the folding rate (the correlation coefficient is −0.77) [16]. It was found that the structural parameters, depending on the protein length (L), correlated well with the logarithm of the folding rate [16]. In the set of papers [13,15,16,21,22], the authors considered the different structural parameters of protein globule compactness: radius of gyration (Rg); normalized radius of gyration (Rg/Rg*, where Rg* is the radius Biomolecules 2020, 10, 197; doi:10.3390/biom10020197 www.mdpi.com/journal/biomolecules 2 of 14 Biomolecules 2020, 10, 197 of gyration of the ball with the uniform density and the same volume); compactness as the relationship between the solvent accessible surface area to the solvent accessible surface of sphere with the same volume (Sasa/S*); and the relationship between the solvent accessible volume to the solvent accessible surface (Vasa/Sasa). The solvent accessible volume (Vasa) is the volume surrounded by the solvent accessible surface (Sasa). The radius of the cross-section (Vasa/Sasa) is proportional but not equal to the radius of the smallest cross-section of the protein globule [16]. It was demonstrated that Vasa/Sasa correlates good with the logarithm of the folding rate (the correlation coefficient is 0.74) [16]. We have shown that the less compact the proteins, the faster they fold. Therefore, α-structural proteins are less compact and the most rapidly folding proteins, while α/β-structural proteins are the most compact and the most slowly folding proteins [15]. We have previously shown that bacterial proteins with two-state kinetics (further, bacterial two-state proteins) fold and unfold faster than two-state eukaryotic proteins [23]. 1. Introduction It turned out that the multi-state eukaryotic proteins fold and unfold faster than bacterial ones. It has been shown that there is a “golden triangle” that limits the possible range of the folding rates for single-domain globular proteins of different sizes and stability [24]. This triangle was based only on the biological and physical limitations. In addition, knowledge of the protein unfolding rates is also necessary to estimate their stability. Much less work has been devoted to the study of the relationship between the unfolding rates and structural parameters. It has been shown that the logarithm of the unfolding and folding rates correlate strongly with the AbsCO and the long-range order (parameter defined by contacts between two residues that are close in space and far in the sequence) [3], with correlation coefficient values of 0.75 or higher. The logarithm of the unfolding rate correlated better with thermodynamic stability, in comparison with the folding rate [19]. Early, it has been shown that structural parameters L, ln(AbsCO), Vasa/Sasa, and Rg were in a good correlation with the logarithm of the protein folding rate [16]. The aim of the study was broader than the search for connections between protein unfolding rates and various structural parameters, such as L, ln(AbsCO), Vasa/Sasa, and Rg. We were trying to find out which parameters are most important for the prediction of the unfolding rates for proteins from different organisms. 3.1. Unfolding Rates of 108 Proteins 3.1. Unfolding Rates of 108 Proteins (B) The average values of the logarithm of the unfolding and folding rates for 108 proteins. Structural parameters that better correlate with the logarithm of the unfolding rates are outlined in a rectangle. Figure 1. (A) Logarithm of the unfolding and folding rates for the three domains of chicken brain alpha-spectrin proteins. (B) The average values of the logarithm of the unfolding and folding rates for 108 proteins. Structural parameters that better correlate with the logarithm of the unfolding rates are outlined in a rectangle. The correlation between the logarithm of the unfolding and folding rates is 0.79 for all proteins. Moreover, this correlation is better for two-state (0.78) than for multi-state proteins (0.73). The separation of 108 proteins by structural classes (α, β, α/β, and α + β) revealed that correlation between the logarithm of the folding and unfolding rates is better for proteins from α and β (0.78 and 0.75) classes, in comparison with the proteins from α/β and α + β classes (0.59 and 0.60). Moreover, two-state proteins make the largest contribution to this correlation (see Table 1). The correlation between the logarithm of the unfolding and folding rates is 0.79 for all proteins. Moreover, this correlation is better for two-state (0.78) than for multi-state proteins (0.73). The separation of 108 proteins by structural classes (α, β, α/β and α + β) revealed that correlation between the logarithm of the folding and unfolding rates is better for proteins from α and β (0.78 and 0.75) classes, in comparison with the proteins from α/β and α + β classes (0.59 and 0.60). Moreover, two-state proteins make the largest contribution to this correlation (see Table 1). , p g ( ) The proteins from α class folds and unfolds faster, while proteins from α/β class folds and unfolds slowly, in comparison with proteins from other structural classes (average logarithm of folding rates: 8.49 ± 0.64 for α, 3.42 ± 0.63 for β, −0.02 ± 0.85 for α/β, and 4.71 ± 0.53 for α+β; average logarithm of unfolding rates: 2.03 ± 1.03 for α, −4.51 ± 1.12 for β, −8.34 ± 1.64 for α/β, and −4.76 ± 0.97 for α + β; see also Supplementary Table S2). 3.1. Unfolding Rates of 108 Proteins 3.1. Unfolding Rates of 108 Proteins The goal of this work was to find the relationship between protein unfolding rates and protein structural parameters. For three spectrin domains: R15, R16, and R17, we observed that the faster the protein folds, the faster it unfolds, and vice versa. Domain R15 folds and unfolds faster than its homologues, R16 and R17 (see Figure 1A). In the case of these spectrin repeats, the folding and unfolding rates may be associated with the mechanical stability of the proteins. Previously, it has been shown that domain R15 is less mechanically stable than domains R16 and R17 [26]. The discovered correlation between the unfolding and folding rates suggests that the statement that the faster the protein folds, the faster it unfolds, and vice versa, is also true for a dataset consisting of 108 proteins (Figure 1B). The goal of this work was to find the relationship between protein unfolding rates and protein structural parameters. For three spectrin domains: R15, R16, and R17, we observed that the faster the protein folds, the faster it unfolds, and vice versa. Domain R15 folds and unfolds faster than its homologues, R16 and R17 (see Figure 1A). In the case of these spectrin repeats, the folding and unfolding rates may be associated with the mechanical stability of the proteins. Previously, it has been shown that domain R15 is less mechanically stable than domains R16 and R17 [26]. The discovered correlation between the unfolding and folding rates suggests that the statement that the faster the protein folds, the faster it unfolds, and vice versa, is also true for a dataset consisting of 108 proteins (Figure 1B). Figure 1. (A) Logarithm of the unfolding and folding rates for the three domains of chicken brain alpha-spectrin proteins. (B) The average values of the logarithm of the unfolding and folding rates for 108 proteins. Structural parameters that better correlate with the logarithm of the unfolding rates are outlined in a rectangle. Figure 1. (A) Logarithm of the unfolding and folding rates for the three domains of chicken brain alpha-spectrin proteins. (B) The average values of the logarithm of the unfolding and folding rates for 108 proteins. Structural parameters that better correlate with the logarithm of the unfolding rates are outlined in a rectangle. Figure 1. (A) Logarithm of the unfolding and folding rates for the three domains of chicken brain alpha-spectrin proteins. 2. Materials and Methods Parameter Rg was calculated as Rg = q P mi(ri−RC)2 M , where mi is the mass of the i-th atom, ri is its Cartesian coordinates, M is the mass of the protein, and RC is the coordinate vector of the mass center of the protein, calculated as follows: P mi(ri −RC) = 0. Biomolecules 2020, 9, x 3 of 14 Errors for data from Table 1, Table 3, Table 5, Figure 5 and Figure 9 were calculated as σ = r Pn i=1(xi−x) n(n−1) , where x is the average value of a parameter and n is a number of proteins. Errors for data from Table 1, Table 3, Table 5, Figure 5 and Figure 9 were calculated as 𝜎= ට ∑ (௫௜ି௫̅) ೙ ೔సభ ௡(௡ିଵ) , where 𝑥̅ is the average value of a parameter and n is a number of proteins. Errors for data from Table 1, Table 3, Table 5, Figure 5 and Figure 9 were calculated as σ = r Pn i=1(xi−x) n(n−1) , where x is the average value of a parameter and n is a number of proteins. Errors for data from Table 1, Table 3, Table 5, Figure 5 and Figure 9 were calculated as 𝜎= ට ∑ (௫௜ି௫̅) ೙ ೔సభ ௡(௡ିଵ) , where 𝑥̅ is the average value of a parameter and n is a number of proteins. r Pn i=1(xi−x) n(n−1) , where x is the average value of a parameter and n is a number of proteins. ට ∑ (௫௜ି௫̅) ೙ ೔సభ ௡(௡ିଵ) , where 𝑥̅ is the average value of a parameter and n is a number of proteins. r P i=1(xi−x) n(n−1) , where x is the average value of a parameter and n is a number of proteins. ට ∑ (௫௜௫) ೔సభ ௡(௡ିଵ) , where 𝑥̅ is the average value of a parameter and n is a number of proteins. 2. Materials and Methods In our study, three databases of proteins were examined. The first database consisted of 108 proteins. Among them, 38 proteins demonstrated multi-state kinetics, and 70 proteins showed two-state kinetics. The second database consisted of 42 bacterial (29 two-state and 13 multi-state) and 53 eukaryotic (32 two-state and 21 multi-state) proteins. The third database consisted of 42 bacterial proteins. Among them, there were 10 thermophilic (8 two-state and 2 multi-state) and 32 mesophilic (21 two-state and 11 multi-state) proteins. Proteins that have experimentally measured folding and unfolding rates were taken into consideration. The database of such proteins has begun to be collected since 2003. At that time, there were only 57 proteins [7]. In 2009, there were already 84 proteins [16]. Now, this database consists of 108 proteins [19,24]. Data on folding and unfolding rates and structural parameters of the proteins (L—length of the protein, Vasa/Sasa, ln(AbsCO) and Rg) are in Supplementary Table S1. Parameters Vasa and Sasa were calculated using YASARA program [25]. 1 N Parameters Vasa and Sasa were calculated using YASARA program [25]. 1 N Parameter ln(AbsCO) was calculated as ln(AbsCO) = ln( 1 N PN k=1 ∆Lij), where N is the number of contacts (within 6 Å) between nonhydrogen atoms in the protein and ∆Lij is the number of residues separating the interacting pair of nonhydrogen atoms (adjacent residues are assumed to be separated by one residue). Bi l l 2020 10 197 3 f 14 3 of 14 Biomolecules 2020, 10, 197 Parameter Rg was calculated as Rg = q P mi(ri−RC)2 M , where mi is the mass of the i-th atom, ri is its Cartesian coordinates, M is the mass of the protein, and RC is the coordinate vector of the mass center of the protein, calculated as follows: P mi(ri −RC) = 0. Errors for data from Table 1, Table 3, Table 5, Figure 5 and Figure 9 were calculated as σ = r Pn i=1(xi−x) n(n−1) , where x is the average value of a parameter and n is a number of proteins. Biomolecules 2020, 9, x 3 of 14 Errors for data from Table 1, Table 3, Table 5, Figure 5 and Figure 9 were calculated as 𝜎= ට ∑ (௫௜ି௫̅) ೙ ೔సభ ௡(௡ିଵ) , where 𝑥̅ is the average value of a parameter and n is a number of proteins. 3.1. Unfolding Rates of 108 Proteins 3.1. Unfolding Rates of 108 Proteins The proteins from α class folds and unfolds faster, while proteins from α/β class folds and unfolds slowly, in comparison with proteins from other structural classes (average logarithm of folding rates: 8.49 ± 0.64 for α, 3.42 ± 0.63 for β, −0.02 ± 0.85 for α/β, and 4.71 ± 0.53 for α + β; average logarithm of unfolding rates: 2.03 ± 1.03 for α, −4.51 ± 1.12 for β, −8.34 ± 1.64 for α/β, and −4.76 ± 0.97 for α + β; see also Supplementary Table S2). β pp y ) Table 1. Correlations between logarithm of unfolding (ln(ku) and folding (ln(kf) rates for four structural classes of proteins α, β, α/β and α + β. It was previously shown that L, ln(AbsCO), Vasa/Sasa, and Rg correlate well with the logarithm of the protein folding rate [16]. Thus, it can be assumed that if these parameters correlate well with the logarithm of the folding rate, then they will also correlate well with the logarithm of the unfolding 4 of 14 Biomolecules 2020, 10, 197 rate. In this case, four parameters were examined: L is a number of amino acid residues in protein, ln(AbsCO) is the logarithm of the absolute contact order, Vasa/Sasa is a radius of cross-section, and Rg is a radius of gyration. rate. In this case, four parameters were examined: L is a number of amino acid residues in protein, ln(AbsCO) is the logarithm of the absolute contact order, Vasa/Sasa is a radius of cross-section, and Rg is a radius of gyration. Table 1. Correlations between logarithm of unfolding (ln(ku) and folding (ln(kf) rates for four structura classes of proteins α, β, α/β and α + β. Correlations with ln(ku) All (Two + Multi) Two-State Multi-State All 0.79* (108) ** 0.78 (70) ** 0.73 (38) ** α 0.78 (31) 0.82 (23) 0.79 (8) β 0.75 (37) 0.74 (25) 0.61 (12) α/β 0.59 (12) - 0.55 (11) α + β 0.60 (28) 0.63 (21) 0.13 (7) * Correlations above 0.75 are shown in bold. ** The number of proteins is indicated in parentheses. Table 1. Correlations between logarithm of unfolding (ln(ku) and folding (ln(kf) rates for four structural classes of proteins α, β, α/β and α + β. The values of structural parameters considered in this paper (L, ln(AbsCO), Vasa/Sasa and Rg) are lower for two-state proteins than for multi-state proteins: 78 ± 5 vs. 3.1. Unfolding Rates of 108 Proteins 3.1. Unfolding Rates of 108 Proteins 130 ± 8 for L, 3.14 ± 0.05 vs. 3.59 ± 0.06 for Vasa/Sasa, 6.91 ± 0.06 vs. 7.22 ± 0.06 for ln(AbsCO), and 12.1 ± 0.3 vs. 14.2 ± 0.3 for Rg (see Table 2). The logarithms of the folding and unfolding rates are higher for two-state proteins, in comparison with multi-state proteins: 6.08 ± 0.50 vs. 2.51 ± 0.59 for the folding rate and −1.51 ± 0.79 vs. −6.09 ± 1.03 for the unfolding rate, respectively (see Figure 1B). Table 2. Average values of structural parameters for 108 proteins. Vasa/Sasa = radius of cross-section. Rg = radius of gyration. ln(AbsCO) = logarithm of the absolute contact order. L = length of the protein. Table 2. Average values of structural parameters for 108 proteins. Vasa/Sasa = radius of cross-section. Rg = radius of gyration. ln(AbsCO) = logarithm of the absolute contact order. L = length of the protein. Average Value All (Two + Multi) Two-State Multi-State Number of proteins 108 70 38 <L> 96 ± 5 78 ± 5 130 ± 8 <Vasa/Sasa> 3.30 ± 0.04 3.14 ± 0.05 3.59 ± 0.06 <ln(AbsCO)> 7.02 ± 0.05 6.91 ± 0.06 7.22 ± 0.06 <Rg> 12.8 ± 0.2 12.1 ± 0.3 14.2 ± 0.3 For 108 proteins, the correlations between the logarithm of the unfolding rate (ln(ku)) and structural parameters such as L, Vasa/Sasa, ln(AbsCO), and Rg were calculated (Table 3 and Figure 2). Vasa/Sasa and ln(AbsCO) are better correlated with the logarithm of the unfolding rate of two-state proteins. For two-state proteins, these correlations are −0.79 and −0.87, in comparison with −0.63 and −0.69 for multi-state proteins. The correlation between Rg and the logarithm of the unfolding rate is almost the same for two-state and multi-state proteins (−0.61 vs. −0.60, respectively). Moreover, L is better correlated with the logarithm of the unfolding rate of multi-state proteins. Good correlation (0.79) between the protein stability (−(lnkf −lnku)) and the logarithm of the unfolding rate has been observed. Table 3. Correlations logarithm of the unfolding rate (ln(ku)) with protein stability (−(lnkf −lnku)) and structural parameters (L, Vasa/Sasa, ln(AbsCO) and Rg) for 108 proteins. Table 3. Correlations logarithm of the unfolding rate (ln(ku)) with protein stability (−(lnkf −lnku)) and structural parameters (L, Vasa/Sasa, ln(AbsCO) and Rg) for 108 proteins. * Correlations above 0.75 are shown in bold. 3.1. Unfolding Rates of 108 Proteins 3.1. Unfolding Rates of 108 Proteins The exception is only for correlation between ln(ku) and L for proteins from β class This correlation is higher for multi-state proteins (−0 86) in comparison with two-state After the separation of 108 proteins by structural classes (α, β, α/β and α + β), we observed that correlations between the logarithm of the unfolding rate (ln(ku)) and L, Vasa/Sasa, ln(AbsCO), and Rg are better for proteins from α and β classes (see Table 4). These correlations are the highest for proteins from β class (higher than 0.8). The largest contribution to these correlations made two-state proteins (see Table 4). The exception is only for correlation between ln(ku) and L for proteins from β class. This correlation is higher for multi-state proteins (−0.86), in comparison with two-state proteins (−0.84). oteins (−0.84). T bl 4 C l i b h l i h f h f ldi (l (k ) d l Table 4. Correlations between the logarithm of the unfolding rate (ln(ku) and structural parameters (L, Vasa/Sasa, ln(AbsCO) and Rg) for four structural classes of proteins (α, β, α/β and α + β). teins (−0.84). T bl 4 C l ti b t th l ith f th f ldi t (l (k ) d t t l t Table 4. Correlations between the logarithm of the unfolding rate (ln(ku) and structural parameters (L, Vasa/Sasa, ln(AbsCO) and Rg) for four structural classes of proteins (α, β, α/β and α + β). Table 4. Correlations between the logarithm of the unfolding rate (ln(ku) and structural parameters (L, Vasa/Sasa, ln(AbsCO), and Rg) for four structural classes of proteins (α, β, α/β, and α + β). elations with ln(ku) All (two + multi) Two-state Multi-state α β α/β α+β α β α/β α+β α β α/β α ber of proteins 31 37 12 28 23 25 1 21 8 12 11 L −0.71 −0.84 −0.50 −0.60 −0.72 −0.84 − −0.77 −0.61 −0.86 −0.53 −0 Vasa/Sasa −0.78 −0.82 −0.11 −0.78 −0.84 −0.83 − −0.83 −0.60 −0.66 −0.28 −0 n(AbsCO) −0.80 −0.89 −0.30 −0.73 −0.85 −0.90 − −0.83 −0.48 −0.77 −0.29 −0 Rg −0.73 −0.83 −0.69 −0.50 −0.79 −0.84 − −0.57 −0.41 −0.74 −0.69 −0 * Correlations above 0.75 are shown in bold. 3.1. Unfolding Rates of 108 Proteins 3.1. Unfolding Rates of 108 Proteins Correlations with ln(ku) All (Two + Multi) Two-State Multi-State Number of proteins 108 70 38 Stability (−(lnkf −lnku)) 0.79 0.79 0.83 L −0.71 −0.65 −0.71 Vasa/Sasa −0.77 −0.79 −0.63 ln(AbsCO) −0.84 −0.87 −0.69 Rg −0.65 −0.61 −0.60 * Correlations above 0.75 are shown in bold. 5 of 14 5 f 14 Biomolecules 2020, 10, 197 Figure 2. Correlations of the logarithm of the unfolding rates of 108 proteins with their structural parameters: L—length of the protein, Vasa/Sasa—radius of the cross-section, ln(AbsCO)— logarithm of the absolute contact order, and Rg (radius of gyration). There is a line approximation of points and its equation: orange line corresponds to two-state proteins and purple line to multi-state proteins. R2 is a linear approximation reliability. Figure 2. Correlations of the logarithm of the unfolding rates of 108 proteins with their structural parameters: L—length of the protein, Vasa/Sasa—radius of the cross-section, ln(AbsCO)— logarithm of the absolute contact order, and Rg (radius of gyration). There is a line approximation of points and its equation: orange line corresponds to two-state proteins and purple line to multi-state proteins. R2 is a linear approximation reliability. Figure 2. Correlations of the logarithm of the unfolding rates of 108 proteins with their structural parameters: L—length of the protein, Vasa/Sasa—radius of the cross-section, ln(AbsCO)— logarithm of the absolute contact order, and Rg (radius of gyration). There is a line approximation of points and its equation: orange line corresponds to two-state proteins and purple line to multi-state proteins. R2 is a linear approximation reliability. Figure 2. Correlations of the logarithm of the unfolding rates of 108 proteins with their structural parameters: L—length of the protein, Vasa/Sasa—radius of the cross-section, ln(AbsCO)— logarithm of the absolute contact order, and Rg (radius of gyration). There is a line approximation of points and its equation: orange line corresponds to two-state proteins and purple line to multi-state proteins. R2 is a linear approximation reliability. After the separation of 108 proteins by structural classes (α, β, α/β, and α + β), we observed that correlations between the logarithm of the unfolding rate (ln(ku)) and L, Vasa/Sasa, ln(AbsCO), and Rg are better for proteins from α and β classes (see Table 4). These correlations are the highest for proteins from β class (higher than 0.8). The largest contribution to these correlations made two-state proteins (see Table 4). 3.1. Unfolding Rates of 108 Proteins 3.1. Unfolding Rates of 108 Proteins Correlations with ln(ku) All (Two + Multi) Two-State Multi-State α β α/β α + β α β α/β α + β α β α/β α + β Number of proteins 31 37 12 28 23 25 1 21 8 12 11 7 L −0.71 −0.84 −0.50 −0.60 −0.72 −0.84 − −0.77 −0.61 −0.86 −0.53 −0.20 Vasa/Sasa −0.78 −0.82 −0.11 −0.78 −0.84 −0.83 − −0.83 −0.60 −0.66 −0.28 −0.54 ln(AbsCO) −0.80 −0.89 −0.30 −0.73 −0.85 −0.90 − −0.83 −0.48 −0.77 −0.29 −0.34 Rg −0.73 −0.83 −0.69 −0.50 −0.79 −0.84 − −0.57 −0.41 −0.74 −0.69 −0.11 * Correlations above 0.75 are shown in bold. 69 0.50 0.79 0.84 orrelations above 0 75 are shown in bold * Correlations above 0.75 are shown in bold. 3.2. Unfolding Rates of Bacterial and Eukaryotic Proteins 3.2. Unfolding Rates of Bacterial and Eukaryotic Proteins The average values of the logarithm of the unfolding and folding rates for bacterial and eukaryotic two- and multi-state proteins. Structural parameters that better correlate with the logarithm of the unfolding rate are outlined in a rectangle. Figure 3. The average values of the logarithm of the unfolding and folding rates for bacterial and eukaryotic two- and multi-state proteins. Structural parameters that better correlate with the logarithm of the unfolding rate are outlined in a rectangle. Figure 3. The average values of the logarithm of the unfolding and folding rates for bacterial and eukaryotic two- and multi-state proteins. Structural parameters that better correlate with the logarithm of the unfolding rate are outlined in a rectangle. Figure 3. The average values of the logarithm of the unfolding and folding rates for bacterial and eukaryotic two- and multi-state proteins. Structural parameters that better correlate with the logarithm of the unfolding rate are outlined in a rectangle. Figure 3. The average values of the logarithm of the unfolding and folding rates for bacterial and eukaryotic two- and multi-state proteins. Structural parameters that better correlate with the logarithm of the unfolding rate are outlined in a rectangle. Table 5. Average values of structural parameters for two-state and multi-state bacterial and eukaryotic proteins. Table 5. Average values of structural parameters for two-state and multi-state bacterial and eukaryotic proteins. Table 5. Average values of structural parameters for two-state and multi-state bacterial and eukaryotic proteins. Table 5. Average values of structural parameters for two-state and multi-state bacterial and eukaryotic proteins. 3.2. Unfolding Rates of Bacterial and Eukaryotic Proteins 3.2. Unfolding Rates of Bacterial and Eukaryotic Proteins f g f y To find the dependence of the unfolding rates on the origin of the proteins, the 42 bacterial and 53 eukaryotic proteins from our database were separately studied. Two-state bacterial proteins fold and unfold faster than two-state eukaryotic proteins. For multi-state proteins, we observed that To find the dependence of the unfolding rates on the origin of the proteins, the 42 bacterial and 53 eukaryotic proteins from our database were separately studied. Two-state bacterial proteins fold and unfold faster than two-state eukaryotic proteins. For multi-state proteins, we observed that bacterial 6 of 14 Biomolecules 2020, 10, 197 proteins fold and unfold slower than eukaryotic proteins (see Figures 3 and 4). The same result was observed when the dataset consisted of 35 bacterial and 38 eukaryotic proteins [23]. The correlation between the logarithm of the unfolding and folding rates is 0.73 for bacterial and 0.75 for eukaryotic proteins. Moreover, for bacterial proteins, this correlation is better for two-state (0.69) than for multi-state proteins (0.45). For eukaryotic proteins, this correlation is better for multi-state (0.81) than for two-state proteins (0.72). Values Vasa/Sasa, ln(AbsCO), and Rg are slightly higher for the bacterial proteins, and this gap increases for multi-state proteins: 3.74 ± 0.07 vs. 3.47 ± 0.07 for Vasa/Sasa, 7.40 ± 0.07 vs. 7.14 ± 0.10 for ln(AbsCO), and 14.8 ± 0.5 vs. 13.9 ± 0.4 for Rg, respectively (Table 5). same result was observed when the dataset consisted of 35 bacterial and 38 eukaryotic proteins [23]. The correlation between the logarithm of the unfolding and folding rates is 0.73 for bacterial and 0.75 for eukaryotic proteins. Moreover, for bacterial proteins, this correlation is better for two-state (0.69) than for multi-state proteins (0.45). For eukaryotic proteins, this correlation is better for multi-state (0.81) than for two-state proteins (0.72). Values Vasa/Sasa, ln(AbsCO), and Rg are slightly higher for the bacterial proteins, and this gap increases for multi-state proteins: 3.74 ± 0.07 vs. 3.47 ± 0.07 for Vasa/Sasa, 7.40 ± 0.07 vs. 7.14 ± 0.10 for ln(AbsCO), and 14.8 ± 0.5 vs. 13.9 ± 0.4 for Rg, respectively (Table 5). Figure 3. The average values of the logarithm of the unfolding and folding rates for bacterial and eukaryotic two- and multi-state proteins. Structural parameters that better correlate with the logarithm of the unfolding rate are outlined in a rectangle. Figure 3. 3.2. Unfolding Rates of Bacterial and Eukaryotic Proteins 3.2. Unfolding Rates of Bacterial and Eukaryotic Proteins Average Value All (two + multi) Two-state Multi-state Bacteria Eukaryota Bacteria Eukaryota Bacteria Eukaryota Number of proteins 42 53 29 32 13 21 <L> 107 ± 9 92 ± 5 87 ± 10 77 ± 4 152 ± 14 115 ± 10 <Vasa/Sasa> 3.37 ± 0.07 3.30 ± 0.04 3.21 ± 0.08 3.20 ± 0.05 3.74 ± 0.07 3.47 ± 0.07 <ln(AbsCO)> 7.10 ± 0.07 7.08 ± 0.06 6.97 ± 0.09 7.04 ± 0.07 7.40 ± 0.07 7.14 ± 0.10 <Rg> 13.1 ± 0.4 12.9 ± 0.3 12.3 ± 0.5 12.3 ± 0.4 14.8 ± 0.5 13.9 ± 0.4 Average Value All (Two + Multi) Two-State Multi-State Bacteria Eukaryota Bacteria Eukaryota Bacteria Eukaryota Number of proteins 42 53 29 32 13 21 <L> 107 ± 9 92 ± 5 87 ± 10 77 ± 4 152 ± 14 115 ± 10 <Vasa/Sasa> 3.37 ± 0.07 3.30 ± 0.04 3.21 ± 0.08 3.20 ± 0.05 3.74 ± 0.07 3.47 ± 0.07 <ln(AbsCO)> 7.10 ± 0.07 7.08 ± 0.06 6.97 ± 0.09 7.04 ± 0.07 7.40 ± 0.07 7.14 ± 0.10 <Rg> 13.1 ± 0.4 12.9 ± 0.3 12.3 ± 0.5 12.3 ± 0.4 14.8 ± 0.5 13.9 ± 0.4 Then, the correlations between the logarithm of the unfolding rate and structural parameters for bacterial and eukaryotic proteins were investigated. The correlations between the logarithm of the unfolding rate and L, Vasa/Sasa, and ln(AbsCO) are almost the same for all bacterial and eukaryotic proteins: −0.67 vs. −0.68 for L, −0.72 vs. −0.69 for Vasa/Sasa, and −0.80 vs. −0.79 for ln(AbsCO), respectively (Table 6 and Figure 4). The difference is observed only for Rg, which correlates better with the logarithm of the unfolding rate of bacterial proteins (−0.71). If we consider these correlations for two-state and multi-state bacterial and eukaryotic proteins separately, we get the following picture. For two-state proteins, the correlation between the logarithm of the unfolding rate and Vasa/Sasa is almost the same for bacterial and eukaryotic proteins (−0.75 vs. −0.77). Rg and ln(AbsCO) better correlate with the logarithm of the unfolding rate of two-state bacterial proteins than with eukaryotic proteins (−0.86 vs. −0.77 for ln(AbsCO) and −0.64 vs. −0.47 for Rg, respectively). L, on the contrary, correlates better with the logarithm of the unfolding rate of two-state eukaryotic proteins (−0.61 vs. −0.75). 3.2. Unfolding Rates of Bacterial and Eukaryotic Proteins 3.2. Unfolding Rates of Bacterial and Eukaryotic Proteins For multi-state proteins, we observed the same picture as for two-state proteins for correlations of L and Rg with the logarithm of the unfolding rate. Both Vasa/Sasa and ln(AbsCO) correlate better with the logarithm of the unfolding rate of multi-state eukaryotic proteins than with bacterial proteins: −0.11 vs. −0.69 for Vasa/Sasa and −0.22 vs. −0.81 for ln(AbsCO), Then, the correlations between the logarithm of the unfolding rate and structural parameters for bacterial and eukaryotic proteins were investigated. The correlations between the logarithm of the unfolding rate and L, Vasa/Sasa, and ln(AbsCO) are almost the same for all bacterial and eukaryotic proteins: −0.67 vs. −0.68 for L, −0.72 vs. −0.69 for Vasa/Sasa, and −0.80 vs. −0.79 for ln(AbsCO), respectively (Table 6 and Figure 4). The difference is observed only for Rg, which correlates better with the logarithm of the unfolding rate of bacterial proteins (−0.71). If we consider these correlations for two-state and multi-state bacterial and eukaryotic proteins separately, we get the following picture. For two-state proteins, the correlation between the logarithm of the unfolding rate and Vasa/Sasa is almost the same for bacterial and eukaryotic proteins (−0.75 vs. −0.77). Rg and ln(AbsCO) better correlate with the logarithm of the unfolding rate of two-state bacterial proteins than with eukaryotic proteins (−0.86 vs. −0.77 for ln(AbsCO) and −0.64 vs. −0.47 for Rg, respectively). L, on the contrary, correlates better with the logarithm of the unfolding rate of two-state eukaryotic proteins (−0.61 vs. −0.75). For multi-state proteins, we observed the same picture as for two-state proteins for correlations of L and Rg with the logarithm of the unfolding rate. Both Vasa/Sasa and ln(AbsCO) correlate better with the logarithm of the unfolding rate of multi-state eukaryotic proteins than with bacterial proteins: −0.11 vs. −0.69 for Vasa/Sasa and −0.22 vs. −0.81 for ln(AbsCO), respectively. Biomolecules 2020, 10, 197 Bi l l 2020 9 7 of 14 7 f 14 molecules 2020, 9, x 7 o Figure 4. Correlations of the logarithm of the unfolding rate of the two- and multi-state bacterial and eukaryotic proteins with structural parameters: L, Vasa/Sasa, ln(AbsCO), and Rg. There is a line approximation of points and its equation: green line corresponds to bacterial proteins and pink line to eukaryotic proteins. R2 is a linear approximation reliability. Figure 4. 3.3. Unfolding Rates of Proteins from Thermophilic and Mesophilic Organisms 3.3. Unfolding Rates of Proteins from Thermophilic and Mesophilic Organisms .3. Unfolding Rates of Proteins from Thermophilic and Mesophilic Organisms .3. Unfolding Rates of Proteins from Thermophilic and Mesophilic Organisms Since a lot of attention was paid to the search for differences between thermophilic and mesophilic proteins — in particular, folding rates — we also decided to conduct our analysis for these proteins. All bacterial proteins were divided into thermophilic and mesophilic groups. Further in the text, we call proteins from thermophilic organisms as thermophilic proteins and proteins from mesophilic organisms as mesophilic proteins. The correlation between the logarithm of the unfolding and folding rates is better for mesophilic (0.76), in comparison with thermophilic (0.73) proteins. Moreover, for mesophilic proteins, this correlation is better for two-state (0.76) than for multi-state proteins (0.12). For thermophilic proteins, it is hard to say something, because there are only two proteins with multi-state kinetics. There is a correlation between stability and the logarithm of the unfolding rate for thermophilic (0.90) and mesophilic (0.73) proteins. The logarithm of the folding rate of thermophilic and mesophilic proteins are almost the same (4.75 ± 1.20 vs. 4.58 ± 0.79) (Figure 6 and Table 7). Still, mesophilic proteins unfold faster than thermophilic proteins (−5.63 ± 2.31 vs. −3.27 ± 1.12). The same picture is observed for two-state thermophilic and mesophilic proteins. Schematic “chevron” plots for thermophilic and mesophilic proteins are presented in Since a lot of attention was paid to the search for differences between thermophilic and mesophilic proteins — in particular, folding rates — we also decided to conduct our analysis for these proteins. All bacterial proteins were divided into thermophilic and mesophilic groups. Further in the text, we call proteins from thermophilic organisms as thermophilic proteins and proteins from mesophilic organisms as mesophilic proteins. The correlation between the logarithm of the unfolding and folding rates is better for mesophilic (0.76), in comparison with thermophilic (0.73) proteins. Moreover, for mesophilic proteins, this correlation is better for two-state (0.76) than for multi-state proteins (0.12). For thermophilic proteins, it is hard to say something, because there are only two proteins with multi-state kinetics. There is a correlation between stability and the logarithm of the unfolding rate for thermophilic (0.90) and mesophilic (0.73) proteins. The logarithm of the folding rate of thermophilic and mesophilic proteins are almost the same (4.75 ± 1.20 vs. 4.58 ± 0.79) (Figure 6 and Table 7). 3.2. Unfolding Rates of Bacterial and Eukaryotic Proteins 3.2. Unfolding Rates of Bacterial and Eukaryotic Proteins Correlations of the logarithm of the unfolding rate of the two- and multi-state bacterial and eukaryotic proteins with structural parameters: L, Vasa/Sasa, ln(AbsCO), and Rg. There is a line approximation of points and its equation: green line corresponds to bacterial proteins and pink line to eukaryotic proteins. R2 is a linear approximation reliability. Figure 4. Correlations of the logarithm of the unfolding rate of the two- and multi-state bacterial and eukaryotic proteins with structural parameters: L, Vasa/Sasa, ln(AbsCO), and Rg. There is a line approximation of points and its equation: green line corresponds to bacterial proteins and pink line to eukaryotic proteins R2 is a linear approximation reliability Figure 4. Correlations of the logarithm of the unfolding rate of the two- and multi-state bacterial and eukaryotic proteins with structural parameters: L, Vasa/Sasa, ln(AbsCO), and Rg. There is a line approximation of points and its equation: green line corresponds to bacterial proteins and pink line to eukaryotic proteins. R2 is a linear approximation reliability. 8 of 14 8 of 14 Biomolecules 2020, 10, 197 Biomolecules 2020, 9, x Table 6. Correlations between the logarithm of the unfolding rate (ln(ku)) and structural parameters (L, Vasa/Sasa, ln(AbsCO) and Rg) for two-state and multi-state bacterial and eukaryotic proteins. g g ( ( )) p (L, Vasa/Sasa, ln(AbsCO), and Rg) for two-state and multi-state bacterial and eukaryotic proteins. Correlations with All (two + multi) Two−state Multi−state Correlations with ln(ku) All (Two + Multi) Two-State Multi-State Bacteria Eukaryota Bacteria Eukaryota Bacteria Eukaryota Number of proteins 42 53 29 32 13 21 L −0.67 −0.68 −0.61 −0.75 −0.43 −0.77 Vasa/Sasa −0.72 −0.69 −0.75 −0.77 −0.11 −0.69 ln(AbsCO) −0.80 −0.79 −0.86 −0.77 −0.22 −0.81 Rg −0.71 −0.46 −0.64 −0.47 −0.58 −0.50 * Correlations above 0.75 are shown in bold. ln(ku) Bacteria Eukaryota Bacteria Eukaryota Bacteria Eukaryota Number of proteins 42 53 29 32 13 21 L −0.67 −0.68 −0.61 −0.75 −0.43 −0.77 Vasa/Sasa −0.72 −0.69 −0.75 −0.77 −0.11 −0.69 ln(AbsCO) −0.80 −0.79 −0.86 −0.77 −0.22 −0.81 Rg −0.71 −0.46 −0.64 −0.47 −0.58 −0.50 * Correlations above 0.75 are shown in bold. * Correlations above 0.75 are shown in bold. orrelations above 0.75 are shown in bold. Amino acid composition of bacterial and eukaryotic proteins was analyzed (Figure 5). The bacterial proteins with two-state kinetics are enriched in Ala, Gly, Lys, and Asn, compared with eukaryotic proteins with two-state kinetics. 3.2. Unfolding Rates of Bacterial and Eukaryotic Proteins 3.2. Unfolding Rates of Bacterial and Eukaryotic Proteins The eukaryotic proteins with two-state kinetics contain more His, Leu, Pro, Arg, Ser, and Trp, compared to the bacterial proteins with two-state kinetics (see Figure 5). Amino acid composition of bacterial and eukaryotic proteins was analyzed (Figure 5). The bacterial proteins with two-state kinetics are enriched in Ala, Gly, Lys, and Asn, compared with eukaryotic proteins with two-state kinetics. The eukaryotic proteins with two-state kinetics contain more His, Leu, Pro, Arg, Ser, and Trp, compared to the bacterial proteins with two-state kinetics (see Figure 5). Figure 5. Amino acid composition of the bacterial and eukaryotic proteins with two- and multi-state kinetics. Figure 5. Amino acid composition of the bacterial and eukaryotic proteins with two- and multi-state kinetics. Figure 5. Amino acid composition of the bacterial and eukaryotic proteins with two- and multi-state kinetics. Figure 5. Amino acid composition of the bacterial and eukaryotic proteins with two- and multi-state kinetics. 3.3. Unfolding Rates of Proteins from Thermophilic and Mesophilic Organisms 3.3. Unfolding Rates of Proteins from Thermophilic and Mesophilic Organisms Still, mesophilic proteins unfold faster than thermophilic proteins (−5.63 ± 2.31 vs. −3.27 ± 1.12). The same picture is observed for two-state thermophilic and mesophilic proteins. Schematic “chevron” plots for thermophilic and mesophilic proteins are presented in Figure 7. Biomolecules 2020, 10, 197 Biomolecules 2020 9 x 9 of 14 9 of 14 9 of 14 9 of 14 Figure 6. The average values of the logarithm of the unfolding and folding rates for thermophilic and mesophilic two- and multi-state proteins. Structural parameters that better correlate with the logarithm of the unfolding rate are outlined in a rectangle. Figure 6. The average values of the logarithm of the unfolding and folding rates for thermophilic and mesophilic two- and multi-state proteins. Structural parameters that better correlate with the logarithm of the unfolding rate are outlined in a rectangle. Figure 6. The average values of the logarithm of the unfolding and folding rates for thermophilic and mesophilic two- and multi-state proteins. Structural parameters that better correlate with the logarithm of the unfolding rate are outlined in a rectangle. Fi 6 Th l f t Figure 6. The average values of the logarithm of the unfolding and folding rates for thermophilic and mesophilic two- and multi-state proteins. Structural parameters that better correlate with the logarithm of the unfolding rate are outlined in a rectangle. Figure 6. The average values of the logarithm of the unfolding and folding rates for thermophilic and mesophilic two- and multi-state proteins. Structural parameters that better correlate with the logarithm of the unfolding rate are outlined in a rectangle. Figure 6. The average values of the logarithm of the unfolding and folding rates for thermophilic and mesophilic two- and multi-state proteins. Structural parameters that better correlate with the logarithm of the unfolding rate are outlined in a rectangle. Figure 7. Schematic “chevron” plots of the constant of observed rate (kapp = kf + ku) versus temperature for thermophilic and mesophilic proteins. Figure 7. Schematic “chevron” plots of the constant of observed rate (kapp = kf + ku) versus temperature for thermophilic and mesophilic proteins. Figure 7. Schematic “chevron” plots of the constant of observed rate (kapp = kf + ku) versus temperature for thermophilic and mesophilic proteins. T bl 7 A l f t t l t f t t t d lti t t th hili d Figure 7. 3.3. Unfolding Rates of Proteins from Thermophilic and Mesophilic Organisms 3.3. Unfolding Rates of Proteins from Thermophilic and Mesophilic Organisms For two−state thermophilic proteins, the correlation between all considered parameters (L, Vasa/Sasa, ln(AbsCO), and R ) is better than for two state mesophilic proteins: 0 92 vs 0 60 for L 0 92 vs 0 78 for <Rg> 13.3 ± 0.8 13.0 ± 0.5 12.5 ± 0.7 12.2 ± 0.6 16.2 ± 1.1 14.5 ± 0.5 Finally, the correlations of the logarithm of the unfolding rate and structural parameters for thermophilic and mesophilic proteins were examined (Table 8 and Figure 8). L and Rg correlate better with the logarithm of the unfolding rate of all thermophilic proteins (−0.83 vs. −0.64 for L and −0.87 vs. −0.66 for Rg), and ln(AbsCO) correlates better with the logarithm of the unfolding rate of all mesophilic proteins (−0.74 vs. −0.83). Vasa/Sasa correlates with the logarithm of the unfolding rate practically the same for all thermophilic and mesophilic proteins (−0.77 vs. −0.75). For two−state thermophilic proteins, the correlation between all considered parameters (L, Vasa/Sasa, ln(AbsCO), and Rg) is better than for two−state mesophilic proteins: −0.92 vs. −0.60 for L, −0.92 vs. −0.78 for Finally, the correlations of the logarithm of the unfolding rate and structural parameters for thermophilic and mesophilic proteins were examined (Table 8 and Figure 8). L and Rg correlate better with the logarithm of the unfolding rate of all thermophilic proteins (−0.83 vs. −0.64 for L and −0.87 vs. −0.66 for Rg), and ln(AbsCO) correlates better with the logarithm of the unfolding rate of all mesophilic proteins (−0.74 vs. −0.83). Vasa/Sasa correlates with the logarithm of the unfolding rate practically the same for all thermophilic and mesophilic proteins (−0.77 vs. −0.75). For two-state thermophilic proteins, the correlation between all considered parameters (L, Vasa/Sasa, ln(AbsCO) and Rg) is better than for two-state mesophilic proteins: −0.92 vs. −0.60 for L, −0.92 vs. −0.78 for Vasa/Sasa, −0.93 vs. −0.85 for ln(AbsCO), and −0.88 vs. −0.60 for Rg. <ln(AbsCO)> 7.13 ± 0.15 7.09 ± 0.08 7.03 ± 0.17 6.95 ± 0.11 7.54 ± 0.07 7.38 ± 0.09 <Rg> 13.3 ± 0.8 13.0 ± 0.5 12.5 ± 0.7 12.2 ± 0.6 16.2 ± 1.1 14.5 ± 0.5 Finally, the correlations of the logarithm of the unfolding rate and structural parameters for thermophilic and mesophilic proteins were examined (Table 8 and Figure 8). L and Rg correlate better with the logarithm of the unfolding rate of all thermophilic proteins (−0.83 vs. −0.64 for L and −0.87 vs. 3.3. Unfolding Rates of Proteins from Thermophilic and Mesophilic Organisms 3.3. Unfolding Rates of Proteins from Thermophilic and Mesophilic Organisms Schematic “chevron” plots of the constant of observed rate (kapp = kf + ku) versus t t f th hili d hili t i Figure 7. Schematic “chevron” plots of the constant of observed rate (kapp = kf + ku) versus temperature for thermophilic and mesophilic proteins. Figure 7. Schematic “chevron” plots of the constant of observed rate (kapp = kf + ku) versus temperature for thermophilic and mesophilic proteins. temperature for thermophilic and mesophilic proteins. Table 7. Average values of structural parameters for two-state and multi-state thermophilic and Table 7. Average values of structural parameters for two-state and multi-state thermophilic and mesophilic proteins Table 7. Average values of structural parameters for two-state and multi-state thermophilic and mesophilic proteins. temperature for thermophilic and mesophilic proteins. Table 7. Average values of structural parameters for two-state and multi-state thermophilic and Table 7. Average values of structural parameters for two-state and multi-state thermophilic and mesophilic proteins Table 7. Average values of structural parameters for two-state and multi-state thermophilic and mesophilic proteins. Table 7. Average values of structural parameters for two state and multi state thermophilic and mesophilic proteins. Average Value All (two + multi) Two-state Multi-state Thermophile Mesophile Thermophile Mesophile Thermophile Mesophile Number of proteins 10 32 8 21 2 11 <L> 105 ± 18 108 ± 11 82 ± 11 89 ± 13 197 ± 22 144 ± 14 <Vasa/Sasa> 3.30 ± 0.14 3.39 ± 0.08 3.13 ± 0.10 3.23 ± 0.10 3.97 ± 0.01 3.70 ± 0.08 <ln(AbsCO)> 7.13 ± 0.15 7.09 ± 0.08 7.03 ± 0.17 6.95 ± 0.11 7.54 ± 0.07 7.38 ± 0.09 <Rg> 13.3 ± 0.8 13.0 ± 0.5 12.5 ± 0.7 12.2 ± 0.6 16.2 ± 1.1 14.5 ± 0.5 Finally, the correlations of the logarithm of the unfolding rate and structural parameters for thermophilic and mesophilic proteins were examined (Table 8 and Figure 8). L and Rg correlate better with the logarithm of the unfolding rate of all thermophilic proteins (−0.83 vs. −0.64 for L and −0.87 vs. −0.66 for Rg), and ln(AbsCO) correlates better with the logarithm of the unfolding rate of all mesophilic proteins (−0.74 vs. −0.83). Vasa/Sasa correlates with the logarithm of the unfolding rate practically the same for all thermophilic and mesophilic proteins (−0.77 vs. −0.75). 3.3. Unfolding Rates of Proteins from Thermophilic and Mesophilic Organisms 3.3. Unfolding Rates of Proteins from Thermophilic and Mesophilic Organisms For two−state thermophilic proteins, the correlation between all considered parameters (L, Vasa/Sasa, ln(AbsCO), and Rg) is better than for two−state mesophilic proteins: −0 92 vs −0 60 for L −0 92 vs −0 78 for mesophilic proteins. Average Value All (two + multi) Two-state Multi-state Thermophile Mesophile Thermophile Mesophile Thermophile Mesophile Number of proteins 10 32 8 21 2 11 <L> 105 ± 18 108 ± 11 82 ± 11 89 ± 13 197 ± 22 144 ± 14 <Vasa/Sasa> 3.30 ± 0.14 3.39 ± 0.08 3.13 ± 0.10 3.23 ± 0.10 3.97 ± 0.01 3.70 ± 0.08 <ln(AbsCO)> 7.13 ± 0.15 7.09 ± 0.08 7.03 ± 0.17 6.95 ± 0.11 7.54 ± 0.07 7.38 ± 0.09 <Rg> 13.3 ± 0.8 13.0 ± 0.5 12.5 ± 0.7 12.2 ± 0.6 16.2 ± 1.1 14.5 ± 0.5 Finally, the correlations of the logarithm of the unfolding rate and structural parameters for thermophilic and mesophilic proteins were examined (Table 8 and Figure 8). L and Rg correlate better with the logarithm of the unfolding rate of all thermophilic proteins (−0.83 vs. −0.64 for L and −0.87 vs. −0.66 for Rg), and ln(AbsCO) correlates better with the logarithm of the unfolding rate of all mesophilic proteins (−0.74 vs. −0.83). Vasa/Sasa correlates with the logarithm of the unfolding rate practically the same for all thermophilic and mesophilic proteins (−0.77 vs. −0.75). For two−state thermophilic proteins, the correlation between all considered parameters (L, Vasa/Sasa, ln(AbsCO), and Rg) is better than for two−state mesophilic proteins: −0.92 vs. −0.60 for L, −0.92 vs. −0.78 for p p Average Value All (Two + Multi) Two-State Multi-State Thermophile Mesophile Thermophile Mesophile Thermophile Mesophile Number of proteins 10 32 8 21 2 11 <L> 105 ± 18 108 ± 11 82 ± 11 89 ± 13 197 ± 22 144 ± 14 <Vasa/Sasa> 3.30 ± 0.14 3.39 ± 0.08 3.13 ± 0.10 3.23 ± 0.10 3.97 ± 0.01 3.70 ± 0.08 <ln(AbsCO)> 7.13 ± 0.15 7.09 ± 0.08 7.03 ± 0.17 6.95 ± 0.11 7.54 ± 0.07 7.38 ± 0.09 <Rg> 13.3 ± 0.8 13.0 ± 0.5 12.5 ± 0.7 12.2 ± 0.6 16.2 ± 1.1 14.5 ± 0.5 Finally, the correlations of the logarithm of the unfolding rate and structural parameters for thermophilic and mesophilic proteins were examined (Table 8 and Figure 8). 3.3. Unfolding Rates of Proteins from Thermophilic and Mesophilic Organisms 3.3. Unfolding Rates of Proteins from Thermophilic and Mesophilic Organisms L and Rg correlate better with the logarithm of the unfolding rate of all thermophilic proteins (−0.83 vs. −0.64 for L and −0.87 vs. −0.66 for Rg), and ln(AbsCO) correlates better with the logarithm of the unfolding rate of all mesophilic proteins (−0.74 vs. −0.83). Vasa/Sasa correlates with the logarithm of the unfolding rate practically the same for all thermophilic and mesophilic proteins (−0.77 vs. −0.75). For two-state thermophilic proteins, the correlation between all considered parameters (L, Vasa/Sasa, ln(AbsCO) and Rg) is better than for two-state mesophilic proteins: −0.92 vs. −0.60 for L, −0.92 vs. −0.78 for Vasa/Sasa, −0.93 vs. −0.85 for ln(AbsCO), and −0.88 vs. −0.60 for Rg. mesophilic proteins. 3.3. Unfolding Rates of Proteins from Thermophilic and Mesophilic Organisms 3.3. Unfolding Rates of Proteins from Thermophilic and Mesophilic Organisms Average Value All (two + multi) Two-state Multi-state Thermophile Mesophile Thermophile Mesophile Thermophile Mesophile Number of proteins 10 32 8 21 2 11 <L> 105 ± 18 108 ± 11 82 ± 11 89 ± 13 197 ± 22 144 ± 14 <Vasa/Sasa> 3.30 ± 0.14 3.39 ± 0.08 3.13 ± 0.10 3.23 ± 0.10 3.97 ± 0.01 3.70 ± 0.08 p p Average Value All (two + multi) Two-state Multi-state Thermophile Mesophile Thermophile Mesophile Thermophile Mesophile Number of proteins 10 32 8 21 2 11 <L> 105 ± 18 108 ± 11 82 ± 11 89 ± 13 197 ± 22 144 ± 14 <Vasa/Sasa> 3.30 ± 0.14 3.39 ± 0.08 3.13 ± 0.10 3.23 ± 0.10 3.97 ± 0.01 3.70 ± 0.08 <ln(AbsCO)> 7 13 ± 0 15 7 09 ± 0 08 7 03 ± 0 17 6 95 ± 0 11 7 54 ± 0 07 7 38 ± 0 09 Average Value All (Two + Multi) Two-State Multi-State Thermophile Mesophile Thermophile Mesophile Thermophile Mesophile Number of proteins 10 32 8 21 2 11 <L> 105 ± 18 108 ± 11 82 ± 11 89 ± 13 197 ± 22 144 ± 14 <Vasa/Sasa> 3.30 ± 0.14 3.39 ± 0.08 3.13 ± 0.10 3.23 ± 0.10 3.97 ± 0.01 3.70 ± 0.08 <ln(AbsCO)> 7.13 ± 0.15 7.09 ± 0.08 7.03 ± 0.17 6.95 ± 0.11 7.54 ± 0.07 7.38 ± 0.09 <Rg> 13.3 ± 0.8 13.0 ± 0.5 12.5 ± 0.7 12.2 ± 0.6 16.2 ± 1.1 14.5 ± 0.5 <ln(AbsCO)> 7.13 ± 0.15 7.09 ± 0.08 7.03 ± 0.17 6.95 ± 0.11 7.54 ± 0.07 7.38 ± 0.09 <Rg> 13.3 ± 0.8 13.0 ± 0.5 12.5 ± 0.7 12.2 ± 0.6 16.2 ± 1.1 14.5 ± 0.5 Finally, the correlations of the logarithm of the unfolding rate and structural parameters for thermophilic and mesophilic proteins were examined (Table 8 and Figure 8). L and Rg correlate better with the logarithm of the unfolding rate of all thermophilic proteins (−0.83 vs. −0.64 for L and −0.87 vs. −0.66 for Rg), and ln(AbsCO) correlates better with the logarithm of the unfolding rate of all mesophilic proteins (−0.74 vs. −0.83). Vasa/Sasa correlates with the logarithm of the unfolding rate practically the same for all thermophilic and mesophilic proteins (−0.77 vs. −0.75). <ln(AbsCO)> 7.13 ± 0.15 7.09 ± 0.08 7.03 ± 0.17 6.95 ± 0.11 7.54 ± 0.07 7.38 ± 0.09 <Rg> 13.3 ± 0.8 13.0 ± 0.5 12.5 ± 0.7 12.2 ± 0.6 16.2 ± 1.1 14.5 ± 0.5 Finally, the correlations of the logarithm of the unfolding rate and structural parameters for thermophilic and mesophilic proteins were examined (Table 8 and Figure 8). L and Rg correlate better with the logarithm of the unfolding rate of all thermophilic proteins (−0.83 vs. −0.64 for L and −0.87 vs. −0.66 for Rg), and ln(AbsCO) correlates better with the logarithm of the unfolding rate of all mesophilic proteins (−0.74 vs. −0.83). Vasa/Sasa correlates with the logarithm of the unfolding rate practically the same for all thermophilic and mesophilic proteins (−0.77 vs. −0.75). For two−state thermophilic proteins, the correlation between all considered parameters (L, Vasa/Sasa, ln(AbsCO), and R ) is better than for two state mesophilic proteins: 0 92 vs 0 60 for L 0 92 vs 0 78 for ( ) <Rg> 13.3 ± 0.8 13.0 ± 0.5 12.5 ± 0.7 12.2 ± 0.6 16.2 ± 1.1 14.5 ± 0.5 Finally, the correlations of the logarithm of the unfolding rate and structural parameters for thermophilic and mesophilic proteins were examined (Table 8 and Figure 8). L and Rg correlate better with the logarithm of the unfolding rate of all thermophilic proteins (−0.83 vs. −0.64 for L and −0.87 vs. −0.66 for Rg), and ln(AbsCO) correlates better with the logarithm of the unfolding rate of all mesophilic proteins (−0.74 vs. −0.83). Vasa/Sasa correlates with the logarithm of the unfolding rate practically the same for all thermophilic and mesophilic proteins (−0.77 vs. −0.75). For two−state thermophilic proteins, the correlation between all considered parameters (L, Vasa/Sasa, ln(AbsCO), and Rg) is better than for two−state mesophilic proteins: −0.92 vs. −0.60 for L, −0.92 vs. −0.78 for Finally, the correlations of the logarithm of the unfolding rate and structural parameters for thermophilic and mesophilic proteins were examined (Table 8 and Figure 8). L and Rg correlate better with the logarithm of the unfolding rate of all thermophilic proteins (−0.83 vs. −0.64 for L and −0.87 vs. −0.66 for Rg), and ln(AbsCO) correlates better with the logarithm of the unfolding rate of all mesophilic proteins (−0.74 vs. −0.83). Vasa/Sasa correlates with the logarithm of the unfolding rate practically the same for all thermophilic and mesophilic proteins (−0.77 vs. −0.75). For two-state thermophilic proteins, the correlation between all considered parameters (L, Vasa/Sasa, ln(AbsCO) and Rg) is better than for two-state mesophilic proteins: −0.92 vs. −0.60 for L, −0.92 vs. −0.78 for Vasa/Sasa, −0.93 vs. −0.85 for ln(AbsCO), and −0.88 vs. −0.60 for Rg. 3.3. Unfolding Rates of Proteins from Thermophilic and Mesophilic Organisms 3.3. Unfolding Rates of Proteins from Thermophilic and Mesophilic Organisms −0.66 for Rg), and ln(AbsCO) correlates better with the logarithm of the unfolding rate of all mesophilic proteins (−0.74 vs. −0.83). Vasa/Sasa correlates with the logarithm of the unfolding rate practically the same for all thermophilic and mesophilic proteins (−0.77 vs. −0.75). For two−state thermophilic proteins, the correlation between all considered parameters (L, Vasa/Sasa, ln(AbsCO), a d R ) i bette tha fo t o tate e o hili otei 0 92 0 60 fo L 0 92 0 78 fo <Rg> 13.3 ± 0.8 13.0 ± 0.5 12.5 ± 0.7 12.2 ± 0.6 16.2 ± 1.1 14.5 ± 0.5 Finally, the correlations of the logarithm of the unfolding rate and structural parameters for thermophilic and mesophilic proteins were examined (Table 8 and Figure 8). L and Rg correlate better with the logarithm of the unfolding rate of all thermophilic proteins (−0.83 vs. −0.64 for L and −0.87 vs. −0.66 for Rg), and ln(AbsCO) correlates better with the logarithm of the unfolding rate of all mesophilic proteins (−0.74 vs. −0.83). Vasa/Sasa correlates with the logarithm of the unfolding rate practically the same for all thermophilic and mesophilic proteins (−0.77 vs. −0.75). For two−state thermophilic proteins, the correlation between all considered parameters (L, Vasa/Sasa, ln(AbsCO), and Rg) is better than for two−state mesophilic proteins: −0.92 vs. −0.60 for L, −0.92 vs. −0.78 for Finally, the correlations of the logarithm of the unfolding rate and structural parameters for thermophilic and mesophilic proteins were examined (Table 8 and Figure 8). L and Rg correlate better with the logarithm of the unfolding rate of all thermophilic proteins (−0.83 vs. −0.64 for L and −0.87 vs. −0.66 for Rg), and ln(AbsCO) correlates better with the logarithm of the unfolding rate of all mesophilic proteins (−0.74 vs. −0.83). Vasa/Sasa correlates with the logarithm of the unfolding rate practically the same for all thermophilic and mesophilic proteins (−0.77 vs. −0.75). For two-state thermophilic proteins, the correlation between all considered parameters (L, Vasa/Sasa, ln(AbsCO) and Rg) is better than for two-state mesophilic proteins: −0.92 vs. −0.60 for L, −0.92 vs. −0.78 for Vasa/Sasa, −0.93 vs. −0.85 for ln(AbsCO), and −0.88 vs. −0.60 for Rg. Biomolecules 2020, 10, 197 Biomolecules 2020 9 x 10 of 14 10 of 14 Figure 8. 3.3. Unfolding Rates of Proteins from Thermophilic and Mesophilic Organisms 3.3. Unfolding Rates of Proteins from Thermophilic and Mesophilic Organisms Correlations of the logarithm of the unfolding rate of the two- and multi-state thermophilic and mesophilic proteins with structural parameters: L (length of the protein), Vasa/Sasa, ln(AbsCO), and Rg. There is a line approximation of points and its equation: red line corresponds to thermophilic proteins and blue line to mesophilic proteins. R2 is a linear approximation reliability. Figure 8. Correlations of the logarithm of the unfolding rate of the two- and multi-state thermophilic and mesophilic proteins with structural parameters: L (length of the protein), Vasa/Sasa, ln(AbsCO), and Rg. There is a line approximation of points and its equation: red line corresponds to thermophilic proteins and blue line to mesophilic proteins. R2 is a linear approximation reliability. Figure 8. Correlations of the logarithm of the unfolding rate of the two- and multi-state thermophilic and mesophilic proteins with structural parameters: L (length of the protein), Vasa/Sasa, ln(AbsCO), and Rg. There is a line approximation of points and its equation: red line corresponds to thermophilic proteins and blue line to mesophilic proteins. R2 is a linear approximation reliability. Figure 8. Correlations of the logarithm of the unfolding rate of the two- and multi-state thermophilic and mesophilic proteins with structural parameters: L (length of the protein), Vasa/Sasa, ln(AbsCO), and Rg. There is a line approximation of points and its equation: red line corresponds to thermophilic proteins and blue line to mesophilic proteins. R2 is a linear approximation reliability. 11 of 14 with the ore Asp, Biomolecules 2020, 10, 197 The thermophil mesophilic proteins Table 8. Correlations between the logarithm of the unfolding rate (ln(ku)) and structural parameters (L, Vasa/Sasa, ln(AbsCO) and Rg) for two-state and multi-state thermophilic and mesophilic proteins. , , , p p p ( g ) bout two-state thermophilic and mesophilic proteins. These data are also consistent with those we obtained earlier in the study of 373 pairs of structurally similar thermophilic and mesop Table 8. Correlations between the logarithm of the unfolding rate (ln(ku)) and structural parameters (L, Vasa/Sasa, ln(AbsCO) and Rg) for two-state and multi-state thermophilic and mesophilic proteins. Correlations with ln(ku) All (Two + Multi) Two-State Multi-State Thermophile Mesophile Thermophile Mesophile Thermophile Mesophile Number of proteins 10 32 8 21 2 11 L −0.83 −0.64 −0.92 −0.60 − −0.27 Vasa/Sasa −0.77 −0.75 −0.92 −0.78 − 0.09 ln(AbsCO) −0.74 −0.83 −0.93 −0.85 − −0.35 Rg −0.87 −0.66 −0.88 −0.60 − −0.47 * Correlations above 0.75 are shown in bold. 3.3. Unfolding Rates of Proteins from Thermophilic and Mesophilic Organisms 3.3. Unfolding Rates of Proteins from Thermophilic and Mesophilic Organisms about two-state thermophilic and mesophilic proteins. These data are also consistent with those that we obtained earlier in the study of 373 pairs of structurally similar thermophilic and mesophilic proteins [27]. Table 8. Correlations between the logarithm of the unfolding rate (ln(ku)) and structural parameters (L, Vasa/Sasa, ln(AbsCO), and Rg) for two-state and multi-state thermophilic and mesophilic proteins. Correlation s with ln(ku) All (two + multi) Two−state Multi−state Thermophil e Mesophil e Thermophil e Mesophil e Thermophil e Mesophil e Number of 10 32 8 21 2 11 * Correlations above 0.75 are shown in bold. e e e The thermophilic proteins are enriched with Lys, Arg, and Val, in comparison with the mesophilic proteins, and enriched in Lys, Asp, Ala, and the mesophilic proteins contain more Asp, Asn, Ser, and Thr, in comparison with the thermophilic proteins (Figure 9). The same can be said about two-state thermophilic and mesophilic proteins. These data are also consistent with those that we obtained earlier in the study of 373 pairs of structurally similar thermophilic and mesophilic proteins [27]. proteins L −0.83 −0.64 −0.92 −0.60 − −0.27 Vasa/Sasa −0.77 −0.75 −0.92 −0.78 − 0.09 ln(AbsCO) −0.74 −0.83 −0.93 −0.85 − −0.35 Rg −0.87 −0.66 −0.88 −0.60 − −0.47 * Correlations above 0.75 are shown in bold. Figure 9. Amino acid composition of the bacterial thermophilic and mesophilic proteins with two- and multi-state kinetics. Figure 9. Amino acid composition of the bacterial thermophilic and mesophilic proteins with two- and multi-state kinetics. Figure 9. Amino acid composition of the bacterial thermophilic and mesophilic proteins with two- and multi-state kinetics. Figure 9. Amino acid composition of the bacterial thermophilic and mesophilic proteins with two- and multi-state kinetics. 4. Discussion 4. Discussion In this paper, we tried to find parameters that are important for predicting the protein unfolding rates. For this, the database consists of 108 proteins with known unfolding and folding rates, and such structural parameters as L, ln(AbsCO), Vasa/Sasa, and Rg were considered. In this paper, we tried to find parameters that are important for predicting the protein unfolding rates. For this, the database consists of 108 proteins with known unfolding and folding rates, and such structural parameters as L, ln(AbsCO), Vasa/Sasa, and Rg were considered. rates, and such structural parameters as L, ln(AbsCO), Vasa/Sasa, and Rg were considered. The good correlation (0.79) between the logarithm of the unfolding rate and protein stability was observed for 108 proteins. p ( ) g The good correlation (0.79) between the logarithm of the unfolding rate and protein stability was observed for 108 proteins. was obse ved o 08 p otei s. First, we divided the proteins in our database into two-states and multi-states. On average, the logarithms of the folding and unfolding rates are higher for two-state proteins, in comparison with First, we divided the proteins in our database into two-states and multi-states. On average, the logarithms of the folding and unfolding rates are higher for two-state proteins, in comparison with multi-state proteins. A good correlation (not lower than 0.70) for the logarithm of the folding and 12 of 14 Biomolecules 2020, 10, 197 unfolding rates for two- and multi-state proteins was observed. It has been shown that the logarithm of the unfolding rate of two-state proteins correlate better with Vasa/Sasa (−0.79) and ln(AbsCO) (−0.87), and the logarithm of the unfolding rate of multi-state correlates better with L (−0.71). Then, we separately studied bacterial and eukaryotic proteins from our database. It has been shown that two-state bacterial proteins fold and unfold faster than two-state eukaryotic proteins, and multi-state eukaryotic proteins fold and unfold faster than multi-state bacterial proteins. The logarithm of the unfolding rate of two-state bacterial proteins correlates better with ln(AbsCO) (−0.86) and Rg (−0.64), and eukaryotic proteins correlate better with L (−0.75). For multi-state proteins, the following picture is observed: the logarithm of the unfolding rate of bacterial proteins correlates better with Rg (−0.58), and eukaryotic proteins correlate better with L (−0.77), Vasa/Sasa (−0.69), and ln(AbsCO) (−0.81). Finally, we separately studied the thermophilic and mesophilic bacterial proteins from our database. 4. Discussion 4. Discussion There is correlation of the logarithm of the unfolding rate with protein stability for thermophilic (0.90) and mesophilic proteins (0.73). It has been shown that the logarithm of the unfolding rates of thermophilic proteins are about two orders of magnitude lower than that of mesophilic proteins, but the logarithm of the folding rates of thermophilic and mesophilic proteins are almost the same. The logarithm of the unfolding rate of two- and multi-state thermophilic proteins correlate better with all considered structural parameters (L, Vasa/Sasa, ln(AbsCO) and Rg), in comparison with the mesophilic proteins. We have tried to find out which parameters are most important for the prediction of the unfolding rates for proteins from different structural classes (α, β, α/β and α + β); proteins of different origins (bacterial and eukaryotic); and proteins from different organisms (thermophilic and mesophilic). 5. Conclusions Thus, it has been shown that there is a good correlation between the logarithm of the unfolding and folding rates (0.79) and between the logarithm of the unfolding rate and proteins stabilities (0.79) for 108 proteins. The correlation between the unfolding and folding rates is better for: two-state (0.78), in comparison with multi-state (0.73) proteins; α and β proteins (0.78 and 0.75), in comparison with α/β and α + β protein (0.59 and 0.60) structural classes; eukaryotic (0.75), in comparison with bacterial (0.73) proteins; and mesophilic (0.76), in comparison with thermophilic (0.73) proteins. The structural parameter ln(AbsCO) better correlates with the logarithm of the unfolding rate for: all 108 proteins; proteins from α and β structural classes; and bacterial, eukaryotic, and mesophilic proteins, in comparison with other parameters (L, Vasa/Sasa and Rg). Supplementary Materials: The following are available online: http://www.mdpi.com/2218-273X/10/2/197/s1. Table S1. Logarithm of folding and unfolding rates and some structural parameters for 108 proteins. Table S2. Average values of different parameters for four structural classes of proteins (α, β, α/β and α + β). Author Contributions: Conceptualization and design of the experiments, O.V.G. and A.V.G.; software, A.V.G.; formal analysis, A.V.G. and O.V.G.; investigation, A.V.G. and O.V.G.; data curation, A.V.G. and O.V.G.; visualization, A.V.G.; writing—original draft preparation, A.V.G. and O.V.G.; funding acquisition, A.V.G.; supervision, O.V.G.; and validation, A.V.G. and O.V.G. All authors have read and agreed to the published version of the manuscript. Funding: This research was funded by the Russian Science Foundation, grant number 19-74-10051. Funding: This research was funded by the Russian Science Foundation, grant number 19-74-10051. Conflicts of Interest: The authors declare no conflicts of interest. ln(kf)—logarithm of the folding rate, ln(ku)—logarithm of the unfolding rate, ln(kmt)—logarithm of the mid-transition folding/unfolding rate, L—length of the protein, rCO—relative contact order, ln(AbsCO)—logarithm of the absolute contact order, Vasa/Sasa—radius of cross-section, and Rg—radius of gyration. Abbreviations ln(kf)—logarithm of the folding rate, ln(ku)—logarithm of the unfolding rate, ln(kmt)—logarithm of the mid-transition folding/unfolding rate, L—length of the protein, rCO—relative contact order, ln(AbsCO)—logarithm of the absolute contact order, Vasa/Sasa—radius of cross-section, and Rg—radius of gyration. 13 of 14 Biomolecules 2020, 10, 197 References 1. Jackson, S.E. How do small single-domain proteins fold? Fold. Des. 1998, 3, R81–R91. [CrossRef] 2. Plaxco, K.W.; Simons, K.T.; Baker, D. Contact order, transition state placement and the refolding rates of single domain proteins. J. Mol. Biol. 1998, 277, 985–994. [CrossRef] [PubMed] 3. Gromiha, M.M.; Selvaraj, S. Comparison between long-range interactions and contact order in determining the folding rate of two-state proteins: Application of long-range order to folding rate prediction. J. Mol. Biol. 2001, 310, 27–32. [CrossRef] [PubMed] 4. Zhou, H.; Zhou, Y. Folding rate prediction using total contact distance. Biophys. 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When null hypothesis significance testing is unsuitable for research: a reassessment
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Edited by: Satrajit S. Ghosh, Massachusetts Institute of Technology, United States Edited by: Satrajit S. Ghosh, Massachusetts Institute of Technology, United States Reviewed by: Bertrand Thirion, Institut National de Recherche en Informatique et en Automatique (INRIA), France Cyril R. Pernet, University of Edinburgh, United Kingdom Keywords: replication crisis, false positive findings, research methodology, null hypothesis significance testing, Bayesian methods Reviewed by: Bertrand Thirion, Institut National de Recherche en Informatique et en Automatique (INRIA), France Cyril R. Pernet, University of Edinburgh, United Kingdom Reviewed by: Bertrand Thirion, Institut National de Recherche en Informatique et en Automatique (INRIA), France Cyril R. Pernet, University of Edinburgh, United Kingdom “What used to be called judgment is now called prejudice and what used to be called prejudice is now called a null hypothesis. In the social sciences, particularly, it is dangerous nonsense (dressed up as the “scientific method”) and will cause much trouble before it is widely appreciated as such.” (Edwards, 1972; p.180.) p “...the mathematical rules of probability theory are not merely rules for calculating frequencies of random variables; they are also the unique consistent rules for conducting inference (i.e., plausible reasoning)” *Correspondence: Denes Szucs ds377@cam.ac.uk *Correspondence: Denes Szucs ds377@cam.ac.uk (Jaynes, 2003; p. xxii). (Jaynes, 2003; p. xxii). When Null Hypothesis Significance Testing Is Unsuitable for Research: A Reassessment 1 Department of Psychology, University of Cambridge, Cambridge, United Kingdom, 2 Meta-Research Innovation Center at Stanford and Department of Medicine, Department of Health Research and Policy, and Department of Statistics, Stanford University, Stanford, CA, United States Null hypothesis significance testing (NHST) has several shortcomings that are likely contributing factors behind the widely debated replication crisis of (cognitive) neuroscience, psychology, and biomedical science in general. We review these shortcomings and suggest that, after sustained negative experience, NHST should no longer be the default, dominant statistical practice of all biomedical and psychological research. If theoretical predictions are weak we should not rely on all or nothing hypothesis tests. Different inferential methods may be most suitable for different types of research questions. Whenever researchers use NHST they should justify its use, and publish pre-study power calculations and effect sizes, including negative findings. Hypothesis-testing studies should be pre-registered and optimally raw data published. The current statistics lite educational approach for students that has sustained the widespread, spurious use of NHST should be phased out. THE REPLICATION CRISIS AND NULL HYPOTHESIS SIGNIFICANCE TESTING (NHST) Received: 03 February 2017 Accepted: 13 July 2017 Published: 03 August 2017 There is increasing discontent that many areas of psychological science, cognitive neuroscience, and biomedical research (Ioannidis, 2005; Ioannidis et al., 2014) are in a crisis of producing too many false positive non-replicable results (Begley and Ellis, 2012; Aarts et al., 2015). This wastes research funding, erodes credibility and slows down scientific progress. Since more than half a century many methodologists have claimed repeatedly that this crisis may at least in part be related to problems with Null Hypothesis Significance Testing (NHST; Rozeboom, 1960; Bakan, 1966; Meehl, 1978; Gigerenzer, 1998; Nickerson, 2000). However, most scientists REVIEW published: 03 August 2017 doi: 10.3389/fnhum.2017.00390 REVIEW published: 03 August 2017 doi: 10.3389/fnhum.2017.00390 Citation: Szucs D and Ioannidis JPA (2017) When Null Hypothesis Significance Testing Is Unsuitable for Research: A Reassessment. Front. Hum. Neurosci. 11:390. doi: 10.3389/fnhum.2017.00390 August 2017 | Volume 11 | Article 390 Frontiers in Human Neuroscience | www.frontiersin.org 1 Null Hypothesis Significance Testing: A Reassessment Szucs and Ioannidis (and in particular psychologists, biomedical scientists, social scientists, cognitive scientists, and neuroscientists) are still near exclusively educated in NHST, they tend to misunderstand and abuse NHST and the method is near fully dominant in scientific papers (Chavalarias et al., 1990-2015). Here we provide an accessible critical reassessment of NHST and suggest that while it may have legitimate uses when there are precise quantitative predictions and/or as a heuristic, it should be abandoned as the cornerstone of research. (and in particular psychologists, biomedical scientists, social scientists, cognitive scientists, and neuroscientists) are still near exclusively educated in NHST, they tend to misunderstand and abuse NHST and the method is near fully dominant in scientific papers (Chavalarias et al., 1990-2015). Here we provide an accessible critical reassessment of NHST and suggest that while it may have legitimate uses when there are precise quantitative predictions and/or as a heuristic, it should be abandoned as the cornerstone of research. Hence, a single significant result should not represent a “scientific fact” but should merely draw attention to a phenomenon which seems worthy of further investigation including replication (Goodman, 2008). In contrast to the above, until recently replication studies have been very rare in many scientific fields; lack of replication efforts has been a particular problem in the psychological sciences (Makel et al., 2012), but this may hopefully change now with the wide attention that replication has received (Aarts et al., 2015). Our paper does not concern specifically the details of neuro-imaging methodology, many papers dealt with such details recently (Pernet and Poline, 2015; Nichols et al., 2016, 2017). Rather, we take a more general view in discussing fundamental problems that can affect any scientific field, including neuroscience and neuro-imaging. In relation to this it is important to see that non-invasive neuroscience data related to behavioral tasks cannot be interpreted if task manipulations did not work and/or behavior is unclear. This is because most measured brain activity changes can be interpreted in many different ways on their own (Poldrack, 2006; see Section 2.7 in Nichols et al., 2016). NHST as a Weak Heuristic Based on the p-Value: Fisher p-values were popularized by Fisher (1925). In the context of the current NHST approach Fisher only relied on the concepts of the null hypothesis (H0) and the exact p-value (hereafter p will refer to the p-value and “pr” to probability; see Appendix 1 in Supplementary Material for terms). He thought that experiments should aim to reject (or “nullify”; henceforth the name “null hypothesis”) H0 which assumes that the data demonstrates random variability according to some distribution around a certain value. Discrepancy from H0 is measured by a test statistic whose values can be paired with one or two-tailed p-values which tell us how likely it is that we would have found our data or more extreme data if H0 was really correct. Formally we will refer to the p-value as: pr(data or more extreme data|H0). It is important to realize that the p-value represents the “extremeness” of the data according to an imaginary data distribution assuming there is no bias in data sampling. Crucially, the Neyman–Pearson approach is designed to work efficiently (Neyman and Pearson, 1933) in the context of long-run repeated testing (exact replication). Hence, there is a major difference between the p-value which is computed for a single data set and α, β, power, Type I, and Type II error which are so called “frequentist” concepts and they make sense in the context of a long-run of many repeated experiments. If we only run a single experiment all we can claim is that if we had run a long series of experiments we would have had 100α% false positives (Type I error) had H0 been true and 100β% false negatives (Type II error) had H1 been true provided we got the power calculations right. Note the conditionals. The late Fisher viewed the exact p-value as a heuristic piece of inductive evidence which gives an indication of the plausibility of H0 together with other available evidence, like effect sizes (see Hubbard and Bayarri, 2003; Gigerenzer et al., 2004). Fisher recommended that H0 can usually be rejected if p ≤0.05 but in his system there is no mathematical justification for selecting a particular p-value for the rejection of H0. Rather, this is up to the substantively informed judgment of the experimenter. Fisher thought that a hypothesis is demonstrable only when properly designed experiments “rarely fail” to give us statistically significant results (Gigerenzer et al., 1989, p. 96; Goodman, 2008). Citation: So, as most behavioral data are analyzed by NHST statistics NHST based inference from behavioral data also plays a crucial role in interpreting brain data. Neyman and Pearson: A Decision Mechanism Optimized for the Long-Run The concepts of the alternative hypothesis (H1), α, power, β, Type I, and Type II errors were introduced by Neyman and Pearson (Neyman and Pearson, 1933; Neyman, 1950) who set up a formal decision procedure motivated by industrial quality control problems (Gigerenzer et al., 1989). Their approach aimed to minimize the false negative (Type II) error rate to an acceptable level (β) and consequently to maximize power (1-β) subject to a bound (α) on false positive (Type I) errors (Hubbard and Bayarri, 2003). α can be set by the experimenter to an arbitrary value and Type-II error can be controlled by setting the sample size so that the required effect size can be detected (see Figure 1 for illustration). In contrast to Fisher, this framework does not use the p-value as a measure of evidence. We merely determine the critical value of the test statistic associated with α and reject H0 whenever the test statistic is larger than the critical value. The exact p-value is irrelevant because the sole objective of the decision framework is long- run error minimization and only the critical threshold but not the exact p-value plays any role in achieving this goal (Hubbard and Bayarri, 2003). Neyman and Pearson rejected the idea of inductive reasoning and offered a reasoning-free inductive behavioral rule to choose between two behaviors, accepting or rejecting H0, irrespective of the researcher’s belief about whether H0 and H1 are true or not (Neyman and Pearson, 1933). Frontiers in Human Neuroscience | www.frontiersin.org NHST as a Weak Heuristic Based on the p-Value: Fisher In the Neyman–Pearson framework optimally setting α and β assures long-term decision-making efficiency in light of our costs and benefits by committing Type I and Type II errors. However, optimizing α and β is much easier in industrial quality control than in research where often there is no reason to expect a specific effect size associated with H1 (Gigerenzer et al., 1989). August 2017 | Volume 11 | Article 390 2 Null Hypothesis Significance Testing: A Reassessment Szucs and Ioannidis FIGURE 1 | NHST concepts make sense in the context of a long run of studies. 3 × 10,000 studies with normally distributed data were simulated for 3 situations (A: True H0 situation: Mean = 0; SD = 1; n = 16. B: Mean = 0.5; SD = 1; n = 16; Power = 0.46. C: Mean = 0.5; SD = 1; n = 32; Power = 0.78.). One sample two-tailed t-tests determined whether the sample means were zero. The red dots in the top panels show t scores for 3 × 1,000 studies (not all studies are shown for better visibility). The vertical dashed lines mark the critical rejection thresholds for H0, t(α/2) for the two-tailed test. The studies producing a t statistic more extreme than these thresholds are declared statistically significant. The middle panels show the distribution of t scores for all 3 × 10,000 studies (bins = 0.1). The bottom panels show the distribution of p-values for all 3 × 10,000 studies (bins = 0.01) and state the proportion of significant studies. The inset in the bottom right panel shows the mean absolute effect sizes in standard deviation units for situations A-C from all significant (Sig.) and non-significant (n.s.) studies with 95% bias corrected and accelerated bootstrap confidence intervals (10,000 permutations). The real effect size was 0 in situation (A) and 0.5 in situations (B,C). Note that the less is the power the more statistically significant studies overstate the effect size. Also note that p-values are randomly distributed and the larger is power the more right skewed is the distribution of p-values. In the true H0 situation the distribution of p-values is uniform between 0 and 1. See further explanation of this figure in Appendix 2 in Supplementary Material. FIGURE 1 | NHST concepts make sense in the context of a long run of studies. NHST as a Weak Heuristic Based on the p-Value: Fisher 3 × 10,000 studies with normally distributed data were simulated for 3 situations (A: True H0 situation: Mean = 0; SD = 1; n = 16. B: Mean = 0.5; SD = 1; n = 16; Power = 0.46. C: Mean = 0.5; SD = 1; n = 32; Power = 0.78.). One sample two-tailed t-tests determined whether the sample means were zero. The red dots in the top panels show t scores for 3 × 1,000 studies (not all studies are shown for better visibility). The vertical dashed lines mark the critical rejection thresholds for H0, t(α/2) for the two-tailed test. The studies producing a t statistic more extreme than these thresholds are declared statistically significant. The middle panels show the distribution of t scores for all 3 × 10,000 studies (bins = 0.1). The bottom panels show the distribution of p-values for all 3 × 10,000 studies (bins = 0.01) and state the proportion of significant studies. The inset in the bottom right panel shows the mean absolute effect sizes in standard deviation units for situations A-C from all significant (Sig.) and non-significant (n.s.) studies with 95% bias corrected and accelerated bootstrap confidence intervals (10,000 permutations). The real effect size was 0 in situation (A) and 0.5 in situations (B,C). Note that the less is the power the more statistically significant studies overstate the effect size. Also note that p-values are randomly distributed and the larger is power the more right skewed is the distribution of p-values. In the true H0 situation the distribution of p-values is uniform between 0 and 1. See further explanation of this figure in Appendix 2 in Supplementary Material. For example, if a factory has to produce screw heads with a diameter of 1 ± 0.01 cm than we know that we have to be able to detect a deviation of 0.01 cm to produce acceptable quality output. In this setting we know exactly the smallest effect size we are interested in (0.01 cm) and we can also control the sample size very efficiently because we can easily take a sample of a large number of screws from a factory producing them by the million assuring ample power. Frontiers in Human Neuroscience | www.frontiersin.org NHST as a Weak Heuristic Based on the p-Value: Fisher On the one hand, failing to detect too large or too small screws (Type II error) will result in our customers canceling their orders (or, in other industrial settings companies may deliver faulty cars or exploding laptops to customers exposing themselves to substantial litigation and compensation costs). On the other hand, throwing away false positives (Type I error), i.e., completely good batches of screws which we think are too small or too large, will also cost us a certain amount of money. Hence, we have a very clear scale (monetary value) to weigh the costs and benefits of both types of errors and we can settle on some rationally justified values of α and β so as to minimize our expenses and maximize our profit. In contrast to such industrial settings, controlling the sample size and effect size and setting rational α and β levels is not August 2017 | Volume 11 | Article 390 Frontiers in Human Neuroscience | www.frontiersin.org 3 Null Hypothesis Significance Testing: A Reassessment Szucs and Ioannidis that straightforward in most research settings where the true effect sizes being pursued are largely unknown and deciding about the requested size of a good enough effect can be very subjective. For example, what is the smallest difference of interest between two participant groups in a measure of “fMRI activity”? Or, what is the smallest difference of interest between two groups of participants when we measure their IQ or reaction time? And, even if we have some expectations about the “true effect size,” can we test enough participants to ensure a small enough β? Further, what is the cost of falsely claiming that a vaccine causes autism thereby generating press coverage that grossly misleads the public (Deer, 2011; Godlee, 2011)? What is the cost of running too many underpowered studies thereby wasting perhaps most research funding, boosting the number of false positive papers and complicating interpretation (Schmidt, 1992; Ioannidis, 2005; Button et al., 2013)? More often than not researchers do not know the “true” size of an effect they are interested in, so they cannot assure adequate sample size and it is also hard to estimate general costs and benefits of having particular α and β values. NHST as a Weak Heuristic Based on the p-Value: Fisher While some “rules of thumb” exist about what are small, modest, and large effects (e.g., Cohen, 1962, 1988; Jaeschke et al., 1989; Sedlmeier and Gigerenzer, 1989), some large effects may not be actionable (e.g., a change in some biomarker that is a poor surrogate and thus bears little relationship to major, clinical outcomes), while some small effects may be important and may change our decision (e.g., most survival benefits with effective drugs are likely to be small, but still actionable). FIGURE 2 | The distribution of p-values if the alternative hypothesis (H1) is true. Each line depicts the distribution of p-values resulting from one-sample two-tailed t-tests testing whether the sample mean was zero. Effect sizes (ES) indicate the true sample means for normally distributed data with standard deviation 1. For each effect size one million simulations were run with 16 cases in each simulation. The distribution of the p-value is becoming increasingly right skewed with increasing effect size and power. Note that α, the Type I error rate, is fix irrespective of what p-value is found in an experiment. FIGURE 2 | The distribution of p-values if the alternative hypothesis (H1) is true. Each line depicts the distribution of p-values resulting from one-sample two-tailed t-tests testing whether the sample mean was zero. Effect sizes (ES) indicate the true sample means for normally distributed data with standard deviation 1. For each effect size one million simulations were run with 16 cases in each simulation. The distribution of the p-value is becoming increasingly right skewed with increasing effect size and power. Note that α, the Type I error rate, is fix irrespective of what p-value is found in an experiment. Given the above ambiguity, researchers fall back to the default α = 0.05 level with usually undefined power. So, the unjustified α and β levels completely discredit the originally intended “efficiency” rationale of the creators of the Neyman–Pearson decision mechanism (Neyman and Pearson, 1933). (“positive” and “negative”) is again crucial if we rely on NHST because only then can they inform us about the distribution of p-values. Another point to notice is that both p-values and usual standardized effect size measures (Cohen’s D, correlation values, etc.) are direct functions of NHST test statistics. Hence, for given degrees of freedom NHST test statistics, effect size measures and p-values will have non-linear correspondence as illustrated in Figure 3. Measures Contrary to the fact that in Figure 1 all 10,000 true H0 and 10,000 true H1 samples were simulated from identical H0 and H1 distributions, the t scores and the associated p-values reflect a dramatic spread. That is, p-values are best viewed as random variables which can take on a range of values depending on the actual data (Sterling, 1959; Murdoch et al., 2008). Consequently, it is impossible to tell from the outcome of a single (published) experiment delivering a statistically significant result whether a true effect exist. The only difference between the true H0 and true H1 situations is that when H0 is true in all experiments, the distribution of p-values is uniform between 0 and 1 whereas when H1 is true in all experiments p-values are more likely to fall on the left of the 0–1 interval, that is, their distribution becomes right skewed. The larger is the effect size and power the stronger is this right skew (Figure 2). This fact led to the suggestion that comparing this skew allows us to determine the robustness of findings in some fields by studying “p curves” (Hung et al., 1997; Simonsohn et al., 2014a,b). Hence, from this perspective, replication, and unbiased publication of all results Frontiers in Human Neuroscience | www.frontiersin.org NEGLECTING THE FULL CONTEXT OF NHST LEADS TO CONFUSIONS ABOUT THE P-VALUE NHST completely neglects the above mentioned pre-study information and exclusively deals with rows 2–4 of Table 1. NHST computes the one or two-tailed p-value for a particular data set assuming that H0 is true. Additionally, NHST logic takes long-run error probabilities (α and β) into account conditional on H0 and H1. These long-run probabilities are represented in typical 2 × 2 NHST contingency tables but note that β is usually unknown in real studies. Most textbooks illustrate NHST by partial 2 × 2 tables (see Table 1) which fail to contextualize long-run conditional probabilities and fail to clearly distinguish between long-run probabilities and the p-value which is computed for a single data set (Pollard and Richardson, 1987). This leads to major confusions about the meaning of the p-value (see Appendix 2 in Supplementary Material). As we have seen, NHST never computes the probability of H0 and H1 being true or false, all we have is a decision mechanism hoping for the best individual decision in view of long-run Type I and Type II error expectations. Nevertheless, following the repeated testing logic of the NHST framework, for many experiments we can denote the long-run probability of H0 being true given a statistically significant result as False Report Probability (FRP), and the long-run probability of H1 being true given a statistically significant result as True Report Probability (TRP). FRP and TRP are represented in row 5 of Table 1 and it is important to see that they refer to completely different conditional probabilities than the p-value. pp y First, both H0 and H1 have some usually unknown pre-study or “prior” probabilities, pr(H0) and pr(H1). Nevertheless, these probabilities may be approximated through extensive substantive knowledge. For example, we may know about a single published study claiming to demonstrate H1 by showing a difference between appropriate experimental conditions. However, in conferences we may have also heard about 9 highly powered but failed replication attempts very similar to the original study. In this case we may assume that the odds of H0:H1 are 9:1, that is, pr(H1) is 1/10. Of course, these pre-study odds are usually hard to judge unless we demand to see our colleagues’ “null results” hidden in their drawers because of the practice of not publishing negative findings. NHST in Its Current Form The current NHST merged the approaches of Fisher and Neyman and Pearson and is often applied stereotypically as a “mindless null ritual” (Gigerenzer, 2004). Researchers set H0 nearly always “predicting” zero effect but do not quantitatively define H1. Hence, pre-experimental power cannot be calculated for most tests which is a crucial omission in the Neyman– Pearson framework. Researchers compute the exact p-value as Fisher did but also mechanistically reject H0 and accept the undefined H1 if p ≤(α = 0.05) without flexibility following the behavioral decision rule of Neyman and Pearson. As soon as p ≤α, findings have the supposed right to become a scientific fact defying the exact replication demands of Fisher and the belief neutral approach of Neyman and Pearson. Researchers also interpret the exact p-value and use it as a relative measure of evidence against H0, as Fisher did. A “highly significant” August 2017 | Volume 11 | Article 390 4 Null Hypothesis Significance Testing: A Reassessment Szucs and Ioannidis FIGURE 3 | The relationship of the p-value, test statistic and effect sizes. (A) The relationship of t values, degrees of freedoms and p-values for Pearson correlation studies (df = n–2). (B) The relationship of Pearson correlation (r) values, degrees of freedoms, and p-values [r = t/sqrt(df + t2)]. (C) The relationship of r- and t-value pairs for each degree of freedom at various p-values. The bold black lines mark the usual significance level of α = 0.05. Note that typically only results which exceed the α = 0.05 threshold are reported in papers. Hence, papers mostly report exaggerated effect sizes. FIGURE 3 | The relationship of the p-value, test statistic and effect sizes. (A) The relationship of t values, degrees of freedoms and p-values for Pearson correlation studies (df = n–2). (B) The relationship of Pearson correlation (r) values, degrees of freedoms, and p-values [r = t/sqrt(df + t2)]. (C) The relationship of r- and t-value pairs for each degree of freedom at various p-values. The bold black lines mark the usual significance level of α = 0.05. Note that typically only results which exceed the α = 0.05 threshold are reported in papers. Hence, papers mostly report exaggerated effect sizes. NHST in Its Current Form not allow for accepting it and because researchers erroneously often think that the single published positive study has a very small, acceptable error rate of providing false positive statistically significant results which equals α, or the p-value. So, they often spuriously assume that the negative studies somehow lacked the sensitivity to show an effect while the single positive study is perceived as a well-executed sensitive experiment delivering a “conclusive” verdict rather than being a “lucky” false positive (Bakan, 1966). (See a note on pilot studies in Serious Underestimation of the Proportion of False Positive Findings in NHST). result with a small p-value is perceived as much stronger evidence than a weakly significant one. However, while Fisher was conscious of the weak nature of the evidence provided by the p-value (Wasserstein and Lazar, 2016), generations of scientists encouraged by incorrect editorial interpretations (Bakan, 1966) started to exclusively rely on the p-value in their decisions even if this meant neglecting their substantive knowledge: scientific conclusions merged with reading the p- value (Goodman, 1999). Frontiers in Human Neuroscience | www.frontiersin.org NEGLECTING THE FULL CONTEXT OF NHST LEADS TO CONFUSIONS ABOUT THE P-VALUE FRP = False positives All statistically significant results = 5% of 100 studies 5% of 100 studies + 60% of 100 studies = 5 5 + 60 = 5 65 = 0.0769 FRP = False positives All statistically significant results = 5% of 100 studies 5% of 100 studies + 60% of 100 studies = 5 5 + 60 = 5 65 = 0.0769 That is, we will have 5 false positives out of a total of 65 statistically significant outcomes which means that the proportion of false positive studies amongst all statistically significant results is 7.69%, higher than the usually assumed 5%. However, this example still assumes that we get every second hypothesis right. If we are not as lucky and only get every sixth hypothesis right then if we run 600 studies, 500 of them will have true H0 true situations and 100 of them will have true H1 situations. Hence, the computation will look like: NEGLECTING THE FULL CONTEXT OF NHST LEADS TO CONFUSIONS ABOUT THE P-VALUE Note that the p value does not fit this view as it does not have any long run interpretation besides that it is a random variable (Murdoch et al., 2008). The most important variables are bolded, familiar signal detection categories are also provided. NHST does not deal with the concepts in italics. NHST textbooks typically only present rows 3 and 4 of this table (Alpha level, Power, Confidence level and Type II error). We follow the NHST view and deal with long run probabilities only. Note that the p value does not fit this view as it does not have any long run interpretation besides that it is a random variable (Murdoch et al., 2008). The most important variables are bolded, familiar signal detection categories are also provided. NHST does not deal with the concepts in italics. and in the other 100 studies (true H1) 60% of studies will come up significant. FRP is the ratio of false positive studies to all studies which come up significant: and the only parameter NHST computes is the p-value it is well- documented (Oakes, 1986; Gliner et al., 2002; Castro Sotos et al., 2007, 2009; Wilkerson and Olson, 2010; Hoekstra et al., 2014) that many, if not most researchers confuse FRP with the p-value or α and they also confuse the complement of p-value (1-p) or α (1-α) with TRP (Pollard and Richardson, 1987; Cohen, 1994). These confusions are of major portend because the difference between these completely different parameters is not minor, they can differ by orders of magnitude, the long-run FRP being much larger than the p-value under realistic conditions (Sellke et al., 2001; Ioannidis, 2005). The complete misunderstanding of the probability of producing false positive findings is most probably a key factor behind vastly inflated confidence in research findings and we suggest that this inflated confidence is an important contributor to the current replication crisis in biomedical science and psychology. Frontiers in Human Neuroscience | www.frontiersin.org NEGLECTING THE FULL CONTEXT OF NHST LEADS TO CONFUSIONS ABOUT THE P-VALUE Current scientific practices appreciate the single published “positive” study more than the 9 unpublished negative ones perhaps because NHST logic only allows for rejecting H0 but does Simply put, the p-value is pretty much the only thing that NHST computes but scientists usually would like to know the probability of their theory being true or false in light of their data (Pollard and Richardson, 1987; Goodman, 1993; Jaynes, 2003; Wagenmakers, 2007). That is, researchers are interested in the post-experimental probability of H0 and H1. Most probably, for the reason that researchers do not get what they really want to see August 2017 | Volume 11 | Article 390 5 Null Hypothesis Significance Testing: A Reassessment Szucs and Ioannidis TABLE 1 | “pr” stands for probability. True null effect (H0) True positive effect (H1) Pre-experiment probability of H0 and H1 Long run of experiments pr(H0) pr(H1) The conditional probability of having this data or more extreme data given that H0 is true Single experiment p-value — The conditional probability of having a significant test result given that H0 or H1 are true Long run of experiments Alpha level (α) Type I error False Positive False Alarm Power = 1 −β True Positive Hit The conditional probability of not having a significant test result given that H0 or H1 are true Long run of experiments 1 – α = Confidence level True Negative Correct Rejection β = 1 – Power Type II error False Negative Miss Post-experiment probability of H0 and H1 given a significant test result Long run of experiments FRP pr(H0|significant result) TRP pr(H1|significant result) NHST textbooks typically only present rows 3 and 4 of this table (Alpha level, Power, Confidence level and Type II error). We follow the NHST view and deal with long run probabilities only. Note that the p value does not fit this view as it does not have any long run interpretation besides that it is a random variable (Murdoch et al., 2008). The most important variables are bolded, familiar signal detection categories are also provided. NHST does not deal with the concepts in italics. TABLE 1 | “pr” stands for probability. NHST textbooks typically only present rows 3 and 4 of this table (Alpha level, Power, Confidence level and Type II error). We follow the NHST view and deal with long run probabilities only. Serious Underestimation of the Proportion of False Positive Findings in NHST For example, if α = 0.05, power = 0.6, and H0:H1 odds = 1 then a 10% bias (u = 0.1) will raise FRP to 18.47%. A 20% bias will raise FRP to 26.09%. If H0:H1 odds = 6 then FRP will be 67.92%. Looking at these numbers the replication crisis does not seem surprising: using NHST very high FRP can be expected even with modestly high H0:H1 odds and moderate bias (Etz and Vandekerckhove, 2016). Hence, under realistic conditions FRP not only extremely rarely equals α or the p-value (and TRP extremely rarely equals 1-α and/or 1-p-value) but also, FRP is much larger than the generally assumed 5% and TRP is much lower than the generally assumed 95%. Overall, α or the p-value practically says nothing about the likelihood of our research findings being true or false. experiment is run: We do not know what percent of the published statistically significant findings are lucky false positives explained post-hoc (Kerr, 1998) when in fact researchers could not detect the originally hypothesized effect and/or worked out analyses depending on the data (Gelman and Loken, 2014). However, it is reasonable to assume that only the most risk avoidant studies have lower H0:H1 odds than 1, relatively conservative studies have low to moderate H0:H1 odds (1–10) while H0:H1 odds can be much higher in explorative research (50–100 or even higher; Ioannidis, 2005). The above H0:H1 assumptions are reasonable, as they are supported by empirical data in many different fields. For example, half or more of the drugs tested in large, late phase III trials show higher effectiveness against older comparators (H0:H1 = <1; Soares et al., 2005). Conversely, the vast majority of tested hypotheses in large-scale exploratory research reflect null effects, e.g., in the search of genetic variants associated with various diseases in the candidate gene era where investigators were asking hypotheses one or a few at a time (the same way that investigators continue to test hypotheses in most other biomedical and social science fields) yielded thousands of putative discovered associations, but only 1.2% of them were subsequently validated to be non-null when large-scale consortia with accurate measurements and rigorous analyses plans assessed them (Chanock et al., 2007; Ioannidis et al., 2011). Of the hundreds of thousands to many millions of variables assessed in current agnostic–omics testing, much less than 1% are likely to reflect non-null effects (H0:H1>>100). Serious Underestimation of the Proportion of False Positive Findings in NHST TRP is the ratio of truly positive (H1 is true) statistically significant studies to all statistically significant studies: 60/65 = 0.9231 = 1 −FRP = 1 −0.0769. FIGURE 4 | Illustration of long run False Positive Probability (FRP) and True Positive Probability (TRP) of studies. Let’s assume that we run 2 × 100 studies, H0 is true in 100 studies and H1 is true in 100 studies with α = 0.05 and Power = 1−β = 0.6. (A) Shows the outcome of true H0 studies, 5 of the 100 studies coming up statistically significant. (B) Shows the outcome of true H1 studies, 60 of the 100 studies coming up statistically significant [note that realistically the 60 studies would be scattered around just as in panel (A) but for better visibility they are represented in a block]. (C) Illustrates that true H0 and true H1 studies would be indistinguishable. That is, researchers do not know which study tested a true H0 or true H1 situation (i.e., they could not distinguish studies represented by black and gray squares). All they know is whether the outcome of a particular study out of the 200 studies run was statistically significant or not. FRP is the ratio of false positive (H0 is true) statistically significant studies to all statistically significant studies: 5/65 = 0.0769. TRP is the ratio of truly positive (H1 is true) statistically significant studies to all statistically significant studies: 60/65 = 0.9231 = 1 −FRP = 1 −0.0769. findings have far more difficulty to be published and the researcher may feel that she wasted her efforts. Further, positive findings are more likely to get cited than negative findings (Kjaergard and Gluud, 2002; Jannot et al., 2013; Kivimäki et al., 2014). Consequently, researchers may often be highly biased to reject H0 and publish positive findings. Researcher bias affects FRP even if our NHST decision criteria, α and β, are formally unchanged. Ioannidis (2005) introduced the u bias parameter. The impact of u is that after some data tweaking and selective reporting (see Section NHST May Foster Selective Reporting and Subjectivity) u fraction of otherwise non-significant true H0 results will be reported as significant and u faction of otherwise non-significant true H1 results will be reported as significant. If u increases, FRP increases and TRP decreases. Serious Underestimation of the Proportion of False Positive Findings in NHST g Ioannidis (2005) has shown that most published research findings relying on NHST are likely to be false. The modeling supporting this claim refers to the long-run FRP and TRP which we can compute by applying Bayes’ theorem (see Figure 4 for illustration, see computational details and further illustration in Appendix 3 in Supplementary Material). The calculations must consider α, the power (1-β) of the statistical test used, the pre- study probabilities of H0 and H1, and it is also insightful to consider bias (Berger, 1985; Berger and Delampady, 1987; Berger and Sellke, 1987; Pollard and Richardson, 1987; Lindley, 1993; Sellke et al., 2001; Sterne and Smith, 2001; Ioannidis, 2005). FRP = False positives All statistically significant results = 5% of 500 studies 5% of 500 studies + 60% of 100 studies = 25 25 + 60 = 25 85 = 0.2941 FRP = False positives All statistically significant results = 5% of 500 studies 5% of 500 studies + 60% of 100 studies = 25 25 + 60 = 25 85 = 0.2941 Hence, nearly 1/3 of all statistically significant findings will be false positives irrespective of the p-value. Note that this issue is basically the consequence of running multiple NHST tests throughout the whole literature and FRP can be considered the uncontrolled false discovery rate (FDR) across all studies run (see Section Family-Wise Error Rate (FWER) and FDR Correction in NHST). While NHST neglects the pre-study odds of H0 and H1, these are crucial to take into account when calculating FRP and TRP. For example, let’s assume that we run 200 experiments and in 100 studies our experimental ideas are wrong (that is, we test true H0 situations) while in 100 studies our ideas are correct (that is, we test true H1 situations). Let’s also assume that the power (1-β) of our statistical test is 0.6 and α = 0.05. In this case in 100 studies (true H0) we will have 5% of results significant by chance alone Crucially, estimating pre-study odds is difficult, primarily due to the lack of publishing negative findings and to the lack of proper documentation of experimenter intentions before an August 2017 | Volume 11 | Article 390 6 Null Hypothesis Significance Testing: A Reassessment Szucs and Ioannidis FIGURE 4 | Illustration of long run False Positive Probability (FRP) and True Positive Probability (TRP) of studies. Serious Underestimation of the Proportion of False Positive Findings in NHST Let’s assume that we run 2 × 100 studies, H0 is true in 100 studies and H1 is true in 100 studies with α = 0.05 and Power = 1−β = 0.6. (A) Shows the outcome of true H0 studies, 5 of the 100 studies coming up statistically significant. (B) Shows the outcome of true H1 studies, 60 of the 100 studies coming up statistically significant [note that realistically the 60 studies would be scattered around just as in panel (A) but for better visibility they are represented in a block]. (C) Illustrates that true H0 and true H1 studies would be indistinguishable. That is, researchers do not know which study tested a true H0 or true H1 situation (i.e., they could not distinguish studies represented by black and gray squares). All they know is whether the outcome of a particular study out of the 200 studies run was statistically significant or not. FRP is the ratio of false positive (H0 is true) statistically significant studies to all statistically significant studies: 5/65 = 0.0769. TRP is the ratio of truly positive (H1 is true) statistically significant studies FIGURE 4 | Illustration of long run False Positive Probability (FRP) and True Positive Probability (TRP) of studies. Let’s assume that we run 2 × 100 studies, H0 is true in 100 studies and H1 is true in 100 studies with α = 0.05 and Power = 1−β = 0.6. (A) Shows the outcome of true H0 studies, 5 of the 100 studies coming up statistically significant. (B) Shows the outcome of true H1 studies, 60 of the 100 studies coming up statistically significant [note that realistically the 60 studies would be scattered around just as in panel (A) but for better visibility they are represented in a block]. (C) Illustrates that true H0 and true H1 studies would be indistinguishable. That is, researchers do not know which study tested a true H0 or true H1 situation (i.e., they could not distinguish studies represented by black and gray squares). All they know is whether the outcome of a particular study out of the 200 studies run was statistically significant or not. FRP is the ratio of false positive (H0 is true) statistically significant studies to all statistically significant studies: 5/65 = 0.0769. Frontiers in Human Neuroscience | www.frontiersin.org Serious Underestimation of the Proportion of False Positive Findings in NHST Lower rates of H0:H1 would be incompatible with logical considerations of how many variables are needed to explain all the variance of a disease or outcome risk. At this point it is worth noting that it could be argued that unpublished pilot experiments may prompt us to run studies and hence, often H0:H1 odds would be lower than 1. However, unpublished pilot data often comes from small scale underpowered studies with high FRP, undocumented initial hypotheses and analysis paths. Hence, we doubt that Besides H0:H1 odds bias is another important determinant of FRP and TRP (Ioannidis, 2005). Whenever, H0 is not rejected Frontiers in Human Neuroscience | www.frontiersin.org August 2017 | Volume 11 | Article 390 7 Null Hypothesis Significance Testing: A Reassessment Szucs and Ioannidis TABLE 2 | Potential NHST style argument (based on Pollard and Richardson, 1987). H0 Harold is American H1 Harold is not American Model for H0 If Harold is American (H0), than he is most probably not a member of congress. data Harold is a member of congress. pr(data or more extreme data|H0) Very low Inference Because pr(data or more extreme data|H0) is very low, we reject H0 and accept H1 and conclude: Harold is most probably not American. TABLE 2 | Potential NHST style argument (based on Pollard and Richardson, 1987) statistically significant pilot results inevitably mean low H0:H1 odds. NHST Misleads Because It Neglects Pre-data Probabilities We can assign arbitrary but plausible probabilities: pr(data|H0) = pr(Harold is member of congress | American) = 10−7 pr(data|H1) = pr(Harold is member of congress | not American) = 0 pr(data|H0) = pr(Harold is member of congress | American) = 10−7 pr(data|H1) = pr(Harold is member of congress | not American) = 0 pr(data|H1) = pr(Harold is member of congress | not American) = 0 That is, while the data is indeed rare under H0, its probability is actually zero under H1 (in other words, the data is very unlikely under both the null and the alternative models). So, even if p ≈ 0.0000001, it does not make sense to reject H0 and accept H1 because this data just cannot happen if H1 is true. If we only have these two hypotheses to choose from then it only makes sense to accept H0 because the data is still possible under H0 (Jaynes, 2003). In fact, using Bayes’ theorem we can formally show that The Neglect of Power Reinterpreted g p In contrast to the importance of power in determining FRP and TRP, NHST studies tend to ignore power and β and emphasize α and low p-values. Often, finding a statistically significant effect erroneously seems to override the importance of power. However, statistical significance does not protect us from false positives. FRP can only be minimized by keeping H0:H1 odds and bias low and power high (Pollard and Richardson, 1987; Button et al., 2013; Bayarri et al., 2016). Hence, power is not only important so that we increase our chances to detect true effects but it is also crucial in keeping FRP low. While power in principle can be adjusted easily by increasing sample size, power in many/most fields of biomedical science and psychology has been notoriously low and the situation has not improved much during the past 50 years (Cohen, 1962; Sedlmeier and Gigerenzer, 1989; Rossi, 1990; Hallahan and Rosenthal, 1996; Button et al., 2013; Szucs and Ioannidis, 2017). Clearly, besides making sure that research funding is not wasted, minimizing FRP also provides very strong rationale for increasing the typically used sample sizes in studies. the NHST machinery only computes the p-value conditioned on H0 and it is able to provide this result even if H1 is not specified at all. A widespread misconception flowing from the fuzzy attitude of NHST to H1 is that rejecting H0 allows for accepting a specific H1 (Nickerson, 2000). This is what most practicing researchers do in practice when they reject H0 and argue for their specific H1 in turn. However, NHST only computes probabilities conditional on H0 and it does not allow for the acceptance of either H0, a specific H1 or a generic H1. Rather, it only allows for the rejection of H0. Hence, if we reject H0 we will have no idea about how well our data fits a specific H1. This cavalier attitude to H1 can easily lead us astray even when contrasting H0 just with a single alternative hypothesis as illustrated by the invalid inference based on NHST logic in Table 2 (Pollard and Richardson, 1987). NHST Misleads Because It Neglects Pre-data Probabilities Besides often being subject to conceptual confusion and generating misleading inferences especially in the setting of weak power, NHST has further serious problems. NHST logic is based on the so-called modus tollens (denying the consequent) argumentation (see footnote in Appendix 4 in Supplementary Material): It sets up a H0 model and assumes that if the data fits this model than the test statistic associated with the data should not take more extreme values than a certain threshold (Meehl, 1967; Pollard and Richardson, 1987). If the test statistic contradicts this expectation then NHST assumes that H0 can be rejected and consequently its complement, H1 can be accepted. While this logic may be able to minimize Type I error in well- powered high-quality well-controlled tests (Section Neyman and Pearson: A decision Mechanism Optimized for the Long-Run), it is inadequate if we use it to decide about the truth of H1 in a single experiment, because there is always space for Type I and Type II error (Falk and Greenbaum, 1995). So, our conclusion is never certain and the only way to see how much error we have is to calculate the long-run FRP and TRP using appropriate α and power levels and prior H0:H1 odds. The outcome of the calculation can easily conflict with NHST decisions (see Appendix 4 in Supplementary Material). Our model says that if H0 is true, it is a very rare event that Harold is a member of congress. This rare event then happens which is equivalent to finding a small p-value. Hence, we conclude that H0 can be rejected and H1 is accepted (i.e., Harold is a member of congress and therefore he is not American.). However, if we carefully explicate all probabilities it is easy to see that we are being mislead by NHST logic. First, because we have absolutely no idea about Harold’s nationality we can set pre-data probabilities of both H1 and H0 to 1/2, which means that H0:H1 odds are uninformative, 1:1. Then we can explicate the important conditional probabilities of the data (Harold is a member of congress) given the possible hypotheses. NHST Neglects Predictions under H1 Facilitating Sloppy Research NHST does not require us to specify exactly what data H1 would predict. Whereas, the Neyman–Pearson approach requires researchers to specify an effect size associated with H1 and compute power (1-β), in practice this is easy to neglect because August 2017 | Volume 11 | Article 390 Frontiers in Human Neuroscience | www.frontiersin.org 8 Null Hypothesis Significance Testing: A Reassessment Szucs and Ioannidis the probability of H0 is actually 1 (Appendix 5 in Supplementary Material). the probability of H0 is actually 1 (Appendix 5 in Supplementary Material). because H1 is formally undefined and NHST never tells us anything about it. In light of these problems Jaynes (2003) suggested that the NHST framework addresses an ill-posed problem and provides invalid responses to questions of statistical inference. In most real world problems multiple alternative hypotheses compete to explain the data. However, by using NHST we can only reject H0 and argue for some H1 without any formal justification of why we prefer a particular hypothesis whereas it can be argued that it only makes sense to reject any hypothesis if another one better fits the data (Jaynes, 2003). We only have qualitative arguments to accept a specific H1 and the exclusive focus on H0 makes unjustified inference too easy. For example, if we assume that H0 predicts normally distributed data with mean 0 and standard deviation 1 then we have endless options to pick H1 (Hubbard and Bayarri, 2003): Does H1 imply that the data have a mean other than zero, the standard deviation other than 1 and/or does it represent non-normally distributed data? NHST allows us to consider any of these options implicitly and then accept one of them post-hoc without any quantitative justification of why we chose that particular option. Further, merging all alternative hypotheses into a single H1 is not only too simplistic for most real world problems but it also poses an “inferential double standard” (Rozeboom, 1960): The procedure pits the well-defined H0 against a potentially infinite number of alternatives. It is noteworthy that some may argue that Jaynes’s argument is formally invalid as the NHST approach can be used to reject a low probability H0 in theory. NHST Neglects Predictions under H1 Facilitating Sloppy Research However, recall that (1) NHST does not deliver final objective theoretical decisions, there is no theoretical justification for any α thresholds marking a boundary of informal surprise and NHST merely aims to minimize Type I error on the long run (and in fact, Neyman and Pearson (1933) considered their procedure a theory-free decision mechanism and Fisher considered it a heuristic). (2) NHST can only reject H0 (heuristically or in a theory-free manner) and (3) cannot provide support for any H1. We could also add that many practicing biomedical and social scientists may not have clear quantitative predictions under H0 besides expecting to reject a vague null effect (see Section NHST in Sciences with and without Exact Quantitative Predictions for the difference between sciences with and without exact predictions). Hence, their main (ultimate) objective of using NHST is often actually not the falsification of the exact theoretical predictions of a well-defined theory (H0). Rather, they are more interested in arguing in favor of an alternative theory. For example, with a bit of creativity fMRI “activation” in many different (perhaps post-hoc defined) ROIs can easily be “explained” by some theory when H0 (“no activation”) is rejected in any of the ROIs. However, supporting a specific alternative theory is just not possible in the NHST framework and in this context Jaynes’ comment is perfectly valid: NHST provides an ill-defined framework, after rejecting H0 real-world researchers have no formal hypothesis test outcomes to support their “positive” arguments. Vague H1 definitions (the lack of quantitative predictions) enable researchers to avoid the falsification of their favorite hypotheses by intricately redefining them (especially in fields such as psychology and cognitive neuroscience where theoretical constructs are often vaguely defined) and never providing any definitive assessment of the plausibility of a favorite hypothesis in light of credible alternatives (Meehl, 1967). This problem is reflected in papers aiming at the mere demonstration of often little motivated significant differences between conditions (Giere, 1972) and post-hoc explanations of likely unexpected but statistically significant findings. For example, neuroimaging studies often attempt to explain why an fMRI BOLD signal “deactivation” happened instead of a potentially more reasonable looking “activation” (or, vice versa). Most such findings may be the consequence of the data randomly deviating into the wrong direction relative to zero between-condition difference. Even multiple testing correction will not help such studies as they still rely on standard NHST just with adjusted α thresholds. Frontiers in Human Neuroscience | www.frontiersin.org NHST Neglects Predictions under H1 Facilitating Sloppy Research Similarly, patient studies often try to explain an unexpected difference between patient and control groups (e.g., the patient group is “better” on a measure or shows “more” or “less” brain activation) by some kind of “compensatory mechanism.” In such cases what happens is that “the burden of inference has been delegated to the statistical test,” indeed, and simply because p ≤α odd looking observations and claims are to be trusted as scientific facts (Bakan, 1966, p. 423; Lykken, 1968). NHST Is Unsuitable for Large Datasets NHST Is Unsuitable for Large Datasets In consequence of the recent ‘big data’ revolution access to large databases has increased dramatically potentially increasing power tremendously (though, large data sets with many variables are still relatively rare in neuroscience research). However, NHST leads to worse inference with large databases than with smaller ones (Meehl, 1967; Khoury and Ioannidis, 2014). This is due to how NHST tests statistics are computed, the properties of real data and to the lack of specifying data predicted by H1 (Bruns and Ioannidis, 2016). Most NHST studies rely on nil null hypothesis testing (Nickerson, 2000) which means that H0 expects a true mean difference of exactly zero between conditions with some variation around this true zero mean. Further, NHST machinery guarantees that we can detect any tiny irrelevant effect sizes if sample size is large enough. This is because test statistics are typically computed as the ratio of the relevant between condition differences and associated variability of the data weighted by some function of the sample size [difference/variability × f(sample size)]. The p-value is smaller if the test statistic is larger. Thus, the larger is the difference between conditions and/or the smaller is variability and/or the larger is the sample size the larger Finally, paradoxically, when real life practicing researchers achieve their “goal” and successfully reject H0 they may be left in complete existential vacuum because during the rejection of H0 NHST “saws offits own limb” (Jaynes, 2003; p. 524): If we manage to reject H0 then it follows that pr(data or more extreme data|H0) is useless because H0 is not true. Thus, we are left with nothing to characterize the probability of our data in the real world; we will not know pr(data|H1) for example, August 2017 | Volume 11 | Article 390 9 Null Hypothesis Significance Testing: A Reassessment Szucs and Ioannidis is the test statistic and the smaller is the p-value (see Figure 3 for examples). Consequently, by increasing sample size enough it is guaranteed that H0 can be rejected even with miniature effect sizes (Ziliak and McCloskey, 2008). measurement precision and increased amounts of data increase the difficulties a theory must pass before it is accepted. This is because theoretical predictions are well- defined, numerically precise and it is also easier to control measurements (Lykken, 1968). Hence, NHST may be used to aim to falsify exact theoretical predictions. NHST Is Unsuitable for Large Datasets However, as noted, groups are actually likely to differ and if sample size increases and variability in data decreases it will become easier and easier to reject any kind of H0 when following the NHST approach. In fact, with precise enough measurements, large enough sample size and repeated “falsification” attempts H0 is guaranteed to be rejected on the long run (see Section The Rejection of H0 is Guaranteed on the Long-Run) even if the underlying processes generating the data in two experimental conditions are exactly the same. Hence, ultimately any H1 can be accepted, claiming support for any kind of theory. For example, in an amusing demonstration Carver (1993) used Analysis of Variance to re-analyze the data of Michaelson and Morley (1887) who came up with a “dreaded” null finding and based on this they suggested that the speed of light was constant (H0) thereby providing empirical support for Einstein’s theory of relativity. Carver (1993) found that that the speed of light was actually not constant at p < 0.001. The catch? The effect size as measured by Eta2 was 0.005. While some may feel that Einstein’s theory has now been falsified, perhaps it is also worth considering that here the statistically significant result is essentially insignificant. This example also highlights the fact we are not arguing against the Popperian view of scientific progress by falsifying theories. Rather, we discuss why NHST is a very imperfect method for this falsification (see further arguments as well). The above issues have been demonstrated empirically and by simulations. For example, Bakan (1966; see also Berkson, 1938; Nunnally, 1960; Meehl, 1967) subdivided the data of 60,000 persons according to completely arbitrary criteria, like living east or west of the Mississippi river, living in the north or south of the USA, etc. and found all tests coming up statistically significant. Waller (2004) examined the personality questionnaire data of 81,000 individuals to see how many randomly chosen directional null hypotheses can be rejected. If sample size is large enough, 50% of directional hypothesis tests should be significant irrespective of the hypothesis. As expected, nearly half (46%) of Waller’s (2004) results were significant. Simulations suggest that in the presence of even tiny residual confounding (e.g., some omitted variable bias) or other bias, large observational studies of null effects will generate results that may be mistaken as revealing thousands of true relationships (Bruns and Ioannidis, 2016). NHST Is Unsuitable for Large Datasets For example, a theory may predict that a quantity should be let’s say 8 and the experimental setup can assure that really only very few factors influence measurements—these factors can then be taken into account during analysis. Hence, increased measurement precision will make it easier to demonstrate a departure from numerically exact predictions. So, a “five sigma” deviation rule may make good sense in physics where precise models are giving precise predictions about variables. Parameters of many real data sets are much more likely to differ than to be the same for reasons completely unrelated to our hypotheses (Meehl, 1967, 1990; Edwards, 1972). First, many psychological, social and biomedical phenomena are extremely complex reflecting the contribution of very large numbers of interacting (latent) factors, let it be at the level of society, personality or heavily networked brain function or other biological networks (Lykken, 1968; Gelman, 2015). Hence, if we select any two variables related to these complex networks most probably there will be some kind of at least remote connection between them. This phenomenon is called “crud factor” Meehl (1990) or “ambient correlational noise” (Lykken, 1968) and it is unlikely to reflect a causal relationship. In fact some types of variables, such as intake of various nutrients and other environmental exposures are very frequently correlated among themselves and with various disease outcomes without this meaning that they have anything to do with causing disease outcomes (Patel and Ioannidis, 2014a,b). Second, unlike in physical sciences it is near impossible to control for the relationship of all irrelevant variables which are correlated with the variable(s) of interest (Rozeboom, 1960; Lykken, 1968). Consequently, there can easily be a small effect linking two randomly picked variables even if their statistical connection merely communicates that they are part of a vast complex interconnected network of variables. Only a few of these tiny effects are likely to be causal and of any portend (Siontis and Ioannidis, 2011). In sciences using NHST without clear numerical predictions the situation is the opposite of the above, because NHST does not demand the exact specification of H1, so theories typically only predict a fairly vague “difference” between groups or experimental conditions rather than an exact numerical discrepancy between measures of groups or conditions. NHST Is Unsuitable for Large Datasets Experimental studies may also suffer the same problem, if they have even minimal biases. A typical defense of NHST may be that we actually may not want to increase power endlessly, just as much as we still think that it allows us to detect reasonable effect sizes (Giere, 1972). For example, equivalence testing may be used to reject the hypothesis that a meaningfully large effect exist (e.g., Wellek, 2010) or researchers may check for the sign of expected effects (Gelman and Tuerlinckx, 2000). However, because typically only statistically significant data is published, published studies most probably exaggerate effect sizes. So, estimating true (expected) effect sizes is very difficult. A more reasoned approach may be to consider explicitly what the NHST May Foster Selective Reporting and Subjectivity Because NHST never evaluates H1 formally and it is fairly biased toward the rejection of H0, reporting bias against H0 can easily infiltrate the literature even if formal NHST parameters are fixed (see Section Serious Underestimation of the Proportion of False Positive Findings in NHST about the “u” bias parameter). Overall, a long series of exploratory tools and questionable research practices are utilized in search for statistical significance (Ioannidis and Trikalinos, 2007; John et al., 2012). Researchers can influence their data during undocumented analysis and pre-processing steps and by the mere choice of structuring the data (constituting researcher degrees of freedom; Simmons et al., 2011). This is particularly a problem in neuroimaging where the complexity and idiosyncrasy of analyses is such that it is usually impossible to replicate exactly what happened and why during data analysis (Kriegeskorte et al., 2009; Vul et al., 2009; Carp, 2012). Another term that has been used to describe the impact of diverse analytical choices is “vibration of effects” (Ioannidis, 2008). Different analytical options, e.g., choice of adjusting covariates in a regression model can result in a cloud of results, instead of a single result, and this may entice investigators to select a specific result that is formally significant, while most analytical options would give non-significant results or even results with effects in the opposite direction (“Janus effect”; Patel et al., 2015). Another common mechanism that may generate biased results with NHST is when investigators continue data collection and re-analyse the accumulated data sequentially without accounting for the penalty induced by this repeated testing (DeMets and Lan, 1994; Goodman, 1999; Szucs, 2016). The unplanned testing is usually undocumented and researchers may not even be conscious that it exposes them to Type I error accumulation. Bias may be the key explanation why in most biomedical and social science disciplines, the vast majority of published papers with empirical data report statistically significant results (Kavvoura et al., 2007; Fanelli, 2010; Chavalarias et al., 1990-2015). Overall, it is important to see that NHST can easily be infiltrated by several undocumented subjective decisions. NHST in Sciences with and without Exact Quantitative Predictions Due to the combination of the above properties of real-world data sets and statistical machinery theory testing radically differs in sciences with exact and non-exact quantitative predictions (Meehl, 1967). In physical sciences increased August 2017 | Volume 11 | Article 390 Frontiers in Human Neuroscience | www.frontiersin.org 10 Null Hypothesis Significance Testing: A Reassessment Szucs and Ioannidis the larger are H0:H1 odds and bias and the larger is the long- run FRP. For example, in a large laboratory with 20 post-docs and PhD students, each person running 5 experiments a year implementing 10 significance tests in each experiment we can expect 20 × 5 × 10 × 0.05 = 50 [usually publishable] false results a year at α = 0.05 if H0 is true. Coupled with the fact that a large number of unplanned tests may be run in each study (Simmons et al., 2011; Gelman and Loken, 2014) and that negative results and failed replications are often not published, this leads to “unchallenged fallacies” clogging up the research literature (Ioannidis, 2012; p1; Sterling, 1959; Bakan, 1966; Sterling et al., 1995). Moreover, such published false positive true H0 studies will also inevitably overestimate the effect size of the non-existent effects or of existent, but unimportantly tiny, effects (Schmidt, 1992, 1996; Sterling et al., 1995; Ioannidis, 2008). These effects may even be confirmed by meta-analyses, because meta-analyses typically are not able to incorporate unpublished negative results (Sterling et al., 1995) and they cannot correct many of the biases that have infiltrated the primary studies. For example, such biases may result in substantial exaggeration of measured effect sizes in meta-analyses (see e.g., Szucs and Ioannidis, 2017). consequences (“costs”) are of a false-positive, true-positive, false- negative, and true-negative result. Explicit modeling can suggest that the optimal combination of Type 1 error and power may need to be different depending on what these assumed costs are (Djulbegovic et al., 2014). Different fields may need to operate at different optimal ratios of false-positives to false-negatives (Ioannidis et al., 2011). NHST May Foster Selective Reporting and Subjectivity (Bayesian methods are often blamed such subjectivity, see Section Teach Alternative Approaches Seriously.) Given that the predictions of H1 are rarely precise and that theoretical constructs in many scientific fields (including psychology and cognitive neuroscience) are often poorly defined (Pashler and Harris, 2012), it is easy to claim support for a popular theory with many kinds of data falsifying H0 even if the constructs measured in many papers are just very weakly linked to the original paper, or not linked at all. Overall, the literature may soon give the impression of a steady stream of replications throughout many years. Even when “negative” results appear, citation bias may still continue to distort the literature and the prevailing theory may continue to be based on the “positive” results. Hence, citation bias may maintain prevailing theories even when they are clearly false and unfounded (Greenberg, 2009). NHST Does Not Facilitate Systematic Knowledge Integration Due to high FRP the contemporary research literature provides statistically significant “evidence” for nearly everything (Schoenfeld and Ioannidis, 2012). Because NHST emphasizes all or none p-value based decisions rather than the magnitude of effects, often only p-values are reported for critical tests, effect size reports are often missing and interval estimates and confidence intervals are not reported. In an assessment of the entire biomedical literature in 1990–2015, 96% of the papers that used abstracts reported at least some p-value below 0.05, while only 4% of a random sample of papers presented consistently effect sizes with confidence intervals (Chavalarias et al., 1990-2015). However, oddly enough, the main NHST “measure of evidence,” the p-value cannot be compared across studies. It is a frequent misconception that a lower p-value always means stronger evidence irrespective of the sample size and effect size (Oakes, 1986; Schmidt, 1996; Nickerson, 2000). Besides the non-comparable p-values, NHST does not offer any The Rejection of H0 Is Guaranteed on the Long-Run If H0 is true, with α = 0.05, 5% of our tests will be statistically significant on the long-run. The riskier experiments we run, August 2017 | Volume 11 | Article 390 Frontiers in Human Neuroscience | www.frontiersin.org 11 Null Hypothesis Significance Testing: A Reassessment Szucs and Ioannidis formal mechanism for systematic knowledge accumulation and integration (Schmidt, 1996) unlike Bayesian methods which can take such pre-study information into account. Hence, we end up with many fragmented studies which are most often unable to say anything formal about their favorite H1s (accepted in a qualitative manner). Methods do exist for the meta-analysis of p-values (see e.g., Cooper et al., 2009) and these are still used in some fields. However, practically such meta-analyses still say nothing about the magnitude of the effect size of the phenomenon being addressed. These methods are potentially acceptable when the question is whether there is any non-null signal among multiple studies that have been performed, e.g., in some types of genetic associations where it is taken for granted that the effect sizes are likely to be small anyhow (Evangelou and Ioannidis, 2013). FWER control is a conservative procedure in keeping Type I error rate low but it also sacrifices power increasing Type II error. An alternative to FWER control is False Discovery Rate (FDR) control which allows more Type I errors but assures higher power. Using the same logic as the computation of FRP discussed before, FDR control considers the estimated proportion of false positive statistically significant findings amongst all statistically significant findings (i.e., the proportion of erroneously rejected null hypotheses out of all rejected null hypotheses; Benjamini and Hochberg, 1995). FDR computation is illustrated by Table 3. If we run M hypothesis tests then a certain number of them are likely to test true null effects (M0 in Table 3) and some other number of them are likely to test non-null effects with true alternative hypotheses (M1 in Table 3). Depending on our α level and power (1-β), a certain number of the M0 and M1 tests will reject the null hypothesis (FP and TN, respectively, see Table 3 for abbreviations) while some other number of them will not reject the null hypothesis (TN and FN, respectively). Family-Wise Error Rate (FWER) and FDR Correction in NHST An increasingly important problem is that with the advent of large data sets researchers can use NHST to test multiple, related hypotheses. For example, this problem routinely appears in neuro-imaging where a large amount of non-independent data points are collected and then the same hypothesis test may be run on tens of thousands of observations, for example, from a brain volume, or from 256 electrodes placed on the scalp, each electrode recording voltage 500 times a second. Analysis procedures that generate different views of data (e.g., time- frequency or independent component analyses) may further boost the amount of tests to be run. Of course, in real research settings we do not know how many of our tests test true null effects and we only know how many tests we run and how many of them return statistically significant and non-significant results. So Q can be considered a random variable. However, as Q cannot be controlled directly FDR is defined as the expected value of the proportion of false positive errors: FDR = E[FP/R|R > 0] · pr(R > 0), a variable which can be controlled (see Benjamini and Hochberg, 1995; Curran-Everett, 2000; Nichols and Hayasaka, 2003; Bennett et al., 2009; Benjamini, 2010; Goeman and Solari, 2014). Some FDR estimation procedures can also factor in dependency between tests (Benjamini and Yekutieli, 2001). Regarding these multiple testing situations, a group of statistical tests which are somehow related to each other can be defined as a “family of comparisons.” The probability that a family of comparisons contains at least one false positive error is called the family wise error rate (FWER). If the repeated tests concern independent data sets where H0 is true than the probability of having at least one Type I error in k independent tests, each with significance level α, is αTOTAL = 1 - (1 - α)k. For example if k = 1, 2, 3, 4, 5, and 10 than αTOTAL is 5, 9.75, 14.26, 18.55, 22.62, and 40.13%, respectively (see Curran-Everett, 2000; Szucs, 2016 for graphical illustrations and simulations for non-independent data). j In contrast to FDR, using the notation in Table 3, FWER can be expressed as FWER = pr(FP≥1) = 1 −pr(FP=0) that is, the probability that there is at least one false positive Type I error in a family of observations. The Rejection of H0 Is Guaranteed on the Long-Run If we know the exact numbers in Table 3 then the proportion of false positive statistically significant findings can be computed as the ratio of false positive results to all statistically significant results: Q = FP/(FP + TP) = FP/R (assuming that R̸= 0). Frontiers in Human Neuroscience | www.frontiersin.org Family-Wise Error Rate (FWER) and FDR Correction in NHST That is, 42.86% of statistically significant results will be false positives. FDR = Q · pr(R > 0) = Q · [α · (M0/M) + Power · (M1/M)] = 0.045. If we only test true null effects then Q = false positives/all significant results = α·M / α·M = 1; and FDR = Q · pr(R>0) = Q · α = α = FWER [pr(R>0) = α because all significant results are coming from true null situations]. Note that in real research only R, M, and M-R are known whereas M0, M1, and Q are not known. H0 (leftmost column) stands for the null hypothesis. The proportion of false positive statistically significant test outcomes to all statistically significant test outcomes is Q = FP/(FP + TP) = FP/R (R̸= 0). The numbers give an example for the case when α = 0.05 (so, FWER = α = 0.05) and β =0.4, so Power = 1 −β = 0.6. In the example we run 1,000 null hypothesis tests. We test 9 times as many true null situations than situations with true alternative hypotheses (that is, every 10th of our experimental ideas are correct). In this case Q = 45/105 = 0.4286. That is, 42.86% of statistically significant results will be false positives. FDR = Q · pr(R > 0) = Q · [α · (M0/M) + Power · (M1/M)] = 0.045. If we only test true null effects then Q = false positives/all significant results = α·M / α·M = 1; and FDR = Q · pr(R>0) = Q · α = α = FWER [pr(R>0) = α because all significant results are coming from true null situations]. Note that in real research only R, M, and M-R are known whereas M0, M1, and Q are not known. findings when a single hypothesis is tested simultaneously in many data points (e.g., voxels) but do nothing to protect against many of the other problems discussed in this paper (e.g., generating a high amount of false positives across the literature; being sensitive to undocumented biasing procedures; neglecting predictions under H1; not providing probability statements for H1; neglecting pre-data probabilities; being unable to effectively integrate study results). These problems are valid even when just one single NHST test is run. Family-Wise Error Rate (FWER) and FDR Correction in NHST If the null hypothesis is true in all tests we run then FDR = FWER while if there are situations with true alternative hypotheses then FDR < FWER (see Table 3 for example). Also, various other FDR and FWER measures can be derived (see the above cited reviews). It can be argued that controlling FDR is more useful in research where a very large number of tests are carried out routinely, like neuro- imaging or genetics but less useful in behavioral psychological and social science research where fewer hypotheses may be tested at any one time and accepting any single hypothesis as statistically significant may have large impact on inferences (Gelman et al., 2012). This last statement is also true for behavioral data used to support the interpretation of neuro- imaging findings. There are numerous procedures which can take multiple testing into account by correcting p-values. The simplest of these procedures is Bonferroni correction which computes an adjusted p-value threshold as α/n where α is the statistical significance threshold for a single test and n is the number of tests run. Hence, if we run 5 tests which can be defined as a family of tests and our original α is 0.05 then the Bonferroni corrected adjusted α level is 0.05/5 = 0.01. Any p-values above this threshold should not be considered to demonstrate statistically significant effects. Besides the Bonferroni correction there are other alternative methods of FWER correction, like the Tukey Honestly Significant Difference test, the Scheffe test, Holm’s method, Sidak’s method; Hochberg’s method, etc. Some of these corrections also take the dependency (non-independence) of tests into account (see e.g., Shaffer, 1995; Nichols and Hayasaka, 2003 for review). Most relevant to our paper, both FWER and FDR error rate corrections are based on the same NHST procedure. That is, they do not modify the procedure in any ways other than aiming to decrease Type I error toward initially expected levels when multiple NHST tests are run. That is, these methods can help in constraining the number of random Frontiers in Human Neuroscience | www.frontiersin.org August 2017 | Volume 11 | Article 390 12 Null Hypothesis Significance Testing: A Reassessment Szucs and Ioannidis TABLE 3 | Illustrating the logic behind FDR computation. Family-Wise Error Rate (FWER) and FDR Correction in NHST In addition, empirical analyses of large fMRI data sets found that the most popular fMRI analysis software packages implemented erroneous multiple testing corrections and hence, generate much higher levels of false positive results than expected (Eklund et al., 2012, 2016). This casts doubts on a substantial part of the published fMRI literature. Further, Carp (2012) reported that about 40% of 241 relatively recent fMRI papers actually did not report having used multiple testing correction. So, a very high percentage of fMRI literature may have been exposed to high false positive rates either multiple correction was used or not (see also (Szucs and Ioannidis, 2017) on statistical power). to study model response behavior. This may offer a way to judge the reasonableness of analyses offering richer information than NHST accept/reject decisions (Gelman et al., 2014). The challenge of course is the development of models. However, below we argue that efficient model development can only happen if we refocus our efforts on understanding data patters from the testing of often very vaguely defined hypotheses. In addition, Bayesian methods are also able to formally aggregate data from many experiments (e.g., adding data serially and by hierarchical models; Gelman et al., 2014). This can further maximize large-scale joint efforts for better model specifications. Family-Wise Error Rate (FWER) and FDR Correction in NHST Null hypothesis is true Alternative hypothesis is true Sums H0 is rejected (statistically significant outcome) FP = False Positives 45 (if α = 0.05; 900·0.05 = 45) TP = True Positives 60 (if power = 0.6; 100·0.6 = 60) R 105 H0 is not rejected (statistically non-significant outcome) TN = True Negatives 855 (if α = 0.05; 900·0.95 = 855) FN = False Negatives 40 (if β = 0.4; 100·0.4 = 40) M – R 895 Sums M0 900 M1 100 M 1000 H0 (leftmost column) stands for the null hypothesis. The proportion of false positive statistically significant test outcomes to all statistically significant test outcomes is Q = FP/(FP + TP) = FP/R (R̸= 0). The numbers give an example for the case when α = 0.05 (so, FWER = α = 0.05) and β =0.4, so Power = 1 −β = 0.6. In the example we run 1,000 null hypothesis tests. We test 9 times as many true null situations than situations with true alternative hypotheses (that is, every 10th of our experimental ideas are correct). In this case Q = 45/105 = 0.4286. That is, 42.86% of statistically significant results will be false positives. FDR = Q · pr(R > 0) = Q · [α · (M0/M) + Power · (M1/M)] = 0.045. If we only test true null effects then Q = false positives/all significant results = α·M / α·M = 1; and FDR = Q · pr(R>0) = Q · α = α = FWER [pr(R>0) = α because all significant results are coming from true null situations]. Note that in real research only R, M, and M-R are known whereas M0, M1, and Q are not known. TABLE 3 | Illustrating the logic behind FDR computation. H0 (leftmost column) stands for the null hypothesis. The proportion of false positive statistically significant test outcomes to all statistically significant test outcomes is Q = FP/(FP + TP) = FP/R (R̸= 0). The numbers give an example for the case when α = 0.05 (so, FWER = α = 0.05) and β =0.4, so Power = 1 −β = 0.6. In the example we run 1,000 null hypothesis tests. We test 9 times as many true null situations than situations with true alternative hypotheses (that is, every 10th of our experimental ideas are correct). In this case Q = 45/105 = 0.4286. THE STATE OF THE ART MUST CHANGE NHST Is Unsuitable as the Cornerstone of Scientific Inquiry in Most Fields Frontiers in Human Neuroscience | www.frontiersin.org When and How to Use NHST The currently dominant, NHST influenced approach is that instead of understanding raw data researchers often just focus on the all or nothing rejection of a vaguely defined H0 and shift their attention to interpreting brain “activations” revealed by potentially highly misleading statistical parameter maps. Based on these maps then strong (qualitative) claims may be made about alternative theories whose support may in fact never be tested. So, current approaches seem to reward exuberant theory building based on small and underpowered studies (Szucs and Ioannidis, 2017) much more than meticulous data collection and understanding and modeling extensive raw data patterns. For analogy, in astronomy theories typically built on thousands of years of sky observation data open to everyone. For example, Kepler could identify the correct laws of planetary motion because he had access to the large volume of observational data accumulated by Tycho Brache who devoted decades of his life to much more precise data collection than previously done. Similarly, the crucial tests of Einstein’s theories were precise predictions about data which could be verified or falsified (Smolin, 2006; Chaisson and McMillan, 2017). Importantly, we do not want to ban NHST (Hunter, 1997), we realize that it may be reasonable to use it in some well- justified cases. In all cases when NHST is used its use must be justified clearly rather than used as an automatic default and single cornerstone procedure. On the one hand, NHST can be used when very precise quantitative theoretical predictions can be tested, hence, both power and effect size can be estimated well as intended by Neyman and Pearson (1933). On the other hand, when theoretical predictions are not precise, reasonably powered NHST tests may be used as an initial heuristic look at the data as Fisher (1925) intended. However, in these cases (when well-justified theoretical predictions are lacking) if studies are not pre-registered (see below) NHST tests can only be considered preliminary (exploratory) heuristics. Hence, their findings should only be taken seriously if they are replicated, optimally within the same paper (Nosek et al., 2013). These replications must be well powered to keep FRP low. As discussed, NHST can only reject H0 and can accept neither a generic or specific H1. So, on its own NHST cannot provide evidence “for” something even if findings are replicated. NHST Is Unsuitable as the Cornerstone of Scientific Inquiry in Most Fields q y In summary, NHST provides the illusion of certainty through supposedly ‘objective’ binary accept/reject decisions (Cohen, 1994; Ioannidis, 2012) based on practically not very useful p- values (Bakan, 1966). However, researchers usually never give any formal assessment of how well their theory (a specific H1) fits the facts and, instead of gradual model building (Gigerenzer, 1998) and comparing the plausibility of theories, they can get away with destroying a strawman: they disprove an H0 (which happens inevitably sooner or later) with a machinery biased to disproving it without ever going into much detail about the exact behavior of variables under exactly specified hypotheses (Kranz, 1999; Jaynes, 2003). NHST also does not allow for systematic knowledge accumulation. In addition, both because of its shortcomings and because it is subject to major misunderstandings it facilitates the production of non-replicable false positive reports. Such reports ultimately erode scientific credibility and result in wasting perhaps most of the research funding in some areas (Ioannidis, 2005; Macleod et al., 2014; Kaplan and Irvin, 2015; Nosek et al., 2015). p In the NHST framework the multiple comparison problem is exacerbated by the fact that we may test a very large number of precise null hypotheses (Neath and Cavanaugh, 2006), often without much theoretical justification (e.g., in many explorative whole brain analyses). However, as the H0:H1 odds may be high the NHST mechanism may produce a very large number of falsely significant results. Similarly high numbers of false alarms are produced under realistic conditions even if some rudimentary model is used for the data (e.g., expecting positive or negative difference between conditions; Gelman and Tuerlinckx, 2000). In contrast, while currently there is no standard way to correct for multiple comparisons with Bayesian methods, Bayesian methods have been shown to be more conservative than NHST in some situations (Gelman and Tuerlinckx, 2000) and they offer various methods for correcting for multiple comparisons (e.g., Westfall et al., 1997; Gelman et al., 2014). In addition, Bayesian methods are strongly intertwined with explicit model specifications. These models can then be used to generate simulated data and NHST seems to dominate biomedical research for various reasons. First, it allows for the easy production of a large number of publishable papers (irrespective of their truth value) providing a response to publication pressure. NHST Is Unsuitable as the Cornerstone of Scientific Inquiry in Most Fields Second, NHST August 2017 | Volume 11 | Article 390 Frontiers in Human Neuroscience | www.frontiersin.org 13 Null Hypothesis Significance Testing: A Reassessment Szucs and Ioannidis seems deceptively simple: because the burden of inference (Bakan, 1966) has been delegated to the significance test all too often researchers’ statistical world view is narrowed to checking an inequality: is p ≤0.05 (Cohen, 1994)? After passing this test, an observation can become a “scientific fact” contradicting the random nature of statistical inference (Gelman, 2015). Third, in biomedical and social science NHST is often falsely perceived as the single objective approach to scientific inference (Gigerenzer et al., 1989) and alternatives are simply not taught and/or understood. their variables, make model predictions and then confirm these with likelihood-based and/or Bayesian methods. Making such predictions would probably require intimate familiarity with a lot of raw data. seems deceptively simple: because the burden of inference (Bakan, 1966) has been delegated to the significance test all too often researchers’ statistical world view is narrowed to checking an inequality: is p ≤0.05 (Cohen, 1994)? After passing this test, an observation can become a “scientific fact” contradicting the random nature of statistical inference (Gelman, 2015). Third, in biomedical and social science NHST is often falsely perceived as the single objective approach to scientific inference (Gigerenzer et al., 1989) and alternatives are simply not taught and/or understood. Ways to Change In most biomedical, neuroscience, psychology, and social science fields currently popular analysis methods are based on NHST. It is clear that analysis software and researcher knowledge cannot be changed overnight. Below we summarize some further recommendations which we think can minimize the negative features of NHST even if it continues to be dominant for a while. A very important practical goal would be to change the incentive structure of biomedical and social science to bring it in line with these and similar other recommendations (Wagenmakers et al., 2011; Begley and Ellis, 2012; Nosek et al., 2013; Stodden et al., 2016). Also note that we are not arguing against statistical inference which we consider the “logic of science” (Jaynes, 2003; p. xxii.), quantitative and well justified statistical inference should be at the core of the scientific enterprise. We have now decades of negative experience with NHST which gradually achieved dominance in biomedical and social science since the 1930s (Gigerenzer et al., 1989). Critique of NHST started not much later (Jeffreys, 1939, 1948, 1961) and has been forcefully present since then (Jeffreys, 1939, 1948, 1961; Eysenck, 1960; Nunnally, 1960; Rozeboom, 1960; Clark, 1963; Bakan, 1966; Meehl, 1967; Lykken, 1968) and continues to-date (Wasserstein and Lazar, 2016). The problems are numerous, and as Edwards (1972, p. 179) concluded 44 years ago: “any method which invites the contemplation of a null hypothesis is open to grave misuse, or even abuse.” Time has proven this statement and that problems are unlikely to go away. We suggest that that it is really time for change now. Frontiers in Human Neuroscience | www.frontiersin.org When and How to Use NHST Overall, if we just consider competing “theories” without ever deeply considering extensive raw data patterns it is unlikely that major robust scientific breakthroughs will be done whereas many different plausible looking theories can be promoted. Imagine, for example, a situation where astronomers would have only published the outcomes of their NHST tests, some rejecting that the sun is in the middle of the universe while others rejecting that the earth is in the middle of the universe while publishing no actual raw data. Meta-analyses of published effect sizes would have confirmed both positions as both camps would have only published test statistics which passed the statistical significance threshold. Luckily, real astronomers recorded a lot of data and derived testable theories with precise predictions. For example, if initially researches do not know where to expect experimental effects in a particular experimental task, they could run a whole brain, multiple-testing corrected search for statistical significance in a group of participants. Such a search would provide heuristic evidence if they identify some brain areas reacting to manipulations. In order to confirm these effects they would need to carefully study for example the BOLD signal or EEG amplitude changes in areas or over electrodes of interest, make predictions about the behavior of these variables, replicate measurements and minimally confirm the previous NHST results before the findings can be taken seriously. Much better, if researchers can also provide some model for the behavior of August 2017 | Volume 11 | Article 390 14 Null Hypothesis Significance Testing: A Reassessment Szucs and Ioannidis In basic biomedical and psychology research we often cannot provide very well worked out hypotheses and even a simple directional hypothesis may seem particularly enlightening. Such rudimentary state of knowledge can be respected. However, in such pre-hypothesis stage substantively blind all or nothing accept/reject decisions may be unhelpful and may maintain our ignorance rather than facilitate organizing new information into proper quantitative scientific models. It is much more meaningful to focus on assessing the magnitude of effects along with estimates of uncertainty, let these be error terms, confidence intervals or Bayesian credible intervals (Edwards, 1972; Luce, 1988; Schmidt, 1996; Jaynes, 2003; Gelman, 2013a,b; see Morey et al., 2016 on the difference between classical confidence intervals and Bayesian credible intervals). These provide more direct information on the actual “empirical” behavior of our variables and/or the precision of interval estimation. Publish All Analysis Scripts with Analysis Settings Publish All Analysis Scripts with Analysis Settings Another cost-free improvement is to publish all analysis scripts with the ability to regenerate all figures and tables (Laine et al., 2007; Peng, 2009, 2011; Diggle and Zeger, 2010; Keiding, 2010; Doshi et al., 2013). This does not require large storage space and can also be done in Supplementary Material. If researchers keep this expectation in mind from the start of a project then implementing it becomes relatively straightforward. Program code will often provide information which is missing from papers. With regard to missing analysis information it is important to be conscious of the fact that seemingly innocuous and irrelevant analysis settings (e.g., slightly changing initial filtering parameters) can have major impact on final statistical outcomes at the end of a complicated processing pipeline. For example, modified initial settings may change the statistically significant/non-significant status of final important test statistics. This can be an issue if multiple settings can be justified and/or if some settings leading to significant outcomes are actually less justified than alternative settings. Publishing scripts will also provide more information on potential statistical errors (Bakker and Wicherts, 2001; Nuijten et al., 2016). The change of emphasis suggested here would require that instead of p-values and reporting the outcomes of all/nothing hypothesis tests studies should focus on reporting data in original units of measurement as well as providing derived effect sizes. It is important to publish data summaries (means, standard errors, nowadays extremely rarely plotted empirical data distributions) in original units of measurement as derived measures may be highly biased by some (undocumented) analysis techniques. If we have clear and pre-registered hypotheses then it is relatively straightforward to publish raw data summaries (e.g., mean BOLD signal or ERP amplitude change with standard errors) related to those hypotheses. Clear data presentation usually gets difficult when there are lots of incidental findings. Usually unlimited amount of data summaries can now be published cost free in Supplementary Materials. August 2017 | Volume 11 | Article 390 Frontiers in Human Neuroscience | www.frontiersin.org When and How to Use NHST Gaining enough experience with interval estimates and assuring their robustness by building replication into design (Nosek et al., 2013) may then allow us to describe the behavior of variables by more and more precise scientific models which may provide more clear predictions (Schmidt, 1996; Jaynes, 2003; Gelman, 2013a,b). by high-dimensional neuroscience data (Kriegeskorte et al., 2009; Vul et al., 2009; Carp, 2012) pre-registration seems a necessary pre-condition of robust hypothesis driven neuroscience research. Pre-registration would likely help to cleanse non-replicable “unchallenged fallacies” (Ioannidis, 2012) from the literature. For example, Kaplan and Irvin (2015) found that pre-registering the primary hypotheses of clinical studies decreased the proportion of positive findings from 57% (17 of 30 studies) to 8% (2 out of 25 studies). Hence, another benefit of pre-registration would be to decrease publication volumes. This would require changing the incentive system motivating scientists (Nosek et al., 2013). It is to note that honesty regarding pre-registration and challenging questionable research practices (Simmons et al., 2011) is the shared responsibility of all co-authors. Some fields in medical research have already over 10 years of experience with pre-registration. This experience shows that pre-registration needs to be thorough to be reliable. For example, many observational studies claimed to have been registered but closer scrutiny shows that registration has actually happened after the study/analysis was done (Boccia et al., 2016). In other cases, clinical trials may be seemingly properly pre-registered before they start recruiting patients, but analyses and outcomes were still manipulated after registration (Ioannidis et al., 2017). Hence, proper safeguards should be put in place to ensure that scientists are accountable for any misconduct regarding breaching pre- registration rules. The above problem does not only concern perceived “soft areas” of science where measurement, predictions, control and quantification are thought to be less rigorous than in “hard” areas (Meehl, 1978). In many fields, for example, in cognitive neuroscience, the measurement methods may be “hard” but theoretical predictions and analysis often may be just as “soft” as in any area of “soft” psychology: Using a state of the art fMRI scanner for data collection and novel but extremely complicated and often not well understood analysis paths will not make a badly defined theory well-defined. Publish Data (Summaries) Irrespective of Statistical Significance, Promote Building Good Quality Datasets Including Large Replication Studies It is important to publish data summaries and/or data sets, including the ones not resulting in statistically significant findings. Without these datasets true effect sizes simply cannot be determined. This will require that these datasets become citeable so that their authors can be rewarded if data is used for secondary analyses. Considering for example the above mentioned case of Tycho Brache it is clear that his data collection exercise was a necessary precondition of crowning the Copernican/Newtonian revolution of astronomy (Chaisson and McMillan, 2017). Hence, we should be able to reward the mere collection of large volumes of good quality data: such activity can prove to be an immense service to the whole profession. Initiatives, like registered multi-lab replication studies should also be prioritized when the validity of important proposals is at stake. Funders are currently often reluctant to fund such studies. However, they should realize that the continuous seeking of new results and theories may just waste most of their resources (Ioannidis et al., 2014; Kaplan and Irvin, 2015). All too often statistical understanding is perceived as something external to the subject matter of substantive research. However, it is important to see that statistical understanding influences most decisions about substantive questions, because it underlies the thinking of researchers even if this remains implicit. While common sense “statistics” may be able to cope with simple situations, common sense is not enough to decipher scientific puzzles involving dozens, hundreds, or even thousands of interrelated variables. In such cases well justified applications of probability theory are necessary (Jaynes, 2003). Hence, instead of delegating their judgment to “automatized” but ultimately spurious decision mechanisms, researchers should have confidence in their own informed judgment when they make an inference. Such confidence requires deep study. y Understanding probability is difficult. Common sense is notoriously weak in understanding phenomena based on probabilities (Gigerenzer et al., 2005). We cannot assume that without proper training biomedical and social science graduates would get miraculously enlightened about probability. Pre-registration It is to note that in-house analysis scripts may provide substantial competitive advantage to researchers who are able to programme these. Hence, the unconditional release of these scripts may deprive researchers from an important competitive asset. In such cases in house scripts could be documented in brief methods papers which could be cited when the relevant scripts are used so that researchers benefit from citations. Perhaps specialized methods repository journals could be set up for this purpose. For some recent recommendations for improving computational reproducibility practices see Stodden et al. (2016). In our view one of the most important and virtually cost- free (to researchers) improvement would be to pre-register hypotheses and analysis parameters and approaches (in line with Section 5.2 in Nichols et al., 2016; p11; Gelman and Loken, 2014). Pre-registration can easily be done for example, at the website of the Open Science Foundation (osf.org), also in a manner that it does not immediately become public. Hence, competitors will not be able to scoop good ideas before the study is published. Considering the extreme analysis flexibility offered August 2017 | Volume 11 | Article 390 August 2017 | Volume 11 | Article 390 Frontiers in Human Neuroscience | www.frontiersin.org 15 Null Hypothesis Significance Testing: A Reassessment Szucs and Ioannidis Publish Data (Summaries) Irrespective of Statistical Significance, Promote Building Good Quality Datasets Including Large Replication Studies Some of the best symbolic thinking minds of humanity devoted hundreds of years to the proper understanding of probability and statisticians still do not agree on how best to draw statistical inference (Stigler, 1986; Gigerenzer et al., 1989), e.g., the recent American Statistical Association statement on p-values (Wasserstein and Lazar, 2016) was accompanied by 21 editorials from the statisticians and methodologists who participated in crafting it and who disagreed in different aspects among themselves. Publish Raw Data In an ideal world researchers should publish all raw data. This is easy with small volumes of behavioral data but it has serious monetary and time investment costs with large neural data volumes (see also Nichols et al., 2017). Some repositories have already been set up and it is important that funders cover these costs and optimally provide infrastructure (see Pernet and Poline, 2015; Nichols et al., 2017). Incentives such as a badge system may help promote availability of more raw data (Nosek et al., 2015). In our opinion it is important to publish unprocessed raw data because processed data may already have been distorted/biased in undocumented ways. In general, it is more and more usual to reanalyse data from large repositories, so much further development can be expected in this area (e.g., Eklund et al., 2012). A core problem seems to be that the statistical subject knowledge of many researchers in biomedical and social science has been shown to be poor (Oakes, 1986; Gliner et al., 2002; Castro Sotos et al., 2007, 2009; Wilkerson and Olson, 2010; Hoekstra et al., 2014). NHST perfectly fits with poor understanding because of the perceived simplicity of interpreting its outcome: is p ≤0.05 (Cohen, 1994)? We suggest that the weak statistical understanding is probably due to inadequate “statistics lite” education. This approach does not build up appropriate mathematical fundamentals and does not provide scientifically rigorous introduction into statistics. Hence, students’ knowledge may remain imprecise, patchy, and prone to serious misunderstandings. What this approach achieves, however, is providing students with false confidence of being able to use inferential tools whereas they usually only interpret the p-value provided by black box statistical software. While this educational problem remains unaddressed, poor statistical practices will prevail regardless of what procedures and measures may be favored and/or banned by editorials. Frontiers in Human Neuroscience | www.frontiersin.org August 2017 | Volume 11 | Article 390 Increase Statistical Power and Publish Pre-study Power Calculations In real world research it is usually impossible to determine the statistical power of NHST tests exactly. However, if raw data summaries and/or raw data is published irrespective of statistical significance (Section Pre-registration, Publish Raw Data, and Publish Data (Summaries) Irrespective of Statistical Significance, Promote Building Good Quality Datasets Including Large Replication Studies) then we have a much better chance of trying to determine power. Another option is to determine power to detect pre-defined standardized effect sizes. In any case, power in psychology and neuroscience should be much higher than what it is nowadays (Szucs and Ioannidis, 2017). We hypothesize that pre-registration would facilitate increasing power because researchers could less expect to rely on incidentally finding something statistically significant to report from their studies. Hence, they would have more interest in assuring that they are able to respond their primary, registered hypotheses. There is no reason to assume that understanding twenty first and twenty second century science will require less mathematical and statistical understanding than before. Such as there is no royal road to mathematics, there is no royal road to statistics which is heavily based on mathematics. If statistical understanding does not improve it will not matter whether editorials enforce bootstrapping, likelihood estimation or Bayesian approaches, they will all remain opaque to the August 2017 | Volume 11 | Article 390 16 Null Hypothesis Significance Testing: A Reassessment Szucs and Ioannidis untrained mind and open to abuse such as the NHST of the twentieth century. as they allow for formal knowledge integration from previous studies. One approach would be to phase out the ‘statistics lite’ education approach for all research stream students and teach statistics rigorously. A typical research stream undergraduate training could include, for example, 3–4 semesters of calculus, one semester of introductory statistics, three more semesters of calculus based statistics, and then finally two semesters of more specialized statistics. An alternative and/or complementary approach would be to enhance the training of professional applied statisticians and to ensure that all research involves knowledgeable statisticians or equivalent methodologists. At a minimum, all scientists should be well trained in understanding evidence and statistics and being in a position to recognize that they may need help from a methodologist expert (Marusic and Marusic, 2003; Moharari et al., 2009; Vujaklija et al., 2010). AUTHOR NOTE A previous version of this manuscript was available as a pre- print (http://biorxiv.org/content/early/2016/12/20/095570). The copyright holder for this preprint is the author/funder. It is made available under a CC-BY-NC-ND 4.0 International license. ACKNOWLEDGMENTS It is important to note that Bayesian methods are often accused of subjectivity because they can take prior information into account. However, Bayesian methods are able to consider prior expectations formally and explicitly in their models provided that necessary information (e.g., raw data and/or extensive reporting of data parameters) from previous studies is available. In contrast, as we have discussed NHST can be latently biased by subjectivity at many points without ever revealing any of the biases. In contrast, different reasonable Bayesian priors can be implemented and their impact on outcomes can be debated explicitly and ultimately, the goodness of model predictions can be tested. Hence, we do not see the use of Bayesian priors as a drawback. Rather, explicit priors can represent a strength DS is supported by a twenty-first Century Science Initiative in Understanding Human Cognition Scholar Award (220020370) from the James S. McDonnell Foundation. METRICS is supported from a grant from the Laura and John Arnold Foundation. AUTHOR CONTRIBUTIONS DS wrote the first draft of the manuscript. DS and JI revised successive drafts. Teach Alternative Approaches Seriously Teach Alternative Approaches Seriously It is important that researchers are conscious that NHST only represents a small segment of available statistical techniques. Besides NHST, Bayesian and likelihood based approaches should also be taught, with explanation of the strengths and weaknesses of each inferential method. Hypotheses could be tested by either likelihood ratio testing, and/or Bayesian methods which usually view probability as characterizing the state of our beliefs about the world (Pearl, 1988; Jaynes, 2003; MacKay, 2003; Sivia and Skilling, 2006; Gelman et al., 2014; for neuroscience data see e.g., Lorenz et al., 2017). The above alternative approaches typically require model specifications about alternative hypotheses, they can give probability statements about H0 and alternative hypotheses, they allow for clear model comparison, are insensitive to data collection procedures and do not suffer from problems with large samples. In addition, Bayesian methods can also factor in pre-study (prior) information into model evaluations which may be important for integrating current and previous research findings. Hence, the above alternative approaches seem more suitable for the purpose of scientific inquiry than NHST and ample literature is available on both. The problem is that usually none of these alternative approaches are taught properly in statistics courses for students in psychology, neuroscience, biomedical science and social science. For example, across 1,000 abstracts randomly selected from the biomedical literature of 1990–2015, none reported results in a Bayesian framework (Chavalarias et al., 1990-2015). Frontiers in Human Neuroscience | www.frontiersin.org There Is No Automatic Inference: New-Old Dangers Ahead? g Perhaps the most worrisome false belief about statistics is the belief in automatic statistical inference (Bakan, 1966; Gigerenzer and Marewski, 1998), the illusion that plugging in some numbers into some black box algorithm will give a number (perhaps the p-value or some other metric) that conclusively proves or disproves hypotheses (Bakan, 1966). There is no reason to assume that any kind of “new statistics” (Cumming, 2014) will not suffer the fate of NHST if statistical understanding is inadequate. For example, it has been shown that confidence intervals are misinterpreted just as badly as p-values by undergraduates, graduates, and researchers alike and self-declared statistical experience even slightly positively correlates with the number of errors (Hoekstra et al., 2014). Or, many times black box machine learning algorithms may be run uncritically and/or on relatively small data volumes. However, the more complex is a dataset the more chance such substantively blind search algorithms have to find some relationships where nothing worthy of mention exist. So, uncritical applications are likely to further boost the proportion of false positive findings irrespective of the sophistication of the algorithms (Skokic et al., 2016). Similarly, the proper use of Bayesian methods may require use of advanced simulation methods and a clear understanding and justification of probability distribution models. 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Video observations of treatment administration to children on antiretroviral therapy in rural KwaZulu-Natal
AIDS care
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AIDS CARE, 2016 VOL. 28, NO. S2, 34–41 http://dx.doi.org/10.1080/09540121.2016.1176674 AIDS CARE, 2016 VOL. 28, NO. S2, 34–41 http://dx.doi.org/10.1080/09540121.2016.1176674 AIDS CARE, 2016 VOL. 28, NO. S2, 34–41 http://dx.doi.org/10.1080/09540121.2016.1176674 AIDS CARE, 2016 VOL. 28, NO. S2, 34–41 http://dx.doi.org/10.1080/09540121.2016.1176674 ABSTRACT For children younger than five years, caregivers are responsible for the measurement and administration of antiretroviral medication doses to children. Failure to adhere to the regimen as prescribed may lead to high viral loads (VLs), immune suppression and ultimately drug resistance. In the content of this study, adherence refers to adequate dosing of the medication by a caregiver. Acquired drug resistance to antiretroviral therapy (ART) is prevalent amongst children in South Africa, and poor adherence to the dosing regimen by caregivers may be associated with this problem. In this qualitative study, we purposively recruited 33 caregiver– child dyads from the Hlabisa HIV Treatment and Care Programme database. Children were divided into three groups based on their VL at the time of recruitment. Children with a VL ≥400 cps/ml were grouped as unsuppressed (n = 11); children with a VL ≤400 cps/ml were grouped as suppressed (n = 12); and children with no VL data were grouped as newly initiated (n = 10). Caregiver–child dyads were visited at their households twice to document, by means of video recording, how treatment was administered to the child. Observational notes and video recordings were entered into ATLAS.ti v 7 and analysed thematically. Results were interpreted through the lens of Ecological Systems Theory and the information–motivation–behavioural skills model was used to understand and reflect on several of the factors influencing adherence within the child’s immediate environment as identified in this study. Thematic video analysis indicated context- and medication-related factors influencing ART adherence. Although the majority of children in this sample took their medicine successfully, caregivers experienced several challenges with the preparation and administration of the medications. In the context of emerging drug resistance, efforts are needed to carefully monitor caregiver knowledge of treatment administration by healthcare workers during monthly clinic visits. KEYWORDS HIV; adherence; barriers; facilitators; paediatric; anti- retroviral treatment (Davies et al., 2011; Fox, Ive, Long, Maskew, & Sanne, 2010; Lessells et al., 2014). Video observations of treatment administration to children on antiretroviral therapy in rural KwaZulu-Natal Bronwyne Coetzeea, Ashraf Kageea and Ruth Blandb,c,d aDepartment of Psychology, Stellenbosch University, Stellenbosch, South Africa; bAfrica Centre for Population Health, KwaZulu-Natal, South Africa; cInstitute of Health and Wellbeing, and Royal Hospital for Sick Children, University of Glasgow, Glasgow, UK; dSchool of Public Health, Faculty of Health Sciences, University of Witwatersrand, Johannsburg, South Africa ARTICLE HISTORY Received 29 January 2016 Accepted 23 March 2016 © 2016 The Author(s). Published by Informa UK Limited, trading as Taylor & Francis Group This is an Open Access article distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/by/4.0/), which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited. CONTACT Bronwyne Coetzee bronwyne@sun.ac.za KEYWORDS HIV; adherence; barriers; facilitators; paediatric; anti- retroviral treatment Participants and procedure Purposive sampling was used to select and recruit partici- pants from two primary healthcare clinics in the study area. VL status at the time of recruitment was used to differentiate three groups of children invited to take part in the study. Children on ART for more than a year and with two consecutive VLs ≥400 copies per millilitre (cps/ml) were grouped as unsuppressed (n = 11); children on ART for more than a year with a VL ≤400 cps/ml were grouped as suppressed (n = 12); and children with no VL data were grouped as newly initiated (n = 10). To our knowledge, no study has observed and docu- mented the barriers and facilitators associated with treatment administration of ART to children younger than five years by their caregivers in their homes during medication times in the morning and evening. The aim of this study was to document, by means of video recording, the barriers to and facilitators of adherence to ART among children younger than five years in rural KwaZulu-Natal. g p y Adherence counsellors at each of the clinics invited caregivers to meet with the primary investigator (PI) (BC) and research assistant (RA) in a private room after their clinic visit to inform them of the study and to obtain written informed consent for participation. Caregivers were informed that the PI and an isiZulu- speaking RA (to act as an interpreter) were going to visit each of the recruited caregiver–child dyads at their homes twice (one morning visit and one evening visit) to document, by means of video recording, how children received their ARVs. Participants were not excluded if they did not consent to video recording. All participants gave written informed consent, and none objected to video recording. Furthermore, all participants had indi- cated that household members were aware that the child was taking medications. Following each home visit, participants were handed a food parcel to the value of R150.00 (£6.26) as a token of our appreciation for their willingness to participate and their time in this research. This study was conceptualised, interpreted and under- stood through the lens of Ecological Systems Theory (EST) (Bronfenbrenner, 1979). Ecological theories of health, like EST, consider individual (situated at the micro level) as well as environmental (contextual) factors (meso-, exo- and macro levels) when attempting to under- stand, or when examining, a target behaviour (see Figure 1). Ethics The study received ethical approval from Stellenbosch University (S12/05/135), permission from the local com- munity advisory board and the hospital management before entering into the field. Multiple visits to partici- pants’ households were used to minimise reactivity bias during observations. Further, both the PI and the RA documented the visits by means of field notes and semi-structured observation schedules, separately. A debriefing session was held after each visit to compare and discuss observations. Introduction In the context of limited antiretroviral (ARV) drug options available for adults and children in South Africa (Davies et al., 2011), adherence to a first-line regimen is key to ensure optimal and prolonged benefits of treatment (Bangsberg et al., 2000; Bangsberg et al., 2003). According to the latest guidelines on ART initiation among infants, children and adolescents in South Africa, all children younger than five years old should be initiated on ART and are usually initiated on a first-line ART regimen (Department of Health South Africa, 2014). Second-line regimens, although available, remain costly and often dif- ficult to access (Davies et al., 2011). Particular concern has been raised regarding developing resistance to second-line regimens, especially as monitoring of patient viral loads (VLs) is unavailable in many resource-limited settings For children younger than five years who rely on par- ental or non-parental caregivers to administer their medication to them daily, adherence becomes an even greater task to manage. In the context of this study, adherence refers to adequate dosing of the medication by a caregiver. In addition to having to provide for chil- dren’s most basic needs, caregivers have to integrate and manage complicated regimens into their daily lives (Coetzee, Kagee, & Bland, 2015). For example, in the absence of fixed-dose combinations available to children younger than five years, caregivers of children in this age group are required to carefully and accu- rately measure and administer volumes of liquid drug formulations to children on ART twice a day (World Health Organisation [WHO], 2013). Moreover, some CONTACT Bronwyne Coetzee © 2016 The Author(s). Published by Informa UK Limited, trading as Taylor & Francis Group This is an Open Access article distributed under the terms of the Creative Commons Attribu distribution, and reproduction in any medium, provided the original work is properly cited AIDS CARE 35 35 Trust-funded Africa Centre for Population Health (www.africacentre.ac.za). of the medications have special storage requirements and some are unpalatable and require creative strategies to mask their taste in order to aid administration (Department of Health South Africa, 2014). These fac- tors highlight several characteristics of paediatric ART that complicates adherence. Setting The research was conducted in Northern KwaZulu-Natal (South Africa) under the auspices of the Wellcome Figure 1. The four systems of Bronfenbrenner’s EST. Participants and procedure In addition to EST, we used concepts associated with the information–motivation–behavioural skills (IMB) model to discuss and explain specific observed behaviours in this study (Fisher, Fisher, Amico, & Harman, 2006). Results The median number of adults and children per house- hold was three. Households had up to six occupants in a dwelling consisting of one bedroom and one living area (approximately 30 m2 in total (Hunter & Posel, 2012)). Thus, overcrowded households were common amongst the participants in this study and inhibited the ease with which medication was administered to the child. Medication storage Nearly all of the caregivers stored the medications in the bedroom. Abacavir (ABC) and Lamivudine (3TC) (both liquid ARV formulations) and tablets (such as Efavirenz (EFV), Zidovudine and Stavudine (d4 T)) do not require refrigeration and it was therefore appropriate to store these medications at room temperature. The directions for one of the medications, Lopinavir/ritonavir (LPV/r), state that it should be kept refrigerated (Department of Health South Africa, 2013). Although most caregivers (30 out of 33) had access to a refrigerator, not all caregivers stored LPV/r in the refrigerator. Compared to children with suppressed VLs, fewer caregivers of children newly initiated and with unsuppressed VLs kept LPV/r refriger- ated (Table 1). Caregiver characteristics The median age of the caregivers was 35 years (IQR: 27.5–48), 29 years (IQR: 24–40) and 28.5 (IQR: 25.5– 51) for caregivers of children with suppressed VLs, unsuppressed VLs and newly initiated on ART, respect- ively. A greater proportion of caregivers to children with suppressed VLs (7 out of 12) were married and/or living with a significant other in a permanent union compared to the other two groups (2 out of 11 – unsuppressed, and 2 out of 10 – new enrollers). Furthermore, a greater pro- portion of caregivers to children with suppressed VLs had completed high school (5 out of 12), compared to the other two groups (2 out of 11 – unsuppressed, and 1 out of 10 – new enrollers). Most of the caregivers (27 out of 33) were unemployed and were receiving either a child support grant or disability grant from the South African government. More than half of the caregivers (19 out of 33) were also ART users. Only 24 (73%) had received the pre-ART HIV education sessions before the child was enrolled on treatment. Most caregivers (25 out of 33) indicated that they were not solely respon- sible for treatment administration or for attending clinic visits (11 out of 33), but shared this responsibility with another member of the household, usually an older sib- ling. Caregivers reported that all members of the Medications stored in the bedroom were either hid- den (when kept in plastic bags and stored in a cupboard in the bedroom), or not hidden (when kept visible on a table in the bedroom). Compared to children with supressed and unsuppressed VLs, more of the caregivers of children who were newly initiated (see Table 1) on ART did not keep LPV/r in the fridge and preferred to store medications out of sight. Based on this observation, it is possible that they were hiding the medications from younger children, other household members or visitors. Child characteristics The median age of the children at recruitment was 3.5 years (interquartile range (IQR), 2–4.75) for those with suppressed VLs, 4 years (IQR: 3–5) for those with unsuppressed VLs and 2 years (IQR: 2–5) for those newly initiated on ART. Twelve children were between 0 and 2 years old, 5 children were 3 years old, 4 were 4 years old, and 12 were aged 5 years. The majority of children were female (19 out of 33) and most of them (18 out of 33) had their biological mother as primary caregiver. Other primary caregivers were grandmothers (9 out of 33), fathers (1 out of 33) and aunts (5 out of 33). The average time on ART was 2.3 years, 2.8 years and 0.4 years amongst children with suppressed VLs, unsuppressed VLs and those newly initiated, respectively. Data analysis The sources of data were the video recordings, field notes and data from the semi-structured observation schedules Figure 1. The four systems of Bronfenbrenner’s EST. B. COETZEE ET AL. 36 used during home visits. Demographic data of the chil- dren (such as age, gender, etc.) as well as their caregivers (such as age, marital status, level of education, etc.) were entered and analysed using SPSS v 22. Field notes and video data were analysed thematically using ATLAS.ti v 7 (www.atlasti.com). used during home visits. Demographic data of the chil- dren (such as age, gender, etc.) as well as their caregivers (such as age, marital status, level of education, etc.) were entered and analysed using SPSS v 22. Field notes and video data were analysed thematically using ATLAS.ti v 7 (www.atlasti.com). household were aware that the child was on treatment. However, it was not fully ascertained whether the house- hold members knew this treatment was for HIV, especially in the case of other siblings in the household. Food given before treatment administration During the first visit to the homes, all of the caregivers reported that they usually gave food to the child before treatment administration in the mornings and evenings. However, there were only four morning (three newly initiated, and one suppressed VL) and two evening visits (both newly initiated) in which we had seen a child eat- ing before treatment was administered or had seen vis- ible signs that food had been prepared and given. We did not observe any food given during our visits to the AIDS CARE 37 37 Table 1. Context-related observations. Table 1. Context-related observations. Newly initiated Suppressed VL Unsuppressed VL Morning visit (n = 10) Evening visit (n = 6) Morning visit (n = 12) Evening visit (n = 8) Morning visit (n = 11) Evening visit (n = 9) Medication workspace Lounge 5 4 9 6 2 4 Bedroom 3 2 2 1 7 3 Kitchen 2 0 1 1 2 2 Medication storage In bedroom (hidden) 5 3 5 3 3 5 In bedroom (not hidden) 2 1 6 5 7 4 In lounge (hidden) 3 2 0 0 1 0 In lounge (not hidden) 0 0 1 0 0 LPV/r refrigerateda 4 out of 8 2 out of 8 9 out of 10 7 out of 10 5 out of 9 4 out of 9 Food givenb 3 2 1 0 0 0 a27 (10 out of 12 suppressed; 9 out of 11 unsuppressed; 8 out of 10 newly initiated) children were receiving LPV/r as part of their regimen. bFood given before treatment administration. a27 (10 out of 12 suppressed; 9 out of 11 unsuppressed; 8 out of 10 newly initiated) children were receiving LPV/r as part of th bFood given before treatment administration. households of children with unsuppressed VLs. There was therefore a mismatch between what caregivers had stated during the initial home visit and what we observed during the morning and evening home visits. (usually ranging between 1 and 5 ml) and administer it to the child. Of children newly initiated on ART, 9 received ABC, 10 received 3TC and 8 received LPV/r as part of their regimen. Of children with suppressed VLs, 10 received ABC, 9 received 3TC and 10 received LPV/r as part of their regimen. Of children with unsup- pressed VLs, 7 received ABC, 7 received 3TC and 9 received LPV/r as part of their regimen. Liquid formulations Apart from one child with a suppressed VL, all of the children received liquid ARV formulations as part of their regimen. For children under three years (<10 kgs), a first-line regimen is a combination of ABC, Lami- vudine (3TC) and LPV/r. All three of these medications are available as liquid formulations, and dose changes vary depending on the weight of the child. For children between three and 10 years old (>10 kg), a combination of ABC, 3TC and EFV is recommended (Department of Health South Africa, 2014). Caregivers are required to measure the millilitres prescribed for each medication The majority of caregivers (observed 20 times during morning administration, and 13 times during evening administration) used a separate syringe to measure and administer each dose (Table 2). One way to aid accurate measurements of liquid formulations using syringes is through the use of a syringe nozzle. Syringe nozzles are small plastic attachments that fit into the opening of medication bottles and are made especially for syringes. These devices are typically packaged with the medi- cations ABC and 3TC. The medication LPV/r does not come with such a device. Syringe nozzles were Table 2. Medication-related observations. Newly initiated Suppressed VL Unsuppressed VL Morning visit (n = 10) Evening visit (n = 6) Morning visit (n = 12) Evening visit (n = 8) Morning visit (n = 11) Evening visit (n = 9) Medication measurement tools Syringe 6 3 7 5 7 5 Syringe + nozzle 4 2 4 3 1 1 Measuring cup 0 1 1 0 3 3 Measurement issues Bubble in syringe 1 0 2 1 1 1 Dose checkinga ABC 2 3 2 0 4 3 3TC 2 3 1 1 4 3 LPV/r 3 3 8 6 6 7 Medication administration (directly or indirectly)c Syringe (directly) 6 3 9 7 4 4 Measuring cup (indirectly) 4 3 2 1 7 5 Missed dosesb 1 1 0 0 0 0 aDose checking: caregiver checks the measurement dose amount in the syringe for accuracy. bMissed doses: medication is not taken/given on the day. cMedication administration: directly – medication injected into child’s mouth using syringe. Indirectly – medication transferred into a cup. Table 2. Medication-related observations. Table 2. Medication-related observations. aDose checking: caregiver checks the measurement dose amount in the syringe for accuracy. bMissed doses: medication is not taken/given on the day. Palatability y Most of the children (no problems with palatability in 38 out of 56 observations) ingested their medications with- out much resistance (Table 3). However, we observed four instances (one newly initiated, one suppressed, two unsuppressed) of children spitting medications out; two instances of vomiting (one newly initiated, one unsuppressed); seven instances of coughing (two newly initiated, four suppressed, one unsuppressed) during medication administration or moaning by the child; five instances of child refusal (one instance in which the child ran away at the time of medication administration (newly initiated); and four instances (two newly initiated and two suppressed) where the child pushed the caregiver’s hand away (Table 3). In all instances of palatability difficulty, the caregiver com- pleted dose administration. In both instances of vomiting in particular, the caregivers only administered a partial dose. As such, children who vomited their medications did not receive their full dosage. Figure 2. Dose measurement of 3TC taken at an angle. The image illustrates the caregiver of a suppressed child on ART taking a measurement of 3TC at a horizontal angle. underutilised amongst the caregivers (only used 15 times across 56 observations) (Table 2). Observation revealed that syringe nozzles were used more frequently by care- givers of children newly initiated (observed six times) and suppressed on ART (observed seven times) com- pared to unsuppressed children on ART (observed twice). Fewer instances were observed among those with unsuppressed VLs as a large proportion was receiv- ing LPV/r via syringe only. However, even with a syringe nozzle intact, caregivers who chose to use them did so erroneously. In at least two instances where measurements were taken at a horizon- tal angle, these measurements led to bubble formations in the syringe. Figure 2 shows a caregiver (biological mother) taking a measurement of 3TC with a syringe nozzle intact. The caregiver can be seen holding the bot- tle horizontally while the measurement is taken. The angle at which the doses were measured resulted in bubbles forming within the syringe, suggesting possible dose inaccuracy. The dose was not checked (for accu- racy) by the caregiver after the measurement was made. In four other instances, it was clear from the video data that a large bubble had formed in the syringe (Table 2). In neither of these instances was the bubble removed, indicating possible under-dosing of that medication. Tablet formulations Tablets and capsules were either swallowed whole or dis- solved in water or mixed with a taste distractor (e.g. drinking yoghurt). All the children with suppressed VLs received their tablets/capsules directly and they were subsequently swallowed whole. Discussion To our knowledge, this is the first study to directly observe factors influencing adherence to ART amongst children younger than five years living in a rural area in a resource-constrained environment. Ecological models, like EST, favour behaviour-specific approaches to interventions, such as improving adherence to ART amongst children younger than five years. Thus, by using EST in this study, we were able to identify contex- tual and medication-related factors influencing adher- ence to ART among children in a rural area of South Africa. Liquid formulations cMedication administration: directly – medication injected into child’s mouth using syringe. Indirectly – medication transferred into a cup. 38 B. COETZEE ET AL. B. COETZEE ET AL. B. COETZEE ET AL. 38 Figure 2. Dose measurement of 3TC taken at an angle. The image illustrates the caregiver of a suppressed child on ART taking a measurement of 3TC at a horizontal angle. The tablets of four out of five children in the unsup- pressed group were dissolved in water before being administered. Dissolving tablets into solutions was pro- blematic in cases where the residue of the tablets remained behind in the container (usually a cup) and was discarded by the caregiver when she rinsed out the medication cup. In all four instances where either a tablet or capsule had been dissolved in water, residue of the medications remained behind in glass/cup from which it was administered. Once administered, the caregiver rinsed the administration tool and discarded the residue that remained left over, indicating a possible under-dos- ing of that medication. Context-related factors Caregivers in this study showed a preference for keeping medications hidden. Although caregivers were not AIDS CARE 39 AIDS CARE 39 Table 3. Child-related observations. Table 3. Child-related observations. Newly initiated Suppressed VL Unsuppressed VL Morning visit (n = 10) Evening visit (n = 6) Morning visit (n = 12) Evening visit (n = 8) Morning visit (n = 11) Evening visit (n = 9) Palatability No palatability issues 5 4 10 3 8 8 Spitting 1 0 1 0 2 0 Vomiting 1 0 0 0 0 1 Cough/moans 2 0 0 4 1 0 Refusal 1 2 1 1 0 0 Taste distracter used (yes) 0 1 2 2 1 1 medication, but lacked the necessary skills with which to perform accurate measurements of the doses. In sev- eral instances, erroneous medication preparation appeared to lead to under-dosing. According to the IMB model (Fisher et al., 2006), information is a necess- ary condition for behavioural change, but it is not suffi- cient to bring about that change in itself. Adequate motivation to adhere to the regimen as well as behav- ioural skills to administer the medication effectively are required for behavioural change to occur. While care- givers used medication tools such as syringes and medi- cation cups to measure doses of the medications, they lacked the necessary skills required for performing accu- rate measurements, such as dose checking (especially checking that all of the contents of dissolved tablets and capsules were administered) and removing bubbles in syringes. These findings concur with other studies assessing dosing accuracy among caregivers on ART (Howard et al., 2014; Yin et al., 2008). explicitly asked why they chose to store medications in this way, one might presume that caregivers were hiding the medications from young children or from visitors. Keeping medications hidden adds to the large body of lit- erature on HIV-related stigma (Bogart, Cowgill, & Ken- nedy, 2008; Brown, Macintyre, & Trujillo, 2003; Katz et al., 2013; Rintamaki, Davis, Skripkauskas, Bennett, & Wolf, 2006; Ware, Wyatt, & Tugenberg, 2006). In this instance, perceived stigma prevented caregivers from accessing cue-based strategies, such as visibility of the medications in their homes, for reminder purposes (Marhefka et al., 2008). We also observed little to no food administered prior to treatment administration. However, the lack of food and the subsequent administration of medi- cation suggest that caregivers were willing to adminis- ter treatment without food. Context-related factors Caregivers’ willingness to adhere to ART in the absence of available resources has often been associated with what Olds, Kiwanuka, Ware, Tsai, and Haberer (2015) have referred to as the “Lazarus effect” (p. 586). In their study, based on the observable improvements in children’s health due to ART use, caregivers of children in rural Uganda were less inclined to miss treatment out of fear that the child may become ill again (Olds et al., 2015). Fur- thermore, willingness to provide treatment in the absence of food also contradicts evidence from care- givers and healthcare workers. For example, in our pre- vious work, caregivers to children in the same study area as well as doctors, nurses and counsellors reported that lack of food was one of the most important barriers to adherence to ART(Coetzee et al., 2015). While this barrier has been shown to be true in other contexts as well (Haberer & Mellins, 2009; Simoni et al., 2007; Vreeman, Wiehe, Pearce, & Nyandiko, 2008), our data suggest that this does not always mean that doses are missed. Caregivers to children living in rural areas have low levels of health literacy (Howard et al., 2014), which may contribute to misunderstandings of how best to administer the medication to children. In other research, we showed that adherence counsellors provided little information to caregivers during monthly clinic sessions, which we attributed to the rushed training that adher- ence counsellors received and the lack of follow-up and debriefing (Coetzee et al., 2015). However, caregivers to children with suppressed and unsuppressed VLs tended to use devices such as syringe nozzles less frequently than those newly initiated on ART. Thus, carelessness may also be a consequence of treatment fatigue (Bagenda et al., 2011). While most of the children took their medicines suc- cessfully, several still struggled with the palatability of the formulations. In the few instances where vomiting was observed, this may have been attributable to the lack of food in household. Other studies have shown that lack of food before ART is often accompanied by noxious side effects (Biadgilign, Deribew, Amberbir, & Deribe, 2009; Fetzer et al., 2011). Taste-masking and pill-swal- lowing interventions have contributed suggestions for References Bagenda, A., Barlow-Mosha, L., Bagenda, D., Sakwa, R., Fowler, M. G., & Musoke, P. M. (2011). Adherence to tablet and liquid formulations of antiretroviral medication for paediatric HIV treatment at an urban clinic in Uganda. Annals of Tropical Paediatrics, 31(3), 235–245. doi:10. 1179/1465328111Y.0000000025. Bain-Brickley, D., Butler, L. M., Kennedy, G. E., & Rutherford, G. W. (2011). Interventions to improve adherence to antire- troviral therapy in children with HIV infection. The Cochrane Database of Systematic Reviews, (12), CD009513. doi:10.1002/14651858.CD009513 Conclusions Bangsberg, D. R., Charlebois, E. D., Grant, R., Holodniy, M., Deeks, S., Perry, S., … Moss, A. (2003). High levels of adherence do not prevent accumulation of HIV drug resist- ance mutations. AIDS (London, England), 17(13), 1925– 1932. doi:10.1097/00002030-200309050-00011 The findings from this research demonstrate the impor- tance of understanding context-specific and age-specific factors that influence adherence to ART among infants and young children. By stratifying these factors accord- ing to the micro-, meso-, exo- and macro levels at which they occur, targeted interventions may be devel- oped. To aid accuracy during treatment administration, caregivers’ knowledge of the regimen ought to be moni- tored regularly by adherence counsellors or relevant healthcare staff. Furthermore, in order to facilitate accu- rate transfer of knowledge to caregivers, adherence coun- sellors need to receive regular follow-up training and debriefing sessions. Further research is needed to develop palatable ARV formulations for infants and young chil- dren on ART. Bangsberg, D. R., Hecht, F., Charlebois, E. D., Zolopa, A. R., Holodniy, M., Sheiner, L., … Moss, A. (2000). Adherence to protease inhibitors, HIV-1 viral load, and development of drug resistance in an indigent population. AIDS (London, England), 14(4), 357–366. Biadgilign, S., Deribew, A., Amberbir, A., & Deribe, K. (2009). Barriers and facilitators to antiretroviral medication adher- ence among HIV-infected paediatric patients in Ethiopia: A qualitative study. SAHARA-J: Journal of Social Aspects of HIV/AIDS, 6(4), 148–154. doi:10.1080/17290376.2009. 9724943 Bogart, L. M., Cowgill, B., & Kennedy, D. (2008). HIV-related stigma among people with HIV and their families: a quali- tative analysis. AIDS and Behavior. Retrieved from http:// link.springer.com/article/10.1007/s10461-007-9231-x Medication-related factors Our results demonstrated that caregivers of children on ART were knowledgeable about some aspects of the B. COETZEE ET AL. 40 strategies with which caregivers may deal with these issues (Bain-Brickley, Butler, Kennedy, & Rutherford, 2011). However, these interventions have not been shown to improve adherence to medication in children. Grant 88903 is gratefully acknowledged. Opinions expressed and conclusions arrived at are those of the authors and are not necessarily to be attributed to the NRF. The Africa Centre is funded by the Wellcome Trust, UK, under Grant #097410/Z/ These findings need to be interpreted against several limitations inherent to this research. Although necessary given the methods used, the sample was limited, as only caregivers whose co-household residents knew about the child’s medication taking were recruited. Furthermore, the presence of the PI and RA in participant homes undoubtedly biased some of the observations. Although these findings cannot be generalised, the knowledge is transferable to similar contexts. Acknowledgments Bronfenbrenner, U. (1979). The ecology of human develop- ment: Experiments by nature and design. Cambridge, MA: Harvard University Press. We would like to thank all the participants who took part in this research. We also thank Ms Nokuthula Mgenge for her assistance as research assistant. We would also like to thank the Africa Centre’s community advisory board and the Hlabisa hospital management. Brown, L., Macintyre, K., & Trujillo, L. (2003, February). Interventions to reduce HIV/AIDS stigma: What have we learned? AIDS Education and Prevention. doi:10.1521/ aeap.15.1.49.23844 Coetzee, B. J., Kagee, A., & Bland, R. M. (2015). Barriers and facil- itators to paediatric adherence to antiretroviral therapy in rural South Africa: a multi-stakeholder perspective. AIDS Care, 27 (3), 315–321. doi:10.1080/09540121.2014.967658 Disclosure statement No potential conflict of interest was reported by the authors. Davies, M.-A., Moultrie, H., Eley, B. S., Rabie, H., Van Cutsem, G., Giddy, J., … Boulle, A. (2011). Virologic failure and second-line antiretroviral Therapy in Children in South Africa—The IeDEA Southern Africa Collaboration. JAIDS Journal of Acquired Immune Deficiency Syndromes, 56(3), 270–278. doi:10.1097/QAI.0b013e3182060610 Funding Funding from the Columbia University-Southern African Fogarty AIDS International Training and Research Program (AITRP) funded by the Fogarty International Centre, National Institutes of Health under Grant # D43TW00231 and the US President’s Emergency Plan for AIDS relief (PEPFAR) through HRSA under the terms of T84HA21652 and via the Stellenbosch University Rural Medical Education Partnership Initiative (SUR- MEPI) and the National Research Foundation (NRF), under Department of Health South Africa. (2013). The South African antiretroviral treatment guidelines. South Africa. Retrieved from http://www.doh.gov.za/docs/policy/2013/ART_ Treatment_Guidelines_Final_25March2013.pdf Department of Health South Africa. (2014). National consoli- dated guidelines for the prevention of mother-to-child AIDS CARE 41 AIDS CARE 41 BMC Health Services Research, 14(1), 116. doi:10.1186/ 1472-6963-14-116 transmission of HIV (PMTCT) and the management of HIV in children, adolescents and adults. Pretoria. Retrieved from http://www.health.gov.za/index.php/2014-03-17-09-09-38/ policies-and-guidelines/category/230-2015p Marhefka, S. L., Koenig, L. J., Allison, S. M., Bachanas, P. J., Bulterys, M., Bettica, L., … Abrams, E. J. (2008). Family experiences with pediatric antiretroviral therapy: Responsibilities, barriers, and strategies for remembering medications. AIDS Patient Care and STDs, 22(8), 637– 647. doi:10.1089/apc.2007.0110 Fetzer, B., Mupenda, B., Lusiama, J., Kitetele, F., Golin, C. E., & Behets, F. (2011). Barriers to and facilitators of adherence to pediatric antiretroviral therapy in a Sub-Saharan Setting: Insights from a qualitative study. AIDS Patient Care and STDs, 25(10), 611–621. doi:10.1089/apc.2011.0083 Olds, P. K., Kiwanuka, J. P., Ware, N. C., Tsai, A. C., & Haberer, J. E. (2015). Explaining antiretroviral therapy adherence success among HIV-infected children in rural Uganda: A qualitative study. AIDS and Behavior, 19(4), 584–593. doi:10.1007/s10461-014-0924-7 Fisher, J. D., Fisher, W. A., Amico, K. R., & Harman, J. J. (2006). An information-motivation-behavioral skills model of adherence to antiretroviral therapy. Health Psychology : Official Journal of the Division of Health Psychology, American Psychological Association, 25(4), 462–473. doi:10.1037/0278-6133.25.4.462 Rintamaki, L. S., Davis, T. C., Skripkauskas, S., Bennett, C. L., & Wolf, M. S. (2006). Social stigma concerns and HIV medication adherence. AIDS Patient Care and STDs, 20 (5), 359–368. doi:10.1089/apc.2006.20.359 Fox, M. P., Ive, P., Long, L., Maskew, M., & Sanne, I. (2010). High rates of survival, immune reconstitution, and virologic suppression on second-line antiretroviral therapy in South Africa. Journal of Acquired Immune Deficiency Syndromes (1999), 53(4), 500–506. doi:10.1097/QAI.0b013e3181bcdac1 Simoni, J. M., Montgomery, A., Martin, E., New, M., Demas, P. A., & Rana, S. (2007). Adherence to antiretroviral therapy for pediatric HIV infection: A qualitative systematic review with recommendations for research and clinical manage- ment. Funding PEDIATRICS, 119(6), e1371–e1383. doi:10.1542/ peds.2006-1232 Haberer, J. E., & Mellins, C. A. (2009). Pediatric adherence to HIV antiretroviral therapy. Current HIV/AIDS Reports, 6 (4), 194–200. doi:10.1007/s11904-009-0026-8 Vreeman, R. C., Wiehe, S. E., Pearce, E. C., & Nyandiko, W. M. (2008). A systematic review of pediatric adherence to anti- retroviral therapy in low- and middle-income countries. The Pediatric Infectious Disease Journal, 27(8), 686–691. doi:10.1097/INF.0b013e31816dd325 Howard, L. M., Tique, J. A., Gaveta, S., Sidat, M., Rothman, R. L., Vermund, S. H., & Ciampa, P. J. (2014). Health literacy predicts pediatric dosing accuracy for liquid zidovudine. AIDS, 28(7), 1041–1048. doi:10.1097/QAD.0000000000000 197 Ware, N. C., Wyatt, M., & Tugenberg, T. (2006). Social relationships, stigma and adherence to antiretroviral therapy for HIV/AIDS. AIDS Care, 18(8), 904–910. doi:10. 1080/09540120500330554 Hunter, M., & Posel, D. (2012). Here to work: The socioeco- nomic characteristics of informal dwellers in post-apartheid South Africa. Environment and Urbanization, 24(1), 285– 304. doi:10.1177/0956247811433537 World Health Organisation. (2013). Consolidated ARV guide- lines 2013. Geneva: World Health Organization (Vol. 14). Retrieved from http://www.who.int/hiv/pub/guidelines/ arv2013/ Katz, I., Ryu, A., Onuegbu, A., Psaros, C., Weiser, S. D., Bangsberg, D. R., & Tsai, A. (2013). Impact of HIV-related stigma on treatment adherence: Systematic review and meta-synthesis. Journal of the International AIDS Society, 16 (3(Suppl 2)), 18640. doi:10.7448/IAS.16.3.18640 Yin, H. S., Dreyer, B. P., van Schaick, L., Foltin, G. L., Dinglas, C., & Mendelsohn, A. L. (2008). Randomized controlled trial of a pictogram-based intervention to reduce liquid medication dosing errors and improve adherence among caregivers of young children. Archives of Pediatrics & Adolescent Medicine, 162(9), 814–822. doi:10.1001/archpedi.162.9.814 Lessells, R. J., Stott, K. E., Manasa, J., Naidu, K. K., Skingsley, A., Rossouw, T., & de Oliveira, T. (2014). Implementing antiretroviral resistance testing in a primary health care HIV treatment programme in rural KwaZulu-Natal, South Africa: Early experiences, achievements and challenges.
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Increased structural connectivity in high schizotypy
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a n o p e n a c c e s s j o u r n a l Corresponding Author: Eirini Messaritaki messaritakie2@cardiff.ac.uk Handling Editor: Alex Fornito Copyright: © 2022 Massachusetts Institute of Technology Published under a Creative Commons Attribution 4.0 International (CC BY 4.0) license The MIT Press Keywords: Schizophrenia, Schizotypy, Brain networks, Structural connectivity, Tractography Keywords: Schizophrenia, Schizotypy, Brain networks, Structural connectivity, Tractography RESEARCH a n o p e n a c c e s s j o u r n a l The link between brain structural connectivity and schizotypy was explored in two healthy participant cohorts, collected at two different neuroimaging centres, comprising 140 and 115 participants, respectively. The participants completed the Schizotypal Personality Questionnaire (SPQ), through which their schizotypy scores were calculated. Diffusion-MRI data were used to perform tractography and to generate the structural brain networks of the participants. The edges of the networks were weighted with the inverse radial diffusivity. Graph theoretical metrics of the default mode, sensorimotor, visual, and auditory subnetworks were derived and their correlation coefficients with the schizotypy scores were calculated. To the best of our knowledge, this is the first time that graph theoretical measures of structural brain networks are investigated in relation to schizotypy. A positive correlation was found between the schizotypy score and the mean node degree and mean clustering coefficient of the sensorimotor and the default mode subnetworks. The nodes driving these correlations were the right postcentral gyrus, the left paracentral lobule, the right superior frontal gyrus, the left parahippocampal gyrus, and the bilateral precuneus, that is, nodes that exhibit compromised functional connectivity in schizophrenia. Implications for schizophrenia and schizotypy are discussed. Citation: Messaritaki, E., Foley, S., Barawi, K., Ettinger, U., & Jones, D. K. (2023). Increased structural connectivity in high schizotypy. Network Neuroscience, 7(1), 213–233. https://doi.org/10.1162/netn_a_00279 DOI: https://doi.org/10.1162/netn_a_00279 Supporting Information: https://doi.org/10.1162/netn_a_00279 Received: 8 July 2022 Accepted: 14 September 2022 Competing Interests: The authors have declared that no competing interests exist. Corresponding Author: Eirini Messaritaki messaritakie2@cardiff.ac.uk Handling Editor: Alex Fornito Copyright: © 2022 Massachusetts Institute of Technology Published under a Creative Commons Attribution 4.0 International (CC BY 4.0) license The MIT Press Citation: Messaritaki, E., Foley, S., Barawi, K., Ettinger, U., & Jones, D. K. (2023). Increased structural connectivity in high schizotypy. Network Neuroscience, 7(1), 213–233. https://doi.org/10.1162/netn_a_00279 DOI: https://doi.org/10.1162/netn_a_00279 Supporting Information: https://doi.org/10.1162/netn_a_00279 Received: 8 July 2022 Accepted: 14 September 2022 Competing Interests: The authors have declared that no competing interests exist. Corresponding Author: Eirini Messaritaki messaritakie2@cardiff.ac.uk Handling Editor: Alex Fornito Copyright: © 2022 Massachusetts Institute of Technology Published under a Creative Commons Attribution 4.0 International (CC BY 4.0) license The MIT Press Citation: Messaritaki, E., Foley, S., Barawi, K., Ettinger, U., & Jones, D. K. (2023). Increased structural connectivity in high schizotypy. Network Neuroscience, 7(1), 213–233. https://doi.org/10.1162/netn_a_00279 DOI: https://doi.org/10.1162/netn_a_00279 Supporting Information: https://doi.org/10.1162/netn_a_00279 Received: 8 July 2022 Accepted: 14 September 2022 Competing Interests: The authors have declared that no competing interests exist. Increased structural connectivity in high schizotypy Eirini Messaritaki1 , Sonya Foley1 , Kali Barawi2, Ulrich Ettinger3 , and Derek K. Jones1 1Cardiff University Brain Research Imaging Centre (CUBRIC), School of Psychology, Cardiff University, Cardiff, UK 2School of Medicine, Cardiff University, Cardiff, UK 3Department of Psychology, University of Bonn, Bonn, Germany Eirini Messaritaki1 , Sonya Foley1 , Kali Barawi2, Ulrich Ettinger3 , and Derek K. Jones1 1Cardiff University Brain Research Imaging Centre (CUBRIC), School of Psychology, Cardiff University, Cardiff, UK 2School of Medicine, Cardiff University, Cardiff, UK 3Department of Psychology, University of Bonn, Bonn, Germany INTRODUCTION Schizophrenia is a serious psychiatric disorder of unknown aetiology, while schizotypy encompasses a set of stable personality traits that are thought to reflect the subclinical expres- sion of schizophrenia. Specifically, people with high levels of schizotypy show cognitive and neural patterns resembling the deficits observed in schizophrenia (Ettinger et al., 2014, 2015). Studying schizotypy is, therefore, important for a number of reasons. Firstly, given that partly overlapping factors underlie both conditions (Fanous et al., 2007), studying schizotypy can shed light on the aetiology of schizophrenia. In addition, people with high schizotypy suffer from lower social, educational, and professional levels of functioning, and high levels of dis- tress (Barrantes-Vidal et al., 2010; Cohen & Davis, 2009); therefore, studying schizotypy can help us better understand these problems and develop interventions to ameliorate them. More- over, studying schizotypy can reveal characteristics (in the brains of healthy high-schizotypy participants) that protect or compensate against schizophrenia; those characteristics may be used to inform the development of novel treatments (Ettinger et al., 2014, 2015). Finally, the study of schizotypy is free from any confounds that stem from pharmacological interven- tions, and as such it is of value to both clinical and nonclinical research. Given this similarity of schizophrenia and schizotypy, in building our aims for this study, we looked to the topological properties of the structural and functional brain networks of schizo- phrenia patients. The functional brain networks of schizophrenia patients are compromised and generally exhibit lower connectivity and less efficient organisation, with few exceptions, compared to healthy individuals. For example, a meta-analysis of 13 studies of functional whole-brain net- works, in which the functional connectivity was measured via functional MRI (fMRI), magne- toencephalography (MEG), or electroencephalography (EEG), showed significantly lower small-worldness and measures of local organisation (Kambeitz et al., 2016). Additional studies that were not included in this meta-analysis corroborate the findings. For example, fMRI- measured functional connectivity was lower within the default mode network in schizophre- nia patients compared to healthy controls (Pankow et al., 2015). Schizophrenia patients showed lower activation compared to healthy controls in the lateral frontal cortex bilaterally, in the left basal ganglia and the cerebellum (Landin-Romero et al., 2014). The lateral default mode network exhibited lower functional connectivity with the sensorimotor cortex, but higher connectivity with association areas in schizophrenia patients (Wang et al., 2015). Clustering coefficient: A measure of how interconnected the neighbours of that node are. AUTHOR SUMMARY We investigated the topological organisation of the structural brain networks, derived via tractography, of healthy participants with varying schizotypy scores, in two independent cohorts comprising 140 and 115 participants, respectively. In order to capture the possible impact of myelination or axonal density on the structural networks, we used the inverse radial diffusivity as the edge weight. We observed that participants with the higher schizotypy scores exhibited higher node degree and clustering coefficient in the sensorimotor and default mode networks. This provides evidence of stronger structural connectivity in participants with higher schizotypy, which could indicate a possible protective mechanism against schizophrenia, or imply that structural alterations observed in schizophrenia are a correlate or consequence of the disease rather than its cause. Copyright: © 2022 Massachusetts Institute of Technology Published under a Creative Commons Attribution 4.0 International (CC BY 4.0) license Copyright: © 2022 Massachusetts Institute of Technology Published under a Creative Commons Attribution 4.0 International (CC BY 4.0) license The MIT Press Increased structural connectivity in high schizotypy Tractography: A computational method that uses diffusion-MRI data to reconstruct and visualize the white matter tracts of the brain. INTRODUCTION The anterior and posterior default mode networks exhibited higher and lower connectivity with the right control and lateral visual networks, respectively, in schizophrenia patients (Wang et al., 2015). Analysis of dynamic functional connectivity showed that schizophrenia patients activated the default mode network less frequently than healthy controls, but each activation lasted 4–5 s longer than in the healthy controls (Kottaram et al., 2019). Altered func- tional connectivity was observed in sensorimotor, visual, auditory, social, high level cognitive, and motor processing nodes in schizophrenia patients compared to controls (Berman et al., 2016; Jimenez et al., 2019; Kaufmann et al., 2015), with most of the alterations indicating lower functional connectivity. Schizophrenia patients exhibited lower clustering coefficient, global efficiency and local efficiency, and higher characteristic path length of functional net- works (Zhu et al., 2016). Finally, compared to healthy controls, significant hypoconnectivities were observed between seed regions and the areas in the auditory, default mode, self- referential, and somatomotor network in schizophrenia patients (Li et al., 2019). The structural brain networks of schizophrenia patients have been shown predominantly to have a less central and less efficient organisation compared to those of healthy controls, but Network Neuroscience Network Neuroscience Network Neuroscience 214 Increased structural connectivity in high schizotypy there are some exceptions. For example, the global efficiency, local efficiency, clustering coef- ficient, and mean connectivity strength of structural networks weighted by the connectivity probability were significantly lower in schizophrenia patients compared to controls, while the mean betweenness centrality was significantly higher (Shon et al., 2018). Significantly lon- ger path lengths of frontal and temporal regions were observed in structural networks (with edges weighted by the magnetisation-transfer ratio) of schizophrenia patients compared to controls, and frontal hubs of patients showed a significant reduction of betweenness centrality (van den Heuvel et al., 2010). Structural networks of schizophrenia patients with auditory visual hallucinations showed higher characteristic path length compared with those of healthy controls (Zhu et al., 2016). Additionally, studies have shown that schizophrenia patients have higher radial diffusivity and mean diffusivity in some white matter tracts (left thalamo-occipital tracts, right uncinate fascicle, right middle longitudinal fascicle) compared to healthy controls (Abdul-Rahman et al., 2011; Joo et al., 2018). METHODS Two datasets were collected at the Cardiff University Brain Research Imaging Centre (CUBRIC) between 2014 and 2016 (Cardiff data) and at the University of Munich between 2009 and 2011 (Munich data). The scanners and sequences used were different, and the sensitivity of the data to microstructural parameters is, therefore, different. For that reason, the two datasets were analysed separately. INTRODUCTION Another study showed that schizophrenia patients have higher mean diffusivity in the right anterior thalamic radiations, the forceps minor, the bilateral infe- rior fronto-occipital fasciculus, the left superior longitudinal fasciculus, and the bilateral unci- nate (Clark et al., 2011). Radial diffusivity, although nonspecific in principle, can be influenced by myelination and/or axonal density (Chen et al., 2009; Cykowski et al., 2010; Jones et al., 2013), with higher values potentially indicating lower myelination or axonal density. In this study we used tractography-derived structural brain networks to investigate possible differences in the organisation of the brain networks of healthy participants with varying schi- zotypy scores. Various microstructural metrics, such as the number of reconstructed stream- lines or the fractional anisotropy, have been used in past studies to assign connectivity strength to structural network edges (Caeyenberghs et al., 2016; Messaritaki et al., 2019, 2021). In this study, in order to capture the possible impact of altered myelination or axonal density on the strength of the connections, we used the inverse radial diffusivity to assign edge weights. The aims of the study were to explore whether previously reported alterations of white matter con- nectivity in schizophrenia could also be observed in relation to high-schizotypy scores, thereby providing a test of the (dis)continuity between high schizotypy and schizophrenia. 88/netn_a_00279.pdf by guest on 13 April 2023 Downloaded from http://direct.mit.edu/netn/article-pdf/7/1/213/2072088/netn_a_00279.pdf by guest on 13 April 2023 Munich data. One hundred fifteen healthy participants (55 female, aged 18–50 years, mean age = 27.6 years, SD = 8.0 years) were recruited through advertisements in and around Munich, Germany. All experimental procedures were approved by the research ethics com- mittee of the Faculty of Medicine at the University of Munich, in accordance with the current division of the Declaration of Helsinki. Participants were included after thorough clinical screening and only if they did not meet any of the exclusion criteria: any DSM-IV Axis I dis- orders, first-grade relatives with psychotic disorders, former or current neurological disorders, current physical conditions, current medication except for contraceptives, uncorrected visual impairments. Further inclusion criteria were age between 18 and 55 and German as a first language. All subjects volunteered to take part in the study, gave written informed consent, and received financial compensation for their participation. All participants completed the German version (Klein et al., 1997) of the SPQ (Raine, 1991) to yield their total schizotypy score. In the Munich data, the SPQ subscales showed acceptable to good reliability; the Cronbach’s alpha for the total score was 0.882. High-resolution T1-weighted structural images were acquired on a 3T MAGNETOM Verio scanner (Siemens, Erlangen, Germany) using a 12-channel head matrix Rx-coil. A three- dimensional MPRAGE sequence was used, with repetition time TR = 2,400 ms, echo time TE = 3.06 ms, flip angle = 9° with 160 slices, slice thickness = 1.0 mm, voxel size = 1.0 × 1.0 × 1.0 mm, FOV = 256 mm. Diffusion-weighted data were acquired using a diffusion- weighted interleaved spin-echo, echo planar imaging sequence with the following parameters: 25 axial slices; slice thickness = 5.2 mm; TE = 107 ms; TR = 5 s; gap between slices = 5.2 mm; FOV = 230 mm; matrix = 128 × 128; flip angle = 90; bandwidth = 1,395 Hz/Px; voxel size = 1.8 × 1.8 × 5.2 mm. Diffusion gradients were applied in 20 directions with b = 1,000 s/mm2, with each image acquired three times. Three images with no diffusion weighting were also acquired. Data Collection Cardiff data. One hundred forty healthy participants (92 female, 19–55 years old, mean = 24.7 years, SD = 5.0 years) were recruited in and around Cardiff, UK. All procedures were given ethical approval by the Cardiff University School of Psychology Ethics Committee. All participants gave written informed consent before taking part and received financial compen- sation for their participation. All participants had normal or corrected-to-normal vision and had no history of neurological or neuropsychiatric disorders. All participants completed the English version of the Schizotypal Personality Questionnaire (SPQ) (Raine, 1991), through which their total schizotypy score was calculated. The schizo- typy score is the sum score of nine subscales: ideas of reference, odd beliefs or magical Network Neuroscience 215 Increased structural connectivity in high schizotypy thinking, unusual perceptual experiences, suspiciousness, excessive social anxiety, no close friends, constricted affect, odd or eccentric behaviour, odd speech. The higher the schizotypy score, the more pronounced the schizotypal traits in a participant. For the questionnaire, ade- quate internal consistency and criterion validity have been demonstrated (Fonseca-Pedrero et al., 2018; Klein et al., 2001). In the Cardiff data, the SPQ subscales showed acceptable to good reliability; the Cronbach’s alpha for the total score was 0.815. MRI was carried out on a GE Signa HDx 3T scanner (GE Healthcare, Milwaukee, WI). T1- weighted structural data were acquired using an axial three-dimensional fast spoiled gradient recalled sequence with the following parameters: TR = 8 ms, TE = 3 ms, TI = 450 ms; flip angle = 20°; voxel size = 1 mm; field of view (FOV) ranging from 256 × 192 × 160 mm3 to 256 × 256 × 256 mm3 (anterior-posterior/left-right/superior-inferior). The T1 images were downsampled to 1.5-mm isotropic resolution. Diffusion-weighted MRI data were acquired using a peripherally cardiac-gated sequence with b = 1,200 s/mm2, TR = 20 s, TE = 90 ms, voxel size 2.4 × 2.4 × 2.4 mm3, zero slice gap, FOV = 230 mm. Data were acquired along 30 unique and isotropically distributed gradient orientations. Three images with no diffusion weighting were also acquired. The diffusion images were coregistered to the T1-weighted images and corrected for head movement and eddy current distortions. Free-water correction was also performed (Metzler-Baddeley et al. 2012; Pasternak et al., 2009). Parcellation and Network Construction To validate our analysis against the use of different cortical/subcortical parcellations (Parker et al., 2014), two atlases were employed to identify the areas of the cerebrum that formed the nodes of the structural brain areas: (i) the AAL atlas (Tzourio-Mazoyer et al., 2002), which comprises 90 areas, and (ii) the Desikan–Killiany atlas (Desikan et al., 2006), which comprises 84 areas. FreeSurfer (https://surfer.nmr.mgh.harvard.edu) was used to parcellate the T1- weighted anatomical scans (Fischl et al., 2002, 2004) and the Desikan–Killiany atlas was used to identify the 84 areas of the cerebrum that formed the nodes of the structural brain networks. The AAL parcellation was calculated using ExploreDTI 4.8.6 (Leemans et al., 2009). Each dataset was analysed with both atlas parcellations. However, ExploreDTI failed to give a reli- able parcellation for the Munich data, possibly due to the lower resolution of the MRI images in that dataset. For that reason, the subsequent analysis for the Munich data was done only using the Desikan–Killiany atlas. In order to exclude tracts that are not reliable due to having been recovered with a small number of streamlines, we applied a threshold NSthr and excluded edges with fewer than NSthr streamlines. Because that threshold is arbitrary (Buchanan et al., 2020), we repeated the anal- ysis for thresholds that correspond to mean network sparsity over participants in the range of 0.85 to 0.25. Edges that survived thresholding were weighted with the inverse mean radial diffusivity calculated along the voxels spanning the corresponding white matter tracts. Networks were constructed for the auditory, sensorimotor, visual, and default mode systems, that is, for sub- networks that have consistently been shown to be altered in schizophrenia patients compared to controls. Three graph theoretical metrics, namely the mean node degree, mean clustering coefficient and mean nodal strength, were calculated for all networks using the Brain Connec- tivity Toolbox (Rubinov & Sporns, 2010). These metrics provide insight about different aspects of the topological organisation of the brain networks. Specifically, the node degree and the nodal strength measure the connectivity of a node within the network, while the clustering coefficient measures the connectivity within the neighbours of each node. We also note that the node degree is independent of the edge weighting, while the clustering coefficient and the nodal strength do depend on it. Sparsity: The number of edges in the network divided by the total possible edges in it. Parcellation and Network Construction In order to identify any graph theoretical metrics that were correlated with each other, correlation coefficients were calculated between graph theoretical metrics for each subnetwork. The mean nodal strength exhibited correlation coefficients with absolute values higher than 0.85 with the mean node degree, and for that reason it was removed from the analysis. Node degree: The number of edges that connect the node to the rest of the network. Nodal strength: The sum of the weights of the edges connected to the node. Tractography Probabilistic, anatomically constrained streamline tractography was performed using MRtrix (Tournier et al., 2019), employing the iFOD2 algorithm (Smith et al., 2012, 2015; Tournier Network Neuroscience Network Neuroscience 216 Increased structural connectivity in high schizotypy et al., 2010). The maximum angle between successive steps was 50°, the minimum and max- imum streamline lengths were 30 mm and 25 0mm, respectively, and the FOD amplitude cut- off was 0.06. Two million streamlines were generated for each participant, with the seed points on the interface between grey matter and white matter. Visual inspection of the tractograms was performed to verify that the white matter was adequately covered and that streamlines did not extend outside of the white matter. Nodal strength: The sum of the weights of the edges connected to the node. Demographic Information The schizotypy scores (i.e., SPQ total scores) of the participants in the Cardiff data ranged between 0 and 43 (mean: 12.4, SD: 10.1), while for the Munich data they ranged between 0 and 35 (mean: 8.2, SD: 7.0). No statistically significant correlations were found between schizotypy score and age or sex, or between age and sex of the participants, for the Cardiff or the Munich data. The correlation coefficients and p values between these variables are given in Table 1 for the two datasets. Statistical Analysis Correlation coefficients and the corresponding p values were calculated between the demo- graphic characteristics of the participants, that is, schizotypy score, age, and sex, to verify that those were not correlated with each other. Network Neuroscience Network Neuroscience 217 Increased structural connectivity in high schizotypy Partial correlation coefficients and the corresponding p values were calculated between the schizotypy score and the graph theoretical metrics of the participants, correcting for age and sex. The mean Cook’s distance (Cook, 1977) was used to identify outliers, which were subsequently investigated further. Data points with Cook’s distance greater than four times the mean Cook’s distance were excluded from further analysis, because they either had isolated (unconnected) nodes in their structural connectivity matrices, or were outliers in the degree distribution. The p values that resulted from the correlation analysis were corrected for multiple comparisons using the false discovery rate algorithm (Benjamini & Yekutieli, 2001), applied over all graph theoretical measures of all subnetworks. In order to understand whether any correlations between the graph theoretical metrics and the schizotypy score are due to all the nodes of each subnetwork, or whether they are driven by a few specific nodes of it, we calculated the correlation coefficients between the schizotypy score and the node-specific graph theoretical metrics for those graph theoretical metrics that exhibited statistically significant correlations. We considered that a node is driving the corre- lations if the related p value was lower than 0.05. The analysis pipeline is shown in Figure 1. Cardiff Data: Desikan–Killiany Atlas Significant positive correlations that survived multiple comparison correction and persisted across NSthr were identified between schizotypy score and the mean node degree of the sen- sorimotor network, and between the schizotypy score and the mean clustering coefficient of the default mode network. The related correlation coefficients and p values are shown in Figure 5. The corresponding scatter plots are shown in Figure 6. Cardiff Data: AAL Atlas Significant positive correlations that survived multiple comparison correction and persisted across NSthr were identified between schizotypy score and the mean node degree and mean clustering coefficient for the sensorimotor and the default mode networks. The correlation coefficients and p values across NSthr are shown in Figure 3. Scatter plots for these correlations are shown in Figure 4. Network Analysis The NSthr required to achieve a given mean sparsity for the whole-brain network in the three cases investigated is shown in Figure 2. In order to achieve sparsities as low as 0.25, we use NSthr of [0, 160] for the Cardiff/AAL pair, [0, 60] for the Cardiff/Desikan–Killiany pair, and [0, 120] for the Munich/Desikan–Killiany pair. In the following subsections, in addition to show- ing the correlation coefficients and p values for all these thresholds, we also show scatter plots for the networks that correspond to a sparsity of 0.3, that is, NSthr = 86 for the Cardiff/AAL case, NSthr = 36 for the Cardiff/Desikan–Killiany case, and NSthr = 55 for the Munich/Desikan– Killiany case. Figure 1. Analysis pipeline. Figure 1. Analysis pipeline. 218 Network Neuroscience Increased structural connectivity in high schizotypy Table 1. Correlation coefficients and p values (the latter in parentheses) between the demographic data in the two datasets Table 1. Correlation coefficients and p values (the latter in parentheses) between the demographic data in the two datasets Sz score vs. age Sz score vs. sex Age vs. sex Cardiff data 0.05 (0.59) −0.10 (0.25) −0.07 (0.43) Munich data −0.14 (0.15) 0.17 (0.06) −0.10 (0.29) Munich Data: Desikan–Killiany Atlas Significant positive correlations were identified between the schizotypy score and the mean node degree of the sensorimotor network, and the mean node degree of the visual network. These correlations were nominally significant, that is, did not survive multiple comparison cor- rection, but persisted across NSthr. The correlation coefficients and p values are shown in Figure 7. The corresponding scatter plots are shown in Figure 8. Figure 2. NSthr required to achieve a given sparsity in the whole-brain structural networks for the three dataset/atlas combinations. Figure 2. NSthr required to achieve a given sparsity in the whole-brain structural networks for the three dataset/atlas combinations. Network Neuroscience Network Neuroscience 219 Increased structural connectivity in high schizotypy Figure 3. Correlation coefficients and p values between graph theoretical metrics and schizotypy score for the Cardiff data and the AAL atlas. Only the two subnetworks that showed persistent cor- relations across thresholds are shown. Coloured markers indicate correlation coefficients and p values that survived multiple comparison correction, while blank (fainter coloured) markers indicate correlations that were nominally significant ( p < 0.05), but did not survive multiple comparison correction. Figure 3. Correlation coefficients and p values between graph theoretical metrics and schizotypy score for the Cardiff data and the AAL atlas. Only the two subnetworks that showed persistent cor- relations across thresholds are shown. Coloured markers indicate correlation coefficients and p values that survived multiple comparison correction, while blank (fainter coloured) markers indicate correlations that were nominally significant ( p < 0.05), but did not survive multiple comparison correction. Node-Specific Analysis Cardiff data, AAL atlas. The node-specific analysis for the sensorimotor network showed that the correlations between the schizotypy score and the mean node degree are driven by the right middle frontal gyrus and the right postcentral gyrus, while the correlations between the Figure 4. Scatter plots for the correlations observed in the Cardiff data for the AAL atlas parcella- tion for NSthr = 86. The best fit line and its 95% confidence interval are also shown. Figure 4. Scatter plots for the correlations observed in the Cardiff data for the AAL atlas parcella- tion for NSthr = 86. The best fit line and its 95% confidence interval are also shown. Network Neuroscience Network Neuroscience 220 Increased structural connectivity in high schizotypy Figure 5. Correlation coefficients and p values between graph theoretical metrics and schizotypy score for the Cardiff data and the Desikan–Killiany atlas. Only the two subnetworks that show per- sistent correlations across thresholds are shown. Coloured markers indicate correlation coefficients and p values that survived multiple comparison correction, while blank (fainter coloured) markers indicate correlations that were nominally significant ( p < 0.05), but did not survive multiple com- parison correction. Figure 5. Correlation coefficients and p values between graph theoretical metrics and schizotypy score for the Cardiff data and the Desikan–Killiany atlas. Only the two subnetworks that show per- sistent correlations across thresholds are shown. Coloured markers indicate correlation coefficients and p values that survived multiple comparison correction, while blank (fainter coloured) markers indicate correlations that were nominally significant ( p < 0.05), but did not survive multiple com- parison correction. schizotypy score and the mean clustering coefficient are driven by the right precentral gyrus and the right postcentral gyrus. For the default mode network, the correlations between schi- zotypy score and mean node degree are driven by the left middle frontal gyrus, the right infe- rior frontal gyrus (opercular part), the left inferior frontal gyrus (orbital and triangular parts), the left and right middle cingulate, the left hippocampus, and the left thalamus. Also, for the default mode network, the correlations between schizotypy score and the mean clustering coefficient were driven by the left and right superior frontal gyrus, the right inferior frontal gyrus (triangular part), the left anterior cingulate, the left and right middle cingulate, the left posterior cingulate, the left parahippocampal area, the left middle occipital gyrus, the left Figure 6. Node-Specific Analysis Scatter plots for the correlations observed in the Cardiff data for the Desikan–Killiany parcellation for NSthr = 36. The best fit line and its 95% confidence interval are also shown. Figure 6. Scatter plots for the correlations observed in the Cardiff data for the Desikan–Killiany parcellation for NSthr = 36. The best fit line and its 95% confidence interval are also shown. Network Neuroscience Network Neuroscience 221 Increased structural connectivity in high schizotypy Figure 7. Correlation coefficients and p values between graph theoretical metrics and schizotypy score for the Munich data and the Desikan–Killiany atlas. Only the sensorimotor and the visual subnetworks showed persistent, nominally significant correlations across thresholds. Figure 7. Correlation coefficients and p values between graph theoretical metrics and schizotypy score for the Munich data and the Desikan–Killiany atlas. Only the sensorimotor and the visual subnetworks showed persistent, nominally significant correlations across thresholds. angular gyrus, and the left and right precuneus. These areas are shown in Figure 9. Their correlation coefficients and respective p values are given in Table S1 in the Supporting Infor- mation section. Cardiff data, Desikan–Killiany atlas. The node-specific analysis showed that the correlations between the schizotypy score and the mean node degree of the sensorimotor network were driven by the left paracentral lobule, the right postcentral gyrus, and the right superior frontal gyrus. For the default mode network, the correlations between schizotypy score and the mean clustering coefficient were driven by the left caudal middle frontal gyrus, the left isthmus cin- gulate, the left lateral orbitofrontal gyrus, the left and right parahippocampal area, the left and Figure 8. Scatter plots for the correlations observed in the Munich data for the Desikan–Killiany parcellation for NSthr = 55. The best fit line and its 95% confidence interval are also shown. Figure 8. Scatter plots for the correlations observed in the Munich data for the Desikan–Killiany parcellation for NSthr = 55. The best fit line and its 95% confidence interval are also shown. Network Neuroscience Network Neuroscience 222 222 Increased structural connectivity in high schizotypy Figure 9. Cardiff data: AAL atlas. Nodes that drive the correlations between the schizotypy score and the sensorimotor mean node degree (top left), sensorimotor mean clustering coefficient (top right), default mode mean node degree (bottom left), and default mode mean clustering coeffi- cient (bottom right). Node-Specific Analysis The large markers indicate the nodes of each network that drive the corre- lations; these nodes are also labelled. The small markers indicate the remaining nodes of each network. Downloaded from http://direct.mit.edu/netn/article pdf/7/1/213/2072088/netn_a_00279.pdf by guest on 13 April 2023 Downloaded from http://direct.mit.edu/netn/article-pdf/7/1/213/2072088/netn_a_00279.pdf by guest on 13 April 2023 2088/netn_a_00279.pdf by guest on 13 April 2023 Figure 9. Cardiff data: AAL atlas. Nodes that drive the correlations between the schizotypy score and the sensorimotor mean node degree (top left), sensorimotor mean clustering coefficient (top right), default mode mean node degree (bottom left), and default mode mean clustering coeffi- cient (bottom right). The large markers indicate the nodes of each network that drive the corre- lations; these nodes are also labelled. The small markers indicate the remaining nodes of each network. right precuneus, the left and right thalamus, the right hippocampus, the right pars triangularis, and the right rostral anterior cingulate. These areas are shown in Figure 10. Their correlation coefficients and p values shown in Table S1 in the Supporting Information section. Munich data, Desikan–Killiany atlas. The node-specific analysis showed that the correlations between schizotypy score and the mean node degree of the sensorimotor network were driven by the left paracentral lobule, the left and right postcentral gyrus, the left supramarginal gyrus, the right precentral gyrus, and the right superior frontal gyrus. The correlations between Network Neuroscience 223 Increased structural connectivity in high schizotypy Figure 10. Cardiff data: Desikan–Killiany atlas. Nodes that drive the correlations between the schizotypy score and the sensorimotor mean node degree (left) and default mode mean clustering coefficient (right). The large markers indicate the nodes of each network that drive the correla- tions; these nodes are also labelled. The small markers indicate the remaining nodes of the network. Downloaded from http://direct.mit.edu/netn/article-pdf/7/1/213/2072088/netn_a_00279.pdf by guest on 13 April 2023 Figure 10. Cardiff data: Desikan–Killiany atlas. Nodes that drive the correlations between the schizotypy score and the sensorimotor mean node degree (left) and default mode mean clustering coefficient (right). The large markers indicate the nodes of each network that drive the correla- tions; these nodes are also labelled. The small markers indicate the remaining nodes of the network. schizotypy score and the mean node degree of the visual network were driven by the left lin- gual gyrus, the right cuneus, and the right fusiform area. These areas are shown in Figure 11. DISCUSSION We used graph theoretical metrics to investigate the topology of the structural brain networks of healthy participants with a range of schizotypy scores. To the best of our knowledge, this is the first time these properties are investigated in relation to schizotypy. We used two large datasets, comprising 140 and 115 participants, respectively, collected at different locations, in different MRI scanners and with different scanning parameters. Our results provide evidence that higher levels of schizotypy in healthy participants relate to higher structural connectivity in the sensorimotor and default mode networks, with some increases also possibly present in the visual network. No evidence of any alterations was found for the auditory network. The edges of the networks were weighted by the inverse radial dif- fusivity, which partly reflects differences in myelination or axon density (Chen et al., 2009; Cykowski et al., 2010). Both myelination and axon density support effective communication in the brain (Drakesmith et al., 2019; Lebel & Deoni, 2018). The edge weighting chosen impacts our results because the clustering coefficient (which exhibited statistically significant correlations with the schizotypy score for the sensorimotor and default mode network) depends on the values of the edges. On the other hand, the node degree (which also exhibited statistically significant correlations with the schizotypy score) is independent of the edge weights and is only impacted by the number of edges. This means that had we used a different measure to weight the edges (such as, for example, the number of streamlines or the fractional anisotropy of the diffusion tensor, which are sometimes used in network studies), the results pertaining to the node degree would have been the same. The positive correlations observed between the schizotypy score and the graph theoretical metrics of the sensorimotor network were replicated across the two datasets and atlases. Even though they only reached nominal statistical significance in the Munich data, they were per- sistent across a large range of values of NSthr. On the other hand, the positive correlations observed for the default mode network were not replicated in the Munich data. This could be due to the slightly smaller sample or the lower resolution of the Munich data, or the fact that there were fewer diffusion directions acquired in the diffusion data, as well as a lower b-value—the latter two facts can impact the calculation of the radial diffusivity. Node-Specific Analysis Their correlation coefficients and p values shown in Table S1 in the Supporting Information section. The visualizations of the nodes were done using BrainNet Viewer (Xia et al., 2013). 2088/netn_a_00279.pdf by guest on 13 April 2023 Figure 11. Munich data: Desikan–Killiany atlas. Nodes that drive the correlations between the schizotypy score and the sensorimotor mean node degree (left) and visual mean node degree (right). The large markers indicate the nodes of each network that drive the correlations; these nodes are also labelled. The small markers indicate the remaining nodes of the network. Figure 11. Munich data: Desikan–Killiany atlas. Nodes that drive the correlations between the schizotypy score and the sensorimotor mean node degree (left) and visual mean node degree (right). The large markers indicate the nodes of each network that drive the correlations; these nodes are also labelled. The small markers indicate the remaining nodes of the network. Network Neuroscience 224 Increased structural connectivity in high schizotypy DISCUSSION As mentioned in the Introduction, a pattern of lower functional connectivity has been reported in the literature for schizophrenia patients compared to healthy controls, except for some evidence of higher connectivity in the default mode network. This higher connectivity, however, can be responsible for a failure to completely deactivate the default mode network during tasks (as happens in healthy participants; Raichle et al., 2001), which can lead to com- promised task performance in schizophrenia. Structural connectivity has also been observed to be lower in schizophrenia, although the findings are less coherent, most likely due to dif- ferences in the analysis pipelines used. In this context, there are two possible interpretations for our results. The first possible interpretation is that our results support the notion that higher structural connectivity (in the presence of higher schizotypy scores) is a protective characteristic against schizophrenia. Specifically, the higher structural connectivity in the sensorimotor network of healthy high-schizotypy participants could be enhancing communication among sensorimotor regions, that is, regions implicated in action observation and imitation (Caspers et al., 2010), which are functions crucial for successful social interactions. Additionally, the higher structural connectivity in the default mode network could be leading to more effective communication among regions that are responsible for the emotional engagement during social interactions and the understanding others’ mental states (Li et al., 2014), or enhancing self-referential acti- vation (Davey et al., 2016). This more effective communication could be protecting these participants from the development of schizophrenia. This interpretation also indicates that structural deficiencies in the sensorimotor and default mode networks should be further inves- tigated as potential causes of schizophrenia. We note that such potential compensatory effects have been previously reported. For example, increased frontal grey matter and higher frontal activity in schizotypal personality disorder have been suggested as a possible reason for the lower magnitude of cognitive and deficit symptoms in the disorder compared to schizophrenia (Siever et al., 2002; Siever & Davis, 2004). At the same time, we also note that schizophrenia is normally diagnosed in the late teenage years to the early 20s in men and in the late 20s to early 30s in women. Some participants in the two samples we used were under 30 years old at the time that the data were acquired. DISCUSSION A recent study (Wang et al., 2020a, 2020b) showed that high-schizotypy participants exhib- ited increased white matter connectivity probability between the right middle frontal gyrus and the right insula, and within the default mode network. Higher strength values were found between the left superior frontal gyrus and the right rolandic operculum area, and between the right superior frontal gyrus and the right medial superior frontal gyrus (Wang et al., 2020a, 2020b). This pattern of higher structural connectivity in high schizotypy agrees with our results. The same study also showed that this enhanced structural connectivity appears alongside lower static functional connectivity in the sensorimotor and default mode networks in the higher schizotypy participants. While the general pattern of higher white matter connec- tivity in relation to higher schizotypy agrees with our finding, we note that differences between the studies include that the study of Wang et al. (2020a, 2020b) had participants with higher schizotypy scores than the ones in our study, they did not use graph theory in their analysis, and their connectivity measure is more closely related to the number of streamlines rather than to the myelination or to axonal density in the white matter tracts that we used. Other studies (Hazlett et al., 2012; Nakamura et al., 2005; Pfarr & Nenadic, 2020) have shown altered microstructural properties in white matter tracts in various schizotypy dimen- sions. For example, reduced fractional anisotropy (FA) of the cingulum bundle and the unci- nate fasciculus was related to symptom severity in schizotypal personality disorder (Hazlett Network Neuroscience 225 Increased structural connectivity in high schizotypy et al., 2011, 2012). Also, the FA of the right superior longitudinal fasciculus (SLF) was nega- tively correlated with disorganised schizotypy (Pfarr & Nenadic, 2020). In the same study (Pfarr & Nenadic, 2020), the axial diffusivity of the right SLF, right anterior thalamic radiation (ATR), right uncinate fasciculus (UF), and forceps minor was positively correlated with negative schi- zotypy. Finally, the mean diffusivity of the right UF, the right ATR, and the forceps minor was positively correlated with negative schizotypy. It should be pointed out that these results per- tain to specific white matter tracts, in contrast to our results which pertain to the topological organisation of the entire brain. Those studies also predominantly refer to specific schizotypy dimensions rather than overall schizotypy. DISCUSSION Therefore, some of the higher schizotypy participants in our samples may, at some time after the data were acquired, develop schizophrenia, which would indicate that the increased structural connectivity in the sensorimotor and default mode networks could not fully protect them from developing the disease. A second possible interpretation is that the white matter structural alterations that have been reported in schizophrenia (Abdul-Rahman et al., 2011; Joo et al., 2018; Shon et al., 2018; van den Heuvel et al., 2010; Zhu et al., 2016) may be the pathophysiological correlate or conse- quence of worsening schizophrenia symptoms and/or pharmacological treatment, particularly in chronic schizophrenia, rather than the cause of the disease. This is in agreement with the dysconnection hypothesis (Friston et al., 2016), indicating that functional deficiencies precede structural ones in the disease. Network Neuroscience 226 Increased structural connectivity in high schizotypy Our findings relating to the nodes of the subnetworks that drive the higher structural con- nectivity can also be put in the context of existing findings in the literature. We note that there were some similarities and some differences in the nodes driving the correlations for the three dataset/atlas pairs. For example, the right postcentral gyrus drove the correlations between schizotypy score and mean node degree for the sensorimotor network in all three cases (Cardiff/AAL, Cardiff/Desikan–Killiany, and Munich/Desikan–Killiany), while the left paracen- tral lobule and right superior frontal gyrus drove those correlations for the two cases (Cardiff/ Desikan–Killiany and Munich/Desikan–Killiany). Additionally, the left parahippocampal gyrus and the left and right precuneus drove the correlations between the schizotypy score and the mean clustering coefficient of the default mode network for both the Cardiff/AAL and Cardiff/Desikan–Killiany cases. We now discuss the nodes that consistently drove our observed correlations. s o . n t - l , l - s - y d t e f r - , t - - - l - l h n n Downloaded from http://direct.mit.edu/netn/article-pdf/7/1/213/2072088/netn_a_00279.pdf by guest on 13 April 2023 The right postcentral gyrus has been shown to have lower functional connectivity with its left counterpart in schizophrenia patients (Kaufmann et al., 2015). It has also been shown to have lower grey matter density in schizophrenia patients (Glahn et al., 2008; Li et al., 2015). Additionally, the global brain functional connectivity of the postcentral gyrus was lower in adolescent-onset schizophrenia patients compared to controls (Zhao et al., 2022). DISCUSSION The left paracentral lobule was found to have lower regional homogeneity, a quantity that reflects local functional connectivity and integration of information processing in adolescent- onset schizophrenia patients (Wang et al., 2018). It was also shown to have lower cortical thickness in psychosis patients compared to healthy participants (van Lutterveld et al., 2014), along with the left pars orbitalis, the right fusiform gyrus, and the right inferior temporal gyrus. The resting-state global brain functional connectivity (measured via fMRI) of the right supe- rior frontal gyrus was higher in schizophrenia patients and their siblings compared to controls (Ding et al., 2019). The right superior frontal gyrus also showed lower grey matter in schizo- phrenia patients compared to controls (Xiao et al., 2013). In healthy participants, the grey matter volume of the superior frontal gyrus correlated positively with their SPQ-measured schizotypy scores (Wang et al., 2020a, 2020b). Differential functional performance of the right superior frontal gyrus in low versus high-schizotypy individuals (albeit measured via the Unusual Experiences subscale of the short form of the Oxford and Liverpool Inventory of Feelings and Experiences, O-LIFE; Mason et al., 2005) has also been reported (Premkumar et al., 2012), where it was observed that in viewing rejection scenes, low schizotypy partici- pants activated that gyrus, while high-schizotypy participants deactivated it. The higher node degree of these brain areas (right postcentral gyrus, left paracentral lobule, and right superior frontal gyrus) within their respective subnetworks (sensorimotor and default mode) could be responsible for enhanced neural communication in higher schizotypy individ- uals. This could be protective against some of the functional deficiencies relating to these sub- networks which have been observed in schizophrenia. Abnormalities in the left parahippocampal gyrus region are a ubiquitous finding in schizo- phrenia (Friston et al., 1992). In an EEG study (Soni et al., 2020), the left parahippocampal gyrus was found to have higher power spectral density in patients compared to controls. Sev- eral structural MRI studies have also reported lower volume in the bilateral parahippocampal gyrus in schizophrenia (Honea et al., 2005; Lawrie & Abukmeil, 1998; Sim et al., 2006; Soh et al., 2015; Wright et al., 2000). The precuneus was shown to have higher connectivity with the posterior cingulate cortex in schizophrenia patients (Sendi et al., 2021). DISCUSSION The resting-state functional connectivity between Network Neuroscience 227 Increased structural connectivity in high schizotypy precuneus and hippocampus was found to have deficits in unmedicated patients with schizo- phrenia compared to healthy controls (Kraguljac et al., 2014). The functional connectivity between the precuneus and the bilateral Heschl’s gyri was found to be abnormal in first- episode schizophrenia (Guo et al., 2014). In healthy participants, the schizotypy score was positively associated with gyrification in the precuneus in a study that used the Munich data used in our study (Meller et al., 2019). The precuneus is involved in self-processing, mental imagery, episodic memory retrieval, and discrimination of self versus others (Cavanna & Trimble, 2006); all these processes might be related to inferring the intention of others, which corroborates the possible role of the precuneus for symptoms within the psychosis spectrum. The higher clustering coefficient of the left parahippocampal gyrus and the precuneus within the default mode network implies that their neighbourhoods are better connected in higher schizotypy individuals. This could be compensating for the functional abnormalities observed in these brain areas in schizophrenia. Specifically, if the nodes connected to the pre- cuneus can be used to bypass it, then any functional deficiencies of the precuneus would not affect the brain’s performance in the related functions. The differences in the nodes that drive the correlations for the different atlases used can be attributed to the fact that the two atlases allocate the cortical and subcortical areas differently, with the AAL atlas comprising 90 cortical and subcortical areas while the Desikan–Killiany atlas comprising 82 cortical and subcortical areas with the remaining two areas corresponding to the two hemispheres of the cerebellum. It has been shown that there are differences in the network sparsity (and therefore the node degree) and in the clustering coefficient as a result of the choice of atlas, for example, AAL versus Desikan–Killiany, for the same tractography methods (Parker et al., 2014). We note, however, that the study presented by Parker et al. (2014) used a different edge weight (i.e., connection density) in contrast to the inverse radial diffusivity used in our analysis. A strength of our study stems from the fact that we used two large datasets, comprising 140 and 115 participants. CONCLUSIONS We investigated the topological properties of structural brain subnetworks in healthy partici- pants of varying schizotypy scores. We found evidence of higher structural connectivity of the sensorimotor and default mode networks in participants with high-schizotypy score. This may indicate a possible protective mechanism against schizophrenia or could provide further sup- port for the hypothesis that functional (rather than structural) dysconnection could be the underlying cause of schizophrenia. ACKNOWLEDGMENTS For the purpose of Open Access, the author has applied a CC BY public copyright license to any Author Accepted Manuscript version arising from the submission. AUTHOR CONTRIBUTIONS Eirini Messaritaki: Conceptualization; Formal analysis; Funding acquisition; Investigation; Methodology; Software; Visualization; Writing – original draft; Writing – review & editing. Sonya Foley: Data curation; Formal analysis. Kali Barawi: Data curation. Ulrich Ettinger: Data curation; Methodology; Writing – review & editing. Derek K. Jones: Funding acquisition; Methodology; Supervision; Writing – review & editing. FUNDING INFORMATION Eirini Messaritaki, Wellcome Trust (https://dx.doi.org/10.13039/100010269), Award ID: 204824/Z/16/Z. Derek K. Jones, Wellcome Trust (https://dx.doi.org/10.13039/100010269), Award ID: 096646/Z/11/Z. Derek K. Jones, Wellcome Trust (https://dx.doi.org/10.13039 /100010269), Award ID: 104943/Z/14/Z. Sonya Foley, Wellcome Trust (https://dx.doi.org/10 .13039/100010269), Award ID: 504182. Ulrich Ettinger, Deutsche Forschungsgemeinschaft (https://dx.doi.org/10.13039/501100001659), Award ID: ET 31/2-1. Eirini Messaritaki, Wellcome Trust (https://dx.doi.org/10.13039/100010269), Award ID: 204824/Z/16/Z. Derek K. Jones, Wellcome Trust (https://dx.doi.org/10.13039/100010269), Eirini Messaritaki, Wellcome Trust (https://dx.doi.org/10.13039/100010269), Award ID: 204824/Z/16/Z. Derek K. Jones, Wellcome Trust (https://dx.doi.org/10.13039/100010269), Award ID: 096646/Z/11/Z. Derek K. Jones, Wellcome Trust (https://dx.doi.org/10.13039 .13039/100010269), Award ID: 504182. Ulrich Ettinger, Deutsche Forschungsgemeinschaft (https://dx.doi.org/10.13039/501100001659), Award ID: ET 31/2-1. SUPPORTING INFORMATION Supporting information for this article is available at https://doi.org/10.1162/netn_a_00279. DISCUSSION Despite the differences in scanners, scanning sequences, and the lan- guage in which the SPQ was administered, the findings of stronger structural connectivity in the sensorimotor network were replicated in the two datasets, and employing two different atlases for brain parcellation. Additionally, the finding of stronger structural connectivity in the default mode network was replicated when using the two different atlases in the Cardiff dataset. Our tractography generation is state of the art, utilising an algorithm that limits the presence of false positive tracts. Using the inverse radial diffusivity to assign strength to the structural connections relates them to the myelination and the axonal density of the white matter tracts, which are measures that better reflect efficient brain communication than the more frequently used number of streamlines or FA (Messaritaki et al., 2021). One limitation of our study is the fact that the schizotypy scores went only up to 43, when the maximum possible value of the SPQ total score is 74. This is a typical range for studies of healthy vol- unteers but indicates that our results should be interpreted with regards to that population, while generalisations to individuals with very high-schizotypy levels need to be drawn with caution. In the future, MRI studies should include dedicated sequences with differential sen- sitivity to myelin (e.g., magnetization-transfer imaging (Wolff & Balaban, 1989) or myelin- water-based imaging (Mackay et al., 1994)) and to axonal properties, including higher b-value acquisitions, to provide higher sensitivity to the intra-axonal signal fraction and therefore mea- sures such as apparent axon density (Assaf & Basser, 2005; Zhang et al., 2012) and axon diameter (Barazany et al., 2009). This will allow us to further investigate structural connectiv- ity in high schizotypy. 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The Temporal Relationship Between Terrestrial Gamma‐Ray Flashes and Associated Optical Pulses From Lightning
Journal of geophysical research. Atmospheres
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Citation (APA): Skeie, C. A., Østgaard, N., Mezentsev, A., Bjørge‐Engeland, I., Marisaldi, M., Lehtinen, N., Reglero, V., & Neubert, T. (2022). The temporal relationship between Terrestrial Gamma‐ray flashes and associated optical pulses from lightning. Journal of Geophysical Research: Atmospheres, 127(17), Article e2022JD037128. https://doi.org/10.1029/2022JD037128 Downloaded from orbit.dtu.dk on: Oct 24, 2024 Downloaded from orbit.dtu.dk on: Oct 24, 2024 Downloaded from orbit.dtu.dk on: Oct 24, 2024 General rights Copyright and moral rights for the publications made accessible in the public portal are retained by the authors and/or other copyright owners and it is a condition of accessing publications that users recognise and abide by the legal requirements associated with these rights.  Users may download and print one copy of any publication from the public portal for the purpose of private study or research.  You may not further distribute the material or use it for any profit-making activity or commercial gain  You may freely distribute the URL identifying the publication in the public portal 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. General rights Copyright and moral rights for the publications made accessible in the public portal are retained by the authors and/or other copyright owners and it is a condition of accessing publications that users recognise and abide by the legal requirements associated with these rights. General rights Copyright and moral rights for the publications made accessible in the public portal are retained by the authors and/or other copyright owners and it is a condition of accessing publications that users recognise and abide by the legal requirements associated with these rights.  Users may download and print one copy of any publication from the public portal for the purpose of private study or research.  You may not further distribute the material or use it for any profit-making activity or commercial gain 1.  Introduction Received 16 MAY 2022 Accepted 18 AUG 2022 Terrestrial Gamma-ray Flashes (TGFs) are bursts of hard X- and gamma-rays produced via bremsstrahlung from runaway electrons accelerated in the electric fields of thunderstorms (Dwyer, 2012; Gurevich et al., 1992; Moss et al., 2006; Wilson, 1925). The TGFs are reported to typically last a few tens, up to a few hundred microsec- onds, and have individual photon energies up to ∼40 MeV (Briggs et al., 2013; Fishman et al., 1994; Marisaldi et al., 2010, 2014; Smith et al., 2005). Analysis of the energy spectra of TGFs and lightning radio atmospherics indicate that they are produced below 21 km altitude, most likely between 10 and 15 km (Carlson et al., 2007; Cummer et al., 2014; Dwyer & Smith, 2005; Lindanger et al., 2021; Mailyan et al., 2016; Pu et al., 2019; Stanley et al., 2006; Xu et al., 2012). The underlying mechanism for creating the observed TGF photon fluxes is still unclear. There are two leading models for explaining the observed fluxes based on where and how the electrons are accelerated. Both theories build on electrons being accelerated in electric fields that are strong enough to overcome the friction force of the air, in what is called the runaway process (Wilson, 1925), before being multi- plied in a relativistic runaway electron avalanche process (Gurevich et al., 1992). In the first model, an electron flux is created in an avalanche process developing in the large-scale electric fields within the thunderclouds. Back-scattered X-rays created by bremsstrahlung, and positrons created by pair-production, seed additional elec- tron avalanches in what is called a relativistic feedback mechanism (Dwyer, 2008). In the other model the initial electron flux is created in small, intense, transient overlapping electric fields of streamers, lightning leader and thundercloud electric field. The overlapping electric fields are strong enough to let electrons runaway, and then undergo bremsstrahlung and produce X- and gamma-rays (Celestin & Pasko, 2011; Moss et al., 2006). These two models can also be at play simultaneously, as one does not exclude the other. TGFs were first reported by Fishman et al. (1994), using the Burst and Transient Source Experiment on the Compton Gamma Ray Observatory. © 2022. The Authors. 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. The Temporal Relationship Between Terrestrial Gamma-Ray Flashes and Associated Optical Pulses From Lightning C. A. Skeie1  , N. Østgaard1  , A. Mezentsev1  , I. Bjørge-Engeland1  , M. Marisaldi1,2  , N. Lehtinen1  , V. Reglero3, and T. Neubert4 The Temporal Relationship Between Terrestrial Gamma-Ray Flashes and Associated Optical Pulses From Lightning The temporal relationship between Terrestrial Gamma‐ray flashes and associated optical pulses from lightning The temporal relationship between Terrestrial Gamma‐ray flashes and associated optical pulses from lightning Skeie, C. A.; Østgaard, N.; Mezentsev, A.; Bjørge‐Engeland, I.; Marisaldi, M.; Lehtinen, N.; Reglero, V.; Neubert, T. Published in: Journal of Geophysical Research: Atmospheres Link to article, DOI: 10.1029/2022JD037128 Published in: Journal of Geophysical Research: Atmospheres Document Version Publisher's PDF, also known as Version of record Link back to DTU Orbit Citation (APA): Skeie, C. A., Østgaard, N., Mezentsev, A., Bjørge‐Engeland, I., Marisaldi, M., Lehtinen, N., Reglero, V., & Neubert, T. (2022). The temporal relationship between Terrestrial Gamma‐ray flashes and associated optical pulses from lightning. Journal of Geophysical Research: Atmospheres, 127(17), Article e2022JD037128. https://doi.org/10.1029/2022JD037128 Key Points: A., Østgaard, N., Mezentsev, A., Bjørge-Engeland, I., Marisaldi, M., Lehtinen, N., et al. (2022). The temporal relationship between terrestrial gamma-ray flashes and associated optical pulses from lightning. Journal of Geophysical Research: Atmospheres, 127, e2022JD037128. https://doi. org/10.1029/2022JD037128 Received 16 MAY 2022 Accepted 18 AUG 2022 Correspondence to: C. A. Skeie, Chris.Skeie@uib.no Skeie, C. A., Østgaard, N., Mezentsev, A., Bjørge-Engeland, I., Marisaldi, M., Lehtinen, N., et al. (2022). The temporal relationship between terrestrial gamma-ray flashes and associated optical pulses from lightning. Journal of Geophysical Research: Atmospheres, 127, e2022JD037128. https://doi. org/10.1029/2022JD037128 Skeie, C. A., Østgaard, N., Mezentsev, A., Bjørge-Engeland, I., Marisaldi, M., Lehtinen, N., et al. (2022). The temporal relationship between terrestrial gamma-ray flashes and associated optical pulses from lightning. Journal of Geophysical Research: Atmospheres, 127, e2022JD037128. https://doi. org/10.1029/2022JD037128 Received 16 MAY 2022 Accepted 18 AUG 2022 Key Points: C. A. Skeie1  , N. Østgaard1  , A. Mezentsev1  , I. Bjørge-Engeland1  , M. Marisaldi1,2  , N. Lehtinen1  , V. Reglero3, and T. Neubert4 C. A. Skeie1  , N. Østgaard1  , A. Mezentsev1  , I. Bjørge-Engeland1  , M. Marisald N. Lehtinen1  , V. Reglero3, and T. Neubert4 • Terrestrial Gamma-ray Flashes (TGFs) are produced before or simultaneously with the onset of an optical pulse 1Birkeland Centre for Space Science, Institute of Physics and Technology, University of Bergen, Bergen, Norway, 2INAF-OAS Bologna, Bologna, Italy, 3Imaging Processing Laboratory, University of Valencia, Valencia, Spain, 4National Space Institute, Technical University of Denmark, Kongens Lyngby, Denmark • For some events the delay of the optical pulse is too long to be explained by the light scattering in the cloud Abstract  We present 221 Terrestrial Gamma-ray Flashes (TGFs) and associated optical pulses observed by the Atmosphere-Space Interactions Monitor (ASIM) on board the International Space Station. The events were detected between the end of March 2019 and November 2020 and consist of X- and gamma-ray energy detections, as well as photometer data (180–230, 337, and 777 nm) and optical camera data (337 and 777 nm). Using the available ASIM data and applying a consistency check based on TGF characteristics and lightning detections from lightning radio atmospherics close in time, we determine the most likely position of the TGFs in relation to the photometer field of view (FoV), and the association to the observed optical pulses. Out of the 221 events we find 72 events where the TGF and optical data are determined to be associated and inside the photometer FoV. Using the measured TGF durations and the time between the onsets of the TGFs and optical pulses we find: (a) That the TGF onsets are always before or at the same time as the optical pulse onsets (taking into account cloud scattering). (b) A tendency for longer duration TGFs to have longer delays between onsets. (c) Two groups of events: (a) where there is a possible overlap between the TGFs and the optical emissions, as the TGFs last longer than the delay between onsets and (b) where the TGFs and optical emissions do not overlap, as there are long delays between the onsets, which cannot be explained by cloud scattering. • Longer duration TGFs tend to have longer delays between onsets of the TGFs and the optical pulses Correspondence to: C. A. Skeie, Chris.Skeie@uib.no Citation: Skeie, C. 10.1029/2022JD037128 RESEARCH ARTICLE Key Points: Journal of Geophysical Research: Atmospheres Journal of Geophysical Research: Atmospheres 10.1029/2022JD037128 The atmosphere-space interactions monitor (ASIM) is the first instrument specifically designed to observe TGFs, as well as transient luminous events. ASIM is mounted on the Columbus module of the International Space Station (ISS), and has been gathering data since April 2018. ASIM has multiple detectors consisting of high and low energy X- and gamma-ray detectors, photometers, and optical cameras (Neubert et al., 2019). Past studies using radio data have shown that TGFs likely occur during the early phase of intracloud (IC) lightning (Lu et al., 2010; Pu et al., 2019; Shao et al., 2010; Østgaard et al., 2013, 2021). This is also shown in Lindanger et al. (2022) who used TGF detections paired with optical measurements of lightning activity to show that TGFs are produced during the initial phase of a lightning flash. The sequence of the TGF and optical signal of the flash is still uncertain. Østgaard et al. (2013) were the first to report simultaneous observation of a TGF and optical light from lightning. Using a TGF detected by the Ramaty High Energy Solar Spectroscopic Imager (RHESSI) and optical data from the lightning imaging sensor (LIS) on board the tropical rainfall measuring mission satel- lite, they conclude that the TGF was produced in the initial stage of an IC lightning propagating upwards in the cloud. Gjesteland et al. (2017) reinvestigated the TGF-optical sequence using two TGFs detected by RHESSI and optical data from LIS, as well as lightning radio atmospherics from the World Wide Lightning Location Network. However, due to the relative timing uncertainties of the instruments (±1.6 ms), stemming mainly from the time resolution of LIS, they could not determine the sequence of TGF and optical signal of the flash. Alnussirat et al. (2019) also investigated the sequence of TGF and optical signals, using optical data from the Geostationary Lightning Mapper, with a time resolution of 2 ms and TGFs detected by the Fermi Gamma-ray Burst Monitor. Based on their observations, they find that the TGFs are likely produced during the last stages of the development of the lightning leader channel. However, they could not determine the sequence of TGF-optical emission itself. Journal of Geophysical Research: Atmospheres More recent observations from ASIM have shown that the majority of TGFs occur before or at the onset of the optical emissions, given the uncertainties in the measurements (Heumesser et al., 2021; Neubert et al., 2020; Østgaard, Neubert, et al., 2019). A relevant aspect in the determination of the sequence of the TGF and optical emissions is the cloud scattering of the optical signals. Satellite detection of optical light from lightning including cloud effects such as scattering, has recently been modeled (Luque et al., 2020; Peterson, 2020). The optical light emitted from lightning in different wavelength bands is associated with different processes in a lightning flash, such as the hot channel of a lightning discharge or streamer activity before the discharge (Chanrion et al., 2019). In this work we will investigate the temporal relationship between TGFs and optical emissions from lightning. For this purpose we will use a set of upwards directed TGFs with accompanying optical detections observed from space by ASIM. We start by investigating the sequence of TGF and the main optical pulse (defined in Section 3.6), which has been addressed using ASIM before in Østgaard, Neubert, et al. (2019) and Heumesser et al. (2021). The results of Østgaard, Neubert, et al. (2019) were hampered by the relative timing accuracy (±80 µs) between the Modular X- and Gamma-ray Sensor (MXGS) and modular multi-spectral imaging array (MMIA) instruments. In this paper, we will use a larger data sample from a later period, where the relative accu- racy between the instruments has been improved to ±5 µs, and more sophisticated and accurate methodology. Heumesser et al. (2021) also analyzed TGFs and optical data observed by ASIM, and concluded that the sequence of TGF-optical cannot be addressed due to the uncertainties in timing and the model they used. For this work we have carefully inspected each event and applied a consistency check (outlined in Section 3), where we determine the relationship of each detected TGF and optical pulse. For this purpose we inspected the geolocation source of the radio atmospherics from the lightning discharges, together with the optical detections of lightning and TGF characteristics to determine which of the observed optical pulses are most likely associated with the TGFs. Journal of Geophysical Research: Atmospheres The events where the TGF and optical pulse are found to be associated will then be used to investigate the sequence of the TGF and optical pulse, as well as the relationship between TGF durations and the time delay between TGF and the onset of the optical pulse, to help understanding the processes involved and sequence of events. 1.  Introduction Since then many observations of TGFs have been made, using mostly satellite-based (Briggs et  al.,  2010; Marisaldi et  al.,  2010,  2014; Østgaard, Neubert, et  al.,  2019; Smith et al., 2005), but also aircraft (Bowers et al., 2018; Smith et al., 2011), and ground based instruments (Abbasi et al., 2018; Dwyer et al., 2004, 2012; Hare et al., 2016; Kereszy et al., 2022; Tran et al., 2015; Wada et al., 2019). 1 of 14 1 of 14 SKEIE ET AL. SKEIE ET AL. 3.  Methodology Between end of March 2019 and November 2020 we have observed 221 TGFs, where also optical data from MMIA are available with a relative timing accuracy of ±5 µs. To determine the association between the TGFs and the optical pulses we investigated the photometer data in three main steps. (a) We search for an optical pulse in the 337 and 777 nm optical band within 5 ms of the TGF. If there is no optical pulse the TGF is most likely outside the MMIA FoV and we exclude the event. (b) For the remaining events the MMIA FoV is determined and the surrounding lightning activity within 15 min of the TGF is investigated to determine a possible location of the TGF. (c) A consistency check is performed (as outlined in Section 3.3) using the surrounding lightning activity, camera images (83.3 ms resolution), and TGF characteristics, such as number of counts and their energies. 2.  Instruments and Data The relative timing accuracy between the MXGS and MMIA instrument was ±80 µs until a software update in April 2019, which reduced it to its nominal performance of ±5 µs. Due to a non-optimal timing interface between the ISS and the ASIM instrument, the absolute timing accuracy is found to be ∼−10 to +40 ms (determined using lightning detec- tion location from lightning radio atmospherics). This timing accuracy can be improved  for some events by using lightning detections together with optical data to reduce the absolute timing accuracy to ±1 ms. Figure 1. Illustration of how to determine the corner-point of the modular multi-spectral imaging array (MMIA) field of view (FoV) projected from the International Space Station (ISS) to ground level on Earth in three steps. 1) Rotation due to ISS yaw angle (±Z-direction), 2) move according to roll angle and the 5° tilt of the MMIA instrument in the roll direction (±Y-direction). 3) Move according to the pitch angle (±X-direction). Lightning radio atmospherics used in this work is provided by Vaisala's Global Lightning Detection Network (GLD360) and give us time and loca- tion data of lightning flashes. GLD360 is a ground-based very-low-frequency (VLF) and lower part of low-frequency (LF) radio lightning detection network which employs time of arrival and magnetic direction finding at each sensor to determine the location of individual lightning discharges. The expected GLD360 detection efficiency is ∼75%–85% for cloud-to-ground flashes, ∼40%–50% for IC pulses, with ∼2–6 km uncertainty in median location accuracy (Demetriades et al., 2010; Said & Murphy, 2016). irection). 2.  Instruments and Data The ASIM payload (Neubert et al., 2019) on board the ISS consists of two main instruments: the MMIA and the MXGS. The MMIA (Chanrion et al., 2019) consists of three photometers and two cameras, which are tilted 5° upwards from nadir (toward starboard of ISS) to avoid potential obstructions from payloads on the bottom of the mounting platform. The photometers operate in 180–230 nm (UV), 337 nm (blue) with a 4 nm bandwidth, 777.4 nm (red) with a 5 nm bandwidth, and have a sample rate of 100 kHz. The two optical cameras capture up to 12 frames per second, operate in the 337 and 777 nm bands, and have a 400 × 400 m resolution at nadir. Both the 2 of 14 SKEIE ET AL. SKEIE ET AL. Journal of Geophysical Re Figure 1. Illustration of how to determine the corner-point of the modular multi-spectral imaging array (MMIA) field of view (FoV) projected from the International Space Station (ISS) to ground level on Earth in three steps. 1) Rotation due to ISS yaw angle (±Z-direction), 2) move according to roll angle and the 5° tilt of the MMIA instrument in the roll direction (±Y-direction). 3) Move according to the pitch angle (±X-direction). Journal of Geophysical Research: Atmospheres 10.1029/2022JD037128 337 and 777 nm photometers and cameras have a square field of view (FoV) (80° diagonal), while the UV photometer has a circular FoV (80° diameter). As we are only using the 777 and 337 nm band in this study we will refer to the square FoV as MMIA FoV throughout this paper. To prevent damage by sunlight, the MMIA instrument is only active during night time. The MXGS (Østgaard, Balling, et al., 2019) consists of a high and a low energy detector (LED). The high-energy detector (HED) is always active while outside the South Atlantic Anomaly, detects energies between 300 keV and 30 MeV, and has a time resolution of 28.7 ns. The LED is only active during night, due to optical photon contamination during day time, detects energies between ∼50 and 400 keV, and has a resolution of 1 µs. The ASIM instrument includes a cross triggering system between MXGS and MMIA, such that if either instrument triggers data from both will be kept for a period of ∼2 s, centered on the trigger time. Journal of Geophysical Research: Atmospheres 10.1029/2022JD037128 3.2.  Lightning Detections Using the location data from lightning radio atmospherics (detected by GLD360) in the surrounding area from within ±1,000 s we get three different scenarios. (a) All lightning activity is outside the MMIA FoV, meaning the TGF is most likely from outside the FoV. (b) All lightning activity is inside the MMIA FoV, meaning the TGF is most likely from inside the FoV. (c) There is lightning activity both inside and outside the MMIA FoV. For all of these groups of events we apply the consistency check (see Section 3.3), where the lightning activity is used together with (a) TGFs individual photon energies and fluence and (b) camera images. The GLD360 detections are also used to improve the absolute timing accuracy of ASIM, wh The GLD360 detections are also used to improve the absolute timing accuracy of ASIM, which is found to be −10 to +40 ms before improvement. For many of the events we can improve the absolute timing of ASIM by time alignment of the measured optical pulses and GLD360 detections. This is done by first finding the ISS time of the GLD360 detections, by adding the lights' travel time from source to the ISS, to the given GLD360 detection time. We then use multiple triggers of MMIA data (up to three, consisting of the trigger containing the TGF and one trigger before and after the trigger containing the TGF) to align as many optical pulse peaks as possible to the GLD360 detections. A minimum of two alignments is required, with a minimum of one optical pulse aligned from the MMIA trigger containing the TGF. Aligning the GLD360 detections to the optical pulse peaks is practical, although this approach does not take into account the time delay due to light scattering through the clouds. However, given the typical rise times of the optical pulses, this is well within the error of the method. The lightning detection locations and camera images are then checked for consistency. Using this technique we can get the absolute timing accuracy between MMIA and the GLD360 detections down to ±1 ms. This method of improving absolute timing accuracy has already been implemented in Maiorana et al. (2021), Lindanger et al. (2022), and independently developed and applied in Heumesser et al. (2021). Using the method outlined here we found 95 alignments for the total sample of events. 3.3.  Consistency Check A consistency check is performed to determine if the TGFs are likely to be within the MMIA FoV and have an association with the optical pulses. For this purpose we use the TGF fluence and individual photon energies, and compare to optical camera and lightning activity. For the consistency check we consider a TGF emission half-cone of 30–40° without tilt. The TGF fluence is expected to be reduced as the distance between the TGF and ISS-footpoint increases. This is due to the scattering of photons in the atmosphere and the increasing distance (1/R 2 effect). Furthermore, we expect TGFs observed within the production cone to have more high energy (above energy channel 1,000, which is approximately 10 MeV) counts than the TGFs from outside the production cone. For TGFs observed outside the initial production cone the photons will have undergone Compton scatter- ing and have reduced energies (Carlson et al., 2007; Gjesteland et al., 2011; Lindanger et al., 2021). This means the TGFs with no or very few counts with high energies are more likely to be produced outside the MMIA FoV, because the half-cone angle (30–40°) is similar to the MMIA FoV (diagonal angle of 40°). We also investigate the lightning activity surrounding the TGF and check for a GLD360 detection associated to the TGF and optical pulse. If such a pulse is found we compare the location of the GLD360 detection to the optical camera, as well as the TGF characteristics. We do not use any of these assumptions as hard limitations, but as an indicator to differentiate TGFs inside the MMIA FoV from those outside the MMIA FoV. 3.1.  MMIA Field of View To determine the square MMIA photometer FoV we first interpolate the ISS foot point at the time of the TGF, using the closest ISS locations before and after the TGF, as well as the ISS velocity and the difference in time between the TGF detection and the two points. From the ISS foot-point we map out the 337 and 777 nm photom- eter FoV, which is a square with 80° diagonal for both photometers (Chanrion et al., 2019), with the sides going along and across the ISS direction of travel (X and Y in Figure 1). This is done by using eight points, namely the four corners of the square and points at the middle of each side. These eight points are then shifted using yaw, roll, and pitch angles of the ISS at the time of the TGF, as well as the 5° tilt in the roll direction, as illustrated in Figure 1. The photometer FoV was compared with coastline camera images, acquired specifically to optimize the pointing accuracy. The difference in the determined FoV and coastal camera images was found to be at most ∼20 km, which is acceptable for the purposes of this study. 3 of 14 3 of 14 SKEIE ET AL. SKEIE ET AL. Journal of Geophysical Research: Atmospheres Journal of Geophysical Research: Atmospheres Journal of Geophysical Research: Atmospheres 10.1029/2022JD037128 Figure 2. Example of an event where we have determined the Terrestrial Gamma-ray Flash (TGF) to be inside the modular multi-spectral imaging array (MMIA) field of view (FoV) and associated to the observed optical pulse. (a) Detected counts in high-energy detector (HED). (b–d) Light curves from the photometers, 337, 777, and 180–230 nm. (e) Map of the area around the International Space Station (ISS) footpoint. The blue square shows the MMIA FoV, the black dots are lightning activity within ±15 min of the TGF, the magenta stars are lightning activity within a second of the TGF, and the green star shows the lightning detection aligned to the optical pulse. (f, g) Camera 337 and 777 nm show the optical camera images from the frame containing the TGF (Y-axis is along the flight path of the ISS, which is always eastwards/right in the images). Figure 2. Example of an event where we have determined the Terrestrial Gamma-ray Flash (TGF) to be inside the modular multi-spectral imaging array (MMIA) field of view (FoV) and associated to the observed optical pulse. (a) Detected counts in high-energy detector (HED). (b–d) Light curves from the photometers, 337, 777, and 180–230 nm. (e) Map of the area around the International Space Station (ISS) footpoint. The blue square shows the MMIA FoV, the black dots are lightning activity within ±15 min of the TGF, the magenta stars are lightning activity within a second of the TGF, and the green star shows the lightning detection aligned to the optical pulse. (f, g) Camera 337 and 777 nm show the optical camera images from the frame containing the TGF (Y-axis is along the flight path of the ISS, which is always eastwards/right in the images). that the optical pulses come from the same location as the GLD360 detections. Panel (a) shows the counts, with energies from the HED. The TGF has a hard spectrum (five counts above channel 1,000) with many counts (97) in a short time interval (∼100 µs), which is consistent with a TGF within a small radial distance ∼200–300 km to the ISS foot-point, that is, within the MMIA FoV. ISS is moving eastward and the camera images have Y-axis along the path and X-axis toward starboard. This all indicates that the TGF is from within the MMIA FoV and associated to the observed optical pulse. 3.4.  Event Examples Following are examples that show events where the consistency check is used to help determine the TGF-optical pulse association. Following are examples that show events where the consistency check is used to help determine the TGF-optical pulse association. Figure 2 shows an example of an event where we have determined that the TGF and optical pulse is associated. For this event enough GLD360 detections could be aligned with optical pulses so that the total timing accuracy was reduced to ±1 ms. As seen panel (e) of the figure, most lightning activity is inside the MMIA FoV, with multiple lightning detections within 1 s of the TGF clustered at the same area. The magenta stars show the GLD360 detections within 1 s of the TGF time, with the green star showing the GLD360 detection found to be aligned in time to the optical pulse. No other GLD360 detections were found to be within the −10 to +40 ms window (blue stars), corresponding to the total timing accuracy of ASIM. The camera images (f and g) support 4 of 14 SKEIE ET AL. SKEIE ET AL. Journal of Geophysical Research: Atmospheres Figure 3 shows another example of an event where the TGF is determined to be within the MMIA FoV. For this event there is only lightning activity within the MMIA FoV. There were not enough GLD360 detections to improve the timing accuracy for this event. Two GLD360 detections are seen inside the MMIA FoV within the minimum and maximum of the absolute timing correction (blue stars). One of the detections is most likely an ionospheric reflection, considering the time, distance, and peak currents (opposite but similar magnitude). This is also supported by the camera images, where only one active area is seen. An ELVE (Emission of Light and Very Low Frequency perturbations due to Electromagnetic Pulse Sources) event is observed (UV pulse starting at 0 µs) at the same time as the TGF. Thirteen such ELVEs were found in this data set, these events are also a part of the data set analyzed and presented in Bjørge-Engeland et al. (2022). A total of 72 events was found, where we could determine an association between the TGF and the optical pulse such as the events shown in Figures 2 and 3. Fourty five absolute timing corrections were determined for these 72 events, where 33 ended up with a GLD360 detection aligned with the optical pulse associated to the TGFs. Figure 4 shows an event for which we have determined that the TGF is not from within the MMIA FoV, and therefore the observed optical pulse is not associated with the TGF. The lightning activity map (e) shows there are many centers of activity outside the MMIA FoV, but no lightning activity within 1 s of the TGF (magenta, blue, or green stars). The camera images (f–g) show that the optical pulses come from a location close to the ISS foot-point. The TGF (a) is found to be long, with relatively few counts in HED (19) and only one count above energy channel 1,000. The TGF characteristics in this instance do not match our expectations of a TGF found 5 of 14 5 of 14 SKEIE ET AL. SKEIE ET AL. Journal of Geophysical Research: Atmospheres 10.1029/2022JD037128 Figure 3. Example of an event where we have determined the Terrestrial Gamma-ray Flash (TGF) to be inside the Modular multi-spectral imaging array (MMIA) field of view (FoV) and associated to the observed optical pulse, and there is only lightning activity from within the FoV. Journal of Geophysical Research: Atmospheres (a) Detected counts in high-energy detector (HED) (b–d) Light curves from the photometers, 337, 777, and 180–230 nm. (e) Map of the area around the International Space Station (ISS) footpoint. The blue square show the MMIA FoV, the black dots are lightning activity within ±15 min of the TGF, the magenta stars are lightning activity within a second of the TGF, and the green star shows the lightning detection aligned to the optical pulse. (f, g) Cameras 337 and 777 nm show the optical camera images from the frame containing the TGF (Y-axis i along the flight path of the ISS, which is always eastwards/right in the images). Figure 3. Example of an event where we have determined the Terrestrial Gamma-ray Flash (TGF) to be inside the Modular multi-spectral imaging array (MMIA) field of view (FoV) and associated to the observed optical pulse, and there is only lightning activity from within the FoV. (a) Detected counts in high-energy detector (HED). (b–d) Light curves from the photometers, 337, 777, and 180–230 nm. (e) Map of the area around the International Space Station (ISS) footpoint. The blue square shows the MMIA FoV, the black dots are lightning activity within ±15 min of the TGF, the magenta stars are lightning activity within a second of the TGF, and the green star shows the lightning detection aligned to the optical pulse. (f, g) Cameras 337 and 777 nm show the optical camera images from the frame containing the TGF (Y-axis is along the flight path of the ISS, which is always eastwards/right in the images). inside the MMIA FoV and close to the ISS foot-point. In this case it is likely that the TGF is produced in one of the active areas outside the FoV and is not associated with the optical pulses we observe. We found 57 events of this type, where the TGF is most likely outside the FoV, and consequently not included in our study. The last group of events is the 88 events excluded in the beginning where we have no association, as there is no observed optical pulse within ±5 ms of the TGF. Figure 5 shows one of these events, with no lightning activity within the MMIA FoV as further support. Some lightning activity is observed within 1 s of the TGF just outside the MMIA FoV, shown as the three magenta stars. Journal of Geophysical Research: Atmospheres The TGF has few counts, with energies below channel 300, which is consistent with being produced outside the MMIA FoV. For four events there are difficulties in determining optical pulse onset or TGF association to the optical pulse. This is due to there either being multiple TGFs within the 5 ms time window, but only one optical pulse, or difficulties identifying the pre-activity and determining the onset of the optical pulse. As we do not want to make an assumption on which TGF is associated to the optical pulse, or what is pre-activity and main optical pulse, we decided to exclude these events going forward. Table 1 shows a summary of the number of events in each category. Journal of Geophysical Research: Atmospheres Journal of Geophysical Research: Atmospheres 10.1029/2022JD037128 Figure 4. Example of an event where we have determined the Terrestrial Gamma-ray Flash (TGF) to be outside the modular multi-spectral imaging array (MMIA) field of view (FoV) and is therefore not associated to the observed optical pulse. (a) Detected counts in high-energy detector (HED). (b–d) Light curves from the photometers, 337, 777, and 180–230 nm. (e) Map of the area around the International Space Station (ISS) footpoint. The blue square shows the MMIA FoV, the black dots are lightning activity within ±15 min of the TGF. No lightning detections are within 1 min of the detected TGF (magenta, blue, or green stars). (f, g) Camera 33 and 777 nm show the optical camera images from the frame containing the TGF (up is along the flight path of the ISS, which is always eastwards/right in the images have determined the Terrestrial Gamma-ray Flash (TGF) to be outside the modular multi-spectral imaging array (MMIA) Figure 4. Example of an event where we have determined the Terrestrial Gamma-ray Flash (TGF) to be outside the modular multi-spectral imaging array (MMIA) field of view (FoV) and is therefore not associated to the observed optical pulse. (a) Detected counts in high-energy detector (HED). (b–d) Light curves from the photometers, 337, 777, and 180–230 nm. (e) Map of the area around the International Space Station (ISS) footpoint. The blue square shows the MMIA FoV, the black dots are lightning activity within ±15 min of the TGF. No lightning detections are within 1 min of the detected TGF (magenta, blue, or green stars). (f, g) Camera 337 and 777 nm show the optical camera images from the frame containing the TGF (up is along the flight path of the ISS, which is always eastwards/right in the images). Figure 5. Example of an event where the Terrestrial Gamma-ray Flash (TGF) is outside modular multi-spectral imaging array (MMIA) field of view (FoV), and there is no optical pulse observed in the photometers. (a) Detected counts in high-energy detector (HED). (b–d) Light curves from the photometers, 337, 777, and 180–230 nm. (e) Map of the area around the International Space Station (ISS) footpoint. The blue square shows the MMIA FoV, the black dots are lightning activity within ±15 min of the TGF, the magenta stars show lightning activity within a second of the TGF. Figure 5. 3.5.  TGF Durations The TGF durations were determined using an algorithm to find a “core duration” defined as the shortest time interval which includes 90% of the counts (tcore90). The algorithm works by first binning the counts around TGF time into 40 µs bins and selecting the bin with the most counts as a starting point. Thereafter we go backwards and include any count that is within 100 µs of the previously included count. The first photon of the TGF is then found where there is more than 100 µs between the current count and the one before it. This same approach is also applied from the starting position and forwards in time, to find the end of the TGF. A sliding window is then used to find the shortest duration that contains 90% of the counts (rounded up) between the start and end counts. As this method runs the risk of including cosmic showers and solar protons we perform a manual inspection of the SKEIE ET AL. 6 of 14 3.6.  Optical Pulse Onsets To determine the onset of the optical pulse we determined several separate linear fits to different parts of the optical pulse. We used the intersects of linear fits of the sharpest rise in the optical pulse and the activity before this rise. To make these fits we mainly used the 777 nm light curves, but for some events a good fit could not be made to the 777 nm light curves, so the 337 nm light curves were used instead. The red light (777 nm) is preferred as it is thought to be mainly associ- ated to atomic oxygen (OI) which may only exist (after dissociation of O2) in the heated lightning leader channel (Chanrion et al., 2019), whereas the blue light (337 nm) is associated to the streamer activity, namely N2 second positive band group radiation caused by excitation by supra-thermal electrons. The 337 band might also detect some UV emissions, such as from ELVEs. For each event we separate the optical pulse activity into pre-activity and main rise (sharpest increase). One linear fit was made for the sharpest rise of the optical pulse, while we use three fits for the pre-activity. The pre-activity is thought to be optical emissions from the propagating lightning leader (Østgaard, Neubert, et al., 2019) and can be seen in Figure 6 as the relatively slow increase after −1,000 µs and until ∼+100 µs. For some cases we found it necessary to make two fits to the sharpest increase part, as the pulse was irregular with a separate increase between the sharpest increase and the pre-activity. The average of the intersects between the main rise fit lines and the multiple pre-activity fit lines is used as the optical onset time, with the difference between the minimum and maximum times of these intersects used to define the uncertainties. The uncertainties range from 1 µs to ∼100 µs, with an average of 13 µs. Figure 6 shows an event where we have determined four linear fits. The solid colored lines show the different linear fits: blue for the sharpest increase in the optical pulse, magenta, red, and green for different segments of pre-activity. The colored dots show the start and end of the data used in the fit. The dotted lines show the intersects from the fits of the pre-activity to that of the sharp- est increase. 3.6.  Optical Pulse Onsets As seen in the figure, the pre-activity has a linear trend, but can be broken into three parts for this particular event. The magenta line shows the total pre-activity, while the green line uses the data points at what appears to be a plateau in the pre-activity. Lastly, the red line used the data points in  the last part of the pre-activity where we have a steeper increase than for the previous parts of the pre-activity. Figure 6. Example of optical pulse onset determination. The solid colored lines show the different linear fits: green, purple, and red lines show fits for different parts of the pre-activity data, while the blue line shows a fit to the sharpest increase of the optical pulse. The dots on the fit lines represent the start and end of the data used in the fitting. The dotted lines represent the intersects between the pre-activity fits (green, purple, and red) and the fit for the sharpest increase. Journal of Geophysical Research: Atmospheres 10.1029/2022JD037128 events for the purpose of removing these counts. To determine the uncertain- ties in TGF tcore90 durations we chose to add the closest count in time before and after the determined TGF counts in the method outlined above (these counts will be more than 100 µs before/after the first/last count). We then determine the tcore90 duration of this sample and use the difference in duration compared to the original tcore90 as the uncertainty. Inside FoV (# time alignments) Most likely outside FoV No optical pulse Non-determinable 72 (45) 57 88 4 Note. FoV, field of view. Table 1 Summary of Events Journal of Geophysical Research: Atmospheres Example of an event where the Terrestrial Gamma-ray Flash (TGF) is outside modular multi-spectral imaging array (MMIA) field of view (FoV), and there is no optical pulse observed in the photometers. (a) Detected counts in high-energy detector (HED). (b–d) Light curves from the photometers, 337, 777, and 180–230 nm. (e) Map of the area around the International Space Station (ISS) footpoint. The blue square shows the MMIA FoV, the black dots are lightning activity within ±15 min of the TGF, the magenta stars show lightning activity within a second of the TGF. 7 of 14 7 of 14 SKEIE ET AL. SKEIE ET AL. Journal of Geophysical Research: Atmospheres 4.  Results and Discussion The median determined delay between onsets is 190 µs, with only nine events having delays over ∼500 µs. None of the events are observed to have a negative delay, that is, there are no observed events where the optical pulse onset precedes the onset of the TGF. This has been ambig- uous in previous work with ASIM data, but has been improved here due to the new and larger sample of events with better relative timing accuracy and our method of determining that the location of TGFs is inside the MMIA FoV and association to optical pulses. The sequence of events presented here support the sequence presented in Østgaard, Neubert, et al. (2019). Our determined delay between onsets is a bit different to what has previously bins. (c) Histogram of 10 logarithmic bins showing determined delays between TGF and optical signal. show this relationship, where blue dots have the optical onset determined from 777 nm, and the pink dots have optical onsets determined from 337 nm. The TGFs showing large uncertainties in duration have typically low number of counts, for which our method of determining the TGF duration is less robust. The expected delay of the observed optical onsets due to scatting in the cloud was modeled by Luque et al. (2020). Using a uniform cloud with typical value for particle radius of 10 μm and particle density of 100 cm −3 the delay of the 777 pulse would be 100 µs for a 6 km thick cloud and 200 µs for 8.5 km thick cloud. Considering a cloud top of 15 km, a delay of up to 100 µs seems realistic, with expectations that TGFs from these altitudes (above 9 km) are easily observable. A 200 µs delay due to a 8.5 km thick cloud would place the source very deep in the cloud (∼6.5 km), making it more difficult to observe all but the most powerful TGFs. 200 µs is therefore used as an upper limit of delay due to scattering. Figure 7a shows the lines corresponding to these two cloud scattering delays added to the TGF durations (dotted red and blue lines). The plot shows a tendency for longer-duration TGFs to have longer optical delays. Panel (c) of the figure shows the distribution of the optical onset delays with respect to the TGF tcore90 start time. 4.  Results and Discussion During the period between end of March 2019 and November 2020, 221 events were detected with both MMIA and MXGS data. From these events 88 had no optical pulse within ±5 ms of the TGFs. Using the method outlined in Section 3 we determined that for 72 of the events the TGFs are inside the MMIA FoV, and associated to the observed optical signal. Using the light- ning radio atmospherics from GLD360 we find that the absolute timing can be improved for 95 of the 221 events, which includes 45 timing corrections for the events where TGFs are determined to most likely be inside the MMIA FoV. The mean timing correction was found to be ∼+14 ms, varying from −11.5 to +40 ms. Going forward, all results refer to the sample of 72 events determined to most likely be within the MMIA FoV. Figure 6. Example of optical pulse onset determination. The solid colored lines show the different linear fits: green, purple, and red lines show fits for different parts of the pre-activity data, while the blue line shows a fit to the sharpest increase of the optical pulse. The dots on the fit lines represent the start and end of the data used in the fitting. The dotted lines represent the intersects between the pre-activity fits (green, purple, and red) and the fit for the sharpest increase. Using the TGF tcore90 durations and the delay between onsets we plot the relationship between the two in panel (a) of Figure 7. The blue and pink dots 8 of 14 SKEIE ET AL. 8 of 14 Journal of Geophysical Research: Atmospheres 10.1029/2022JD037128 Figure 7. (a) Plot showing the difference in Terrestrial Gamma-ray Flash (TGF) and optical onset times compared to TGF durations, with accompanying uncertainties. The blue dots show the events with onsets determined from the 777 nm optical pulse and the pink dots show the events where onsets were determined from the 337 nm optical pulse. Green arrows refer to events that are shown in separate Figures. The dotted red line shows the maximum expected optical delay due to cloud scattering, of 200 µs, and the dotted blue line shows the expected value (∼100 µs). (b) TGF tcore90 durations in 10 logarithmic bins. (c) Histogram of 10 logarithmic bins showing determined delays between TGF and optical signal. Figure 7. 4.  Results and Discussion (a) Plot showing the difference in Terrestrial Gamma-ray Flash (TGF) and optical onset times compared to TGF durations, with accompanying uncertainties. The blue dots show the events with onsets determined from the 777 nm optical pulse and the pink dots show the events where onsets were determined from the 337 nm optical pulse. Green arrows refer to events that are shown in separate Figures. The dotted red line shows the maximum expected optical delay due to cloud scattering, of 200 µs, and the dotted blue line shows the expected value (∼100 µs). (b) TGF tcore90 durations in 10 logarithmic bins. (c) Histogram of 10 logarithmic bins showing determined delays between TGF and optical signal. SKEIE ET AL. 9 of 14 show this relationship, where blue dots have the optical onset determined from 777 nm, and the pink dots have optical onsets determined from 337 nm. The TGFs showing large uncertainties in duration have typically low number of counts, for which our method of determining the TGF duration is less robust. The expected delay of the observed optical onsets due to scatting in the cloud was modeled by Luque et al. (2020). Using a uniform cloud with typical value for particle radius of 10 μm and particle density of 100 cm −3 the delay of the 777 pulse would be 100 µs for a 6 km thick cloud and 200 µs for 8.5 km thick cloud. Considering a cloud top of 15 km, a delay of up to 100 µs seems realistic, with expectations that TGFs from these altitudes (above 9 km) are easily observable. A 200 µs delay due to a 8.5 km thick cloud would place the source very deep in the cloud (∼6.5 km), making it more difficult to observe all but the most powerful TGFs. 200 µs is therefore used as an upper limit of delay due to scattering. Figure 7a shows the lines corresponding to these two cloud scattering delays added to the TGF durations (dotted red and blue lines). The plot shows a tendency for longer-duration TGFs to have longer optical delays. Panel (c) of the figure shows the distribution of the optical onset delays with respect to the TGF tcore90 start time. The delays are calculated as the duration from the first photon of the TGF tcore90 duration and the onset of the main optical pulse. The data is binned to 10 logarithmic bins. Journal of Geophysical Research: Atmospheres 10.1029/2022JD037128 been reported using ASIM data. Heumesser et al. (2021) use ASIM data from between June 2018 and October 2019 to determine characteristics of the optical pulses associated to TGFs. Heumesser et al. (2021) shows in Figure 3, panel (a), events with negative delays, with a mean source time of −10 µs and outliers with more than 4 ms delays. We have shown in this work, using the methods outlined that there are no negative values between onsets, albeit with a different methodology and somewhat different data set (we only use ASIM data from after end of March 2019, when the MXGS-MMIA uncertainty was reduced to ±5 µs). However, like Heumesser et al. (2021) we see the same trend with the majority of events having less than ∼2–300 µs delays between onsets and a similar group of outliers at around ∼800–1,200 µs. been reported using ASIM data. Heumesser et al. (2021) use ASIM data from between June 2018 and October 2019 to determine characteristics of the optical pulses associated to TGFs. Heumesser et al. (2021) shows in Figure 3, panel (a), events with negative delays, with a mean source time of −10 µs and outliers with more than 4 ms delays. We have shown in this work, using the methods outlined that there are no negative values between onsets, albeit with a different methodology and somewhat different data set (we only use ASIM data from after end of March 2019, when the MXGS-MMIA uncertainty was reduced to ±5 µs). However, like Heumesser et al. (2021) we see the same trend with the majority of events having less than ∼2–300 µs delays between onsets and a similar group of outliers at around ∼800–1,200 µs. Panel (b) in Figure 7 shows the TGF tcore90 durations of the 72 events within MMIA FoV, binned to 10 logarithmic bins. The average tcore90 duration for the 72 TGFs determined to be within MMIA FoV is found to be ∼100 µs, with a median of 66 µs, which is similar to previously reported ASIM detected TGFs by Østgaard, Neubert, et al. (2019) and Bjørge-Engeland et al. (2022). All events are found to have the optical signal after the TGF onset, with the events below the dotted blue line (100 µs scattering delay) being consistent with optical emissions produced simultaneously with the TGF (taking into account typical delay due to cloud scattering and uncertainties). 4.1.  Events Where Optical Onset Delay Is Less Than 100 µs In Figure 7a there are 37 events (∼51%) where the delay is less than 100 µs, meaning that the TGF and the source of the optical pulse could be simultaneous, when a more realistic delay due to scattering in the cloud is taken into account. For many short TGFs it is difficult to firmly establish whether the TGFs end before the onset of the optical pulse or not, due to the uncertainties of the method. For some events with longer duration the TGFs appear to be continuing after the onset of the optical pulse, suggesting that the conditions for producing runaway electrons as well as X- and gamma-rays (electric field, voltage drop, and seed particles) could still exist after the current through the leader channel occurs. However, most of these events have very weak optical signatures, and we can therefore not make a strong statement based on these outliers. Figure 8 shows one of these events (marked in Figure 7 with a green arrow). Panel (a) shows the HED and LED detections, with the dotted magenta line show- ing the first photon of the TGF tcore90 duration. The three lower panels (b–d) show the photometer data from 337, 777, and 180–230 nm. The dotted magenta line in the 777 nm panel shows the average onset determined from the method described in Section 3.6. As seen in Figure 8 the TGF is not over at the time of the optical pulse onset. The HED counts (black dots) found after the optical pulse onsets are also high energy, so that they cannot be said to be Compton scattered photons (which can be seen in LED, after the end of the black dots). This means that the TGF is still ongoing at the onset of the optical pulse, meaning the large electric field needed for TGF production is still in place when the current surge responsible for the optical pulse starts. Journal of Geophysical Research: Atmospheres There are 37 events (∼51%) which are below the 100 µs delay line (dotted blue), meaning the TGF and the source of the optical pulse could be simultaneous. Fifteen events (∼21%) are above the 200 µs delay line, where the delay of the observed optical pulses cannot be explained by scattering from the cloud alone. Twenty events (∼28%) are between the two delay lines, which means that we cannot rule out that the TGFs and sources of the optical pulses are simultaneous. However, we think it is unlikely that so many powerful TGFs are detected from so deep in the cloud. The next subsections will discuss the events that are below the 100 µs delay line (dotted blue) and the events above the 200 µs delay line (dotted red). 4.  Results and Discussion The delays are calculated as the duration from the first photon of the TGF tcore90 duration and the onset of the main optical pulse. The data is binned to 10 logarithmic bins. The median determined delay between onsets is 190 µs, with only nine events having delays over ∼500 µs. None of the events are observed to have a negative delay, that is, there are no observed events where the optical pulse onset precedes the onset of the TGF. This has been ambig- uous in previous work with ASIM data, but has been improved here due to the new and larger sample of events with better relative timing accuracy and our method of determining that the location of TGFs is inside the MMIA FoV and association to optical pulses. The sequence of events presented here support the sequence presented in Østgaard, Neubert, et al. (2019). Our determined delay between onsets is a bit different to what has previously 9 of 14 SKEIE ET AL. SKEIE ET AL. Journal of Geophysical Research: Atmospheres Journal of Geophysical Research: Atmospheres 10.1029/2022JD037128 Figure 8. Plot showing the detection of a Terrestrial Gamma-ray Flash (TGF) that continues after the onset of the optical pulse. (a) Shows the high-energy detector (HED) and low energy detector (LED) counts given in energy channels. The dotted magenta line shows the first photon of the TGF tcore90 duration. Panels (b–d) show the photometer data of 337, 777, and 180–230 nm respectively. The dotted magenta line in the panel (c) shows the determined onset of the optical pulse. Figure 8. Plot showing the detection of a Terrestrial Gamma-ray Flash (TGF) that continues after the onset of the optical pulse. (a) Shows the high-energy detector (HED) and low energy detector (LED) counts given in energy channels. The dotted magenta line shows the first photon of the TGF tcore90 duration. Panels (b–d) show the photometer data of 337, 777, and 180–230 nm respectively. The dotted magenta line in the panel (c) shows the determined onset of the optical pulse. 4.2.  Events Where Optical Onset Delay Is by More Than 200 µs In Figure 7 there are 15 (∼21%) events that are above the 200 µs delay line (dotted red). This means that the conditions for TGF production are no longer present and a time interval (few hundreds up to a thousand microsec- onds) before the leader current occur is observed. Figure 9 shows one of these 15 events, where panel (a) shows the HED and LED counts of the TGF and the three lower panels (b–d) show the photometer data for 337, 777, and 180–230 nm. The dotted magenta lines show the onset of the TGF tcore90 (a) and optical pulse (c) and ∼1 ms delay between the two can be seen. There is ∼600 µs from the end of the TGF to the onset of the optical pulse. The connection between TGF and the leader current is a complex problem. This study shows that TGFs start before or simultaneous with the optical onset source. The study also shows that there is a group of events where the TGFs end (∼21%) before the leader current occurs. 10 of 14 SKEIE ET AL. SKEIE ET AL. Acknowledgments This study was supported by the Research Council of Norway under contract 223252/F50371 (CoE) and the European Research Council under the European Union's Seventh Framework Programme (FP7/2007–2013)/ERC grant agreement no. 320839. ASIM is a mission of the European Space Agency (ESA) and is funded by ESA and by national grants of Denmark, Norway, and Spain. Journal of Geophysical Research: Atmospheres Journal of Geophysical Research: Atmospheres 10.1029/2022JD037128 Figure 9. Plot showing the detection of a Terrestrial Gamma-ray Flash (TGF) that has an unusual long delay between onset of TGF and optical pulse. (a) Shows the high-energy detector (HED) and low energy detector (LED) counts given in energy channels. The dotted magenta line shows the first photon of the TGF tcore90 duration. Panels (b–d) show the photometer data of 337, 777, and 180–230 nm, respectively. The dotted magenta line in panel (c) shows the determined onset of the optical pulse. Figure 9. Plot showing the detection of a Terrestrial Gamma-ray Flash (TGF) that has an unusual long delay between onset of TGF and optical pulse. (a) Shows the high-energy detector (HED) and low energy detector (LED) counts given in energy channels. The dotted magenta line shows the first photon of the TGF tcore90 duration. Panels (b–d) show the photometer data of 337, 777, and 180–230 nm, respectively. The dotted magenta line in panel (c) shows the determined onset of the optical pulse. Data Availability Statement ASIM data used in this study are publicly available from the ASIM Science Data Center (https://asdc.space.dtu. dk). Values determined in this paper and presentations of the 221 events are available at Zenodo: https://doi. org/10.5281/zenodo.6992464 (Skeie, 2022). ASIM data used in this study are publicly available from the ASIM Science Data Center (https://asdc.space.dtu. dk). Values determined in this paper and presentations of the 221 events are available at Zenodo: https://doi. org/10.5281/zenodo.6992464 (Skeie, 2022). Briggs, M. S., Fishman, G. J., Connaughton, V., Bhat, P. N., Paciesas, W. S., Preece, R. D., et al. (2010). First results on terrestrial gamma ray flashes from the Fermi Gamma-ray burst monitor. Journal of Geophysical Research, 115(A7), A07323. https://doi.org/10.1029/2009JA015242 Briggs, M. S., Xiong, S., Connaughton, V., Tierney, D., Fitzpatrick, G., Foley, S., et  al. (2013). Terrestrial gamma-ray flashes in the Fermi era: Improved observations and analysis methods. Journal of Geophysical Research: Space Physics, 118(6), 3805–3830. https://doi.org/10.1002/jgra.50205 5.  Summary of Results Based on the 72 events where we have optical pulses associated with TGFs, we find the following: 1. All events have TGF onsets before the onset of the optical pulses 1%) of the events are below the 100 µs delay line and therefore have TGF onsets before or simultaneous h f h i l l ki i h li h i i h l d 2. 37 (∼51%) of the events are below the 100 µs delay line and therefore have TGF onsets before or simultane with the onset of the optical pulse, taking into account the light scattering in the cloud • Some of these events could have TGFs ending after the onset of the optical pulse 3. 15 (∼21%) of the events (above the 200 µs delay line) have longer delays than can be explained by cloud scat- tering, which means that the TGFs end before the leader current occurs 4. 20 (∼28%) of the events are between the two delay lines, these events are compatible with TGF onsets before or simultaneous with the onset of the optical pulse, taking into account a 200 µs delay due to light scattering in the cloud 5. 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Het phytopathologisch laboratorium willie commelin scholten van 1895 TOT 1906
Tijdschrift over planteziekten/Tijdschrift over plantenziekten
1,906
public-domain
9,652
28 -- 28 -- HET PHITOPATHOLOGISCH LABORATORIUIII WILLIE COiilHELIH SCHOLTEli VAH 1895 TOF 1906, Met ingang van Januari 1906 vrangde ik ontslag als Directeur van her phytopathologizch laboratorium Willie Commelin Schelten te Amsterdam, om op to fxeden als Directeur van een Instituut veer phytopat.hologie, 't wclk de Reg, eea'J~ng we~rschte to verbinden a.an do Rijks ttoogero Land-, Tuin- en Boschbouwschool to Wageningen. I/et eexsfgenoemde l~boratorium zal nu eerie geheel niettwe l~riMe intredcn, -- niet slochts omd,~t her door her verlies van de f 7000 jaarlijksche tlijk~uhsidie, die her genoot, op veel meet bescheiden veer zal moeten worden voortgezet dan in de laatste jtlren her geval was, maar 66k omdat hot nu zonder twijfel eone gehe~l andere richting zal uitgaan dan onder mijne dil~ctie, gedurealde ct.e eersto tie0a jar~n va.n zijn bosf.~an. Hoewel oorspronke]ijk geen ]and;bouwkundige, ben ik toch dear mijne ongeveer 36-jarige be~.rekking tot her land- bouwonderwijs met de verschillende takken van bodcmkul- tuur langen tijd ten naltwste verbondeal gewec~t; trouwe.ns ik heb van 't begin af aan d.at: ik do nat uurlijke historio nan eene inrichting veer landbouwqnderwijs te doceeren kreeg, getracht, naijn yak zooveel, mogelijk dien~stbanr to maken ann de bGdemkultuur. Her spreekt dus wel van zelf, dat ik, sexlert ik i~ 1895 aaa~ her hoofd we rd gesteld' van her phytopa~hologisch ]aboratorium Willie Commelin Scholten, ook dez~ inriehting in de eersto plaa~s trachtto dlenstbaar te maken ann de belangen der prakf.ijk. Trouwens ook in de statuten dezer st;chting was dit op den voorgi~nd gesteld. Nu echfer aan her hoofd van deze stichting eerie dame is -- 29 -- komen ~e stalin, 4ie hay studie aun de Universitei.t pa,s vo.leindigd h,ee~t, dde zieh w61 eea tijd lang onder do ]ei4ing van mijn' toanmal~ge,n adsistent Dr. van tIall, in zuiver wete,nsehappelijk mye,ologi.sch werken oefe~de, msar zieh aan de eiganlijke ziekte~aleer der planten tot du,sver weinig wijd,de, en die ook stee,d,s buitem a,anraking met de bodem- kultuur is geblevea~, -- nu zal h.et: phytop.atholog~s;eh laboratorium Willie Commelin Seholten zieh noodzake- tijkerwijze op ee~ geheet ande.r gebie,d gaan bew.e,gen. Her zal meer her karakber van een privaat-labora~orium aanne- me,n; er zullen do,or de t.aleatvolle Direc.triee o,nc~e,rzoe,- kingen o.p pl,a.ntkundig, missehien o,ok op i~hygopa.tho~o- giseh g:ebied wo,rden verrieht; maar de inrieht.ing zal zich niet, altharLs nie.t 1,e,eht,st.reeks, met do praktijk beano.eien; al is do mogelijkheid na,tuurlijk niet uitge,sloten d~ som- mige der al, daar verricht, e onderzoeMl~gen op den duur zullen blijken, year d,e praktijk niet onn.ut te zijn. HET PHITOPATHOLOGISCH LABORATORIUIII WILLIE COiilHELIH SCHOLTEli VAH 1895 TOF 1906, Ee~e n~uwe aansluiting aa.n de pra.ktijk, ean w,erken veer de, praktijk, mag men van nu ad aan vaa her phytop~th.ologiseh labora- torium W. C. S. nie4 meet v~rwa:eh~an. En ju~st om4a~ nu m~t mijn a.~treden dit tabera.torium blijkbaar een gehee] am.de,ran weg ~a.1 uitgaan, zooda$ daar- mee a. h. w. e ene bepaalde periode vaa zijne gesehiedeais is afgesleten, is m. i. nu de tijd gekomen, de. gesehiedeais van deze inriehting van a.f hare stiehting t,ot heden in 't ko.rt to besehrijven. De droevige aanleiding tot de stiehting van he* phyto- pathologiseh laboratorium Willie Comme,lin Scholte~ werd in den eer~tan jaargang van dit Tijdsehrift ui~eengezet. (Zie deel I, bl. 7 tot 12). Als eerste Bestuurders traden hadst den tIe6r an Mevrouw Scholtea op de Ileere~ Prof. Hugo de gries, F. B. L6hnis 6n Ern.s~ H. Xrelage. 30 I Nada~ onder~eteekende op 4 :Februari 1895 als Diree- beur was ge,instal]eerd, en du,s a]thans in naam hot ]abor'a- torium was g eopend -- o,nderget,ee.kende a anvaardde eigen- tijk zijne betrekking eerst in den zome,r van ,lit jaar en bleef zoo lung te Wageningen wonen, -- was her ee.rste werk van he.t Bestuur, n aar den toenmaligen Minister van Binnea~- la~udsche Zaken, dan Heer M' van Houten, te gaan, om *.e traehtan eerie sul~sidie voor de stichting te verwerven ; aaa- gezien de middelen, welke door de stiehters waren besehik- baar g~steld, onvoldoande waxen om een phytopatkologiseh laboratorium t~ scheppen, da,t, eenigszins aun ma• eisehen kon vol'dee~a. De Minister was niet ganegen, eene zoo hooge subsidie, te verleenan a]~s word aangevraagd, maar toonde zieh bereid, in 't vervolg de jaarlijk, sche sore van [ 500, die ik in den laatsten tijd te Wageningen als pevsoonlijke toe]age geuoot voor he.t gratis g.even van inliehtingen op phyto- patho]ogi:sch gebied, van a~ 1896 re, stellan ter be,schikking van hot p.hys ltaborato.rium Willie Commelin Seholte.n. Hij meende evenwet dat dan de Heer LShnis, die al.s II~speeteur van hot Landbouwonderwijs toe~l ook in zake subsidies ann partieuliere i.~stellingen ten nutte van den Landbouw den Minister van advies moest die,nan, niet langer in her B estuur van de stichting zitting mocht hebben. Doze word door den Heer P..F.L. Waldock te Loesduinen vervangetu, van wie,ns wer'kzaam leve~ eehter ongelukkige.r- wijze reM~ na eeal paar jaTeal de draad word afgesaedan. HET PHITOPATHOLOGISCH LABORATORIUIII WILLIE COiilHELIH SCHOLTEli VAH 1895 TOF 1906, En zo~staken wij in ~en zomer v,~ 1895 van w al. Her pereeel l~oemer u n ~ we~d door de familie Schol6en aa~ d~ stichting ve.~'huurd; her .diende voor mij e~ mijn gezin als woonhus tevw;.jl 66ne verdiepi~g (be~taaad~ uit twee kamers en suite an ee~ kte;a kamertje) als l aborator~um w~rd ingerieht. Een p l, an~enkasje van den ruin van de familie Scho]f~n werd bij den ruin van m~jn hu~s g~trokk.en, en werd be,sternal o,m di~nen voor h~t he,men van infeetieproe~en. Een aa=tal objec~n vo,ov onderz.oek en voo~ dem~nst~a.tie bij he,t nni- versitair .ondevwij,s, d~or mij t~ Wagenimgen ve~zameld, werden in her laboratorium gedeponeerd ; en ik stelde mijne vrij uitgebreide phytopathologisehe bibli.otheek ter di, spo- sitie van de stichting. I)aa~doo.r wet& he~ mogeaijk ge- maakt, zoowd lint o=derzoek als de college,s ann de. Univ~r- siteit to bcgi_na~en, zonder da~ eenigszins be~angrijke onkos- tea veer instal~ati~ noodig waren. l~aboratori.n~ te ~an.vamxle~. En zo~staken wij in ~en zomer v,~ 1895 van w al. Her pereeel l~oemer u n ~ we~d door de familie Schol6en aa~ d~ stichting ve.~'huurd; her .diende voor mij e~ mijn gezin als woonhus tevw;.jl 66ne verdiepi~g (be~taaad~ uit twee kamers en suite an ee~ kte;a kamertje) als l aborator~um w~rd ingerieht. Een p l, an~enkasje van den ruin van de familie Scho]f~n werd bij den ruin van m~jn hu~s g~trokk.en, en werd be,sternal o,m di~nen voor h~t he,men van infeetieproe~en. Een aa=tal objec~n vo,ov onderz.oek en voo~ dem~nst~a.tie bij he,t nni- versitair .ondevwij,s, d~or mij t~ Wagenimgen ve~zameld, werden in her laboratorium gedeponeerd ; en ik stelde mijne vrij uitgebreide phytopathologisehe bibli.otheek ter di, spo- sitie van de stichting. I)aa~doo.r wet& he~ mogeaijk ge- maakt, zoowd lint o=derzoek als de college,s ann de. Univ~r- siteit to bcgi_na~en, zonder da~ eenigszins be~angrijke onkos- tea veer instal~ati~ noodig waren. Intussche~ bleak alra~ dat de ter be.schikking ge,steldo middel,en ve~l re, geeing w~rem, om bet lab orat~).rium eenigs- zin~ te doe~ zijn ~ut her incest worden. De ruimte was ve~l ~e bepe.rkt, voo.ral teen weldva hn de c.olle~tie~ ~n de bibllotheek (de ~aatste dee~s door a ankoos dee~s door ruiling van her ~ Tij~chri~t ove~ Plantemziekte,n r te,gen aadere, recast buite~lan@sche tijd,schriftea e~ ve,rhanddinge,n) zich sterk uitbmid.dem. Alles ken wddra nie~ m,e~r worden eeborgen ;de n.o~dige instrumenten konde,n nie,t wovdtm aan- geschas ~,n omdat de. middel~n ontbraken ~n omdat ex geen paaa{s veer w.as. HET PHITOPATHOLOGISCH LABORATORIUIII WILLIE COiilHELIH SCHOLTEli VAH 1895 TOF 1906, Met ingang 1899 erl,angde hog ]abor~to.rium eene zooda- rtige verhooging van subsidie, da,t hot voor 't vervolg voor de helft door de Re geering werd beko.sLigd, waarbij eehter de~e --zooals billijk w~s- zich meet invloed op dan g~ng van zaken verzekerde en ~evans den phyt,opa.Lhologisehen dienst in 't loven riep. en ann hot labor~torium verl~and. Pro~. Hugo de Yri,os, die toeal juist midden in zijne belangrijke onder- -- 31 -- zoeking~n omfrealt hot ontstaan der soorien zat, on die vree.sde, d at ]let Be,sttmrslidmaatschap hem na de uitbrei- ding, die hot laboratorium zou onde,rgaan, veel meet yam zijn ko,stbaren tijd zou ontrooven d:an tot du~sve.r he4 geval was, a,eht|c ]ret raadzaaan toem als Voorzltter van he.t Bes"0uur te bedanken. Zijne plaats w~rd ing, enomen door Prof. Went te lN, recht, e,n di~ van den Heer Waldeck door M ~ M. Tyde- man te Breda. tier past mij hier, e,e~ woo,rd van hulde en da:nk te bvengen a, an ProL de Yrie,s, zoowel voor hot werk- zame aande,el, dat hij -- naast de stiehters -- met wijlen den Heer J. H. Krelag~. heeft gehad in hot tot stared komen va.n her laboratorium, als voor de aangename wijze, waaxop hij als Voorzitte,r van hot ]~e,st~ur, st,e,e,cls met mij samenwerktr Toen die Rege,c'ring aan d~e stiehting ee.ne sab,sidie, eerst van / 500, late,r van / 1000 ver]ee~nde, benoemde zij a]s haren vertegenwoordi~e.r in he.t B~stuur den beer LShnis; toen de subsidie tot / 7000 we.rd verh oogd, werde,n twee Re.gee- ringsgedel(,g, eerd,en in hot Bestuur b,en,oemd, n.1. de tIeeren Lovink en LShnis, me,t de bevo'egdhe,id de vergade,ringe,n bij te wmmn e:n (le jaa,rlijksehe rekening van den Pen~ning- me.ester, den tI.e,er Seholte,n, na te zie,n. ()ok n.a.<l.at eel,st- g~mo.emde, hot ambl van Direeteur-Gesleraal van den Land- bouw had aa.nva.ard, b.leef hij, niet~egen,sta:ande, zijne vole a.ndere v.e,ehmlvatt,ende, werkzaaanheden, als ge.delegeerde der ]{eg'eering zit+ing houden in bet Bestuur. -- Bij (le opricbting w,erd door de s.t.iehters een k.a.pitaal van / 100,000 g,estomt: een kapitaal, waarvaxt de re:nte natuurlijk op venue na nie,t voldoende was om hot, labora- torture t.ot sta.nd t,e br'e~ngen en te onde.rho.ttden. Da~.r eehter de. familie Sehol:te:n zich evenwe.1, zij he.t ook niet sehrif- tdijk, verbonden had om ja~arlijks ongeve.e.r (le, re,nto van hog eene sore wm/ 100,000 bij te passen, had ik geen 1)(,zwaar, de betrekking van Direeteur wm hot to stiehten --32 -- l~aboratori.n~ te ~an.vamxle~. HET PHITOPATHOLOGISCH LABORATORIUIII WILLIE COiilHELIH SCHOLTEli VAH 1895 TOF 1906, Er m&dd~n zich v~rschille=de pemsone~ san, die, prak~isch in mijn laboratorium wenschten te wee- ken; nlaar slechts bi] uitzo~deri~g ken aaaa dien we~s.oh wordea gevolg gegoven, g'aar de ruimto z66 belmrkt wa~ d~t, op de uren waarop ik zel~s marx 66n enkel porsoon hag om to 33 -- weN;on, mijne eigen werkplaats daarvoor moest win den inge- mired, lk herhmcr mij, dat ik in die dagen eens een l~ez~){'l; kr~,g v;m een vakgonoot, con hekvndc, gels,re'de. Xa.dal wij evn poos haddcn zitten praie~n, g~,f hij mij ziju" wense}l t.e l.:ennen, mijn lahora.iarium C.pll8 te beziehtigen; hij keek vle;,md q~, h)en hij hoorde, d,t de suit.e, waarin ik h',,m had ontvunge,n, zoo o.ng~we<,r mi,j,~ gelw.clo la.!mra- torium was'.-.- In de evrste j~m,n (retying ik reel bl~z,')eken, niet deehts van l,'avii~.i en van vakgen(,oien, maar vom'al ook va.n kijl~grage nieuwsgie.rigen, die gu'~rne ('~'ns de sli,.h- ting van &, familie Seholteal wensvht~,n h, bezMfligen; m.e.t, her fond, leiden van (lie pct..,oltell gtng %lke.ns reel van mi.in ]mslbaren tijd verloren. Ik kan niet aan andv,r(,n opd,'agen als geleider te funge.eren, omdat ik toen geeu Ixu'sonee]' had, da{ iels van phyiopaihologisdw zakmt afwist ; en boven(lien zmt mij dat niet reel hebben gegvven, daar ik toeh zou hebben moeien werken in helzelfde lokanl, waar he,t bezoek was. ]-)at de llijkssubsidie van / 500 wel.dra op / 1000 wer,l gelmwht., lmatt~, nYt vee.1, a;ulgezien de middelen toeh to beperld l)lcven, .am te krijgen wat ik allereerst noodig had, n.1. uitln'eiding van lokaliteit en van wetenschaplxqijk ge- vormd persone~,l. Torn de tleer .~I ~ Sieke.sz, do toenmalig, o I)ireeteur- Gener;ml van 1,amllmuw, mij d,e opalraeht wihlo verstrekken mn eene reis naar .Xcm'd-Amerika te oadernemen, hm eindo na Iv gaan war mlze l~.egeoring ~e doen stond naar aanIeidhlg w~n hot San aos6 .s(.hihlluisgevaar, wees ik e r gen(u,md(,n l{e(,r op, dat als ik ,le door (le l~geering to verstrekkea Ol)dra.(~ht aa.1..vmmhh', he( labm'at.orium tijdelijk moest wnr- d.(,n gesh)ten, vn dus het ger van inliehtinge.n aan pra(.Ii~.i, zoo hmg ik weg bleef, moes~ worden gestaakt. De ]leer Siekesz, die met mij van meening was, dat eigenlijk reeds 3 m 34 lang de Regeering voor e,en phyto,pa~ho.logiseh Ins6tuut had mo,eten zorgen, gaf mij de. toezegging, daL hij bet e r zou tra,ehten hee.n re. HET PHITOPATHOLOGISCH LABORATORIUIII WILLIE COiilHELIH SCHOLTEli VAH 1895 TOF 1906, leiden, dat aan h.et phytopathotogis, ch labo- ratorium Willie Commelin ScholLen eeme, .Rijkssubsid{e, zo~ worc~e,n verleend, hoog gealoeg om ee,n adsistent te b,eno,e,men, die later--wanne.er hij op de hoogte van he* yak zo~ zijn gekomen -- mij zou kttn~ebn vervangen, als de llege,e'ring eens later v~eer be,slag op mij wenschLe te leggen, o,f als ik om ann,dare reden afwe.zig mo,es,~ zijn. Ik wee,s e r den Heer Sickesz op, dat de be,noeming van ee,n' a:dsistent noodzake- lijke,rwijze met zieh meebracht uitbre,iding va,n lokaliteit en van hulpmiddelen van ondc~ozoek, d aar ~n die. e,eale bn de anderen reeds voor mlj all,ee.n onvoldoende waren. Na mijne terugkom,st mt Amerika werd afgesproke,n, dat de. tIe.er Sicke,sz zou voorsielleJa, aan he.t phy~opatholo,gisch labo.ra- to~rium Willi.e, Commelin Seholte~ eene jaarlijksche sub- s~die van [ 7000 t e ver scha:ffe.n, wa,arvoor dan de, genoe.mde stlehting 66k zou moete~n zorgen voor 4e uitoe~enlag van dean i~tussehen noodig geble.ke,n phytopatho,logischen dienst. De wetgevende maeht keurde den post van f 7000 voor her phytopathologisch laboratorium goed. De, tteer Sickesz wenschte, e ehter niet te tred,en h~ her v erla:nge:n 4er stichters, o,m a.an de. il~riehting voor eenige ja.ren a ehter66.n e,e~ne, zoo- danige ,sub,sidle. ~e ve,rlee.nen ; want bij eerie, eventueele orga- nisati.e van het Laadboawo~derwij.s van den hoogs~en trap w ensehte hij de handen vrij i~ h ebbea, o m d~i~raaa, als zulks noo,dig bleek, een phytot~ath.o]ogisch laboratorium t,~ verbinden, als wanneer uit d,r nard der za~ak de Regeering ~eene ,snbsidie meer zou ver~een,e~ aan e,e~e pa.rticuliero stiehting. De sub,sidle va,n [ 7000 ging in met 1 :l'a.nna.ri 1899. De. loka.li~eiten van her hboratorium we.rden nn aanzienlijk uitgebreid, en we]. doordat van nu a~ aan .deze. stichting van 35 den Heer Scholten twee naast dkander g.d,ege,n huizen h uurde t,ege,n eenen huurprijs van / 3000 pe,r japer, tte4 e.en~, huis (l~>emer Vissch(~rstra,at n ~ 1) werd g.eheel als laborat.orium ingerieht, e,n aan di,t huis we,rde'n verb,onden twev ka.mers van he.t. daa.ra,a.n gre.nze, nd~e pe,ree,e.1 (n ~ 3), het- we,lk overigens al.s mijn woonhuis funge.erd.e. Her sou.ste,r- rain va.n h.et labm,a.toriuni werd als woning van een' eoneie,rge, ingerieht.. La:aIstg~enoemde trad in 't najaar van 1899 in rune.tie; de a.dsi.stent (d.e Hem' C. J. J. wu~ Hqll, to,e,nmaals ~,andidaat in de. plant eu dk!rkunde) aanvaardde zijne betr,ekking op 1 Dcemnb.er 189!1. Met h.et o,og o.p (l.e. HET PHITOPATHOLOGISCH LABORATORIUIII WILLIE COiilHELIH SCHOLTEli VAH 1895 TOF 1906, be~slomme,ringen, die ik ia 't v.ervo]g me.t, de.n phytop,a.t,hologi.svh.e.n dietlst zou }td)b.e.n, on.~l.e.rging mijn saa'aris e,eno, verho~ging. De,z{,gi~lg in in d(m zomer 1899 ; tm-aqjl de salari,ss.en wm den adsisl:e.nt e:n de.n eoneierg.e inginge,n op den ifag, waarop zij hunne be,trekking aanvaardden. D,c, verho.ogde, t{ijks.subsid, ie koa du,s, voor e,en gedee.lt.e w,o,rden b.{>steed voor d.e. aansehatfing van de, noodzakelijke meubelen e,n w.et.en.sehappelijke iitstrume,n• ee,n. ander d.e.el ervan werd gebruiM om verande.ringen nan te bre,ng, en in gas- e.n wate.rle.iding an .a.1 war ve,rder novdig wa,s, om h.et p.ere,e.e.1 Ro.emer Visseherstra.at n o 1 tot ]abora.torium in te richten. Van ],u af aan e,ers.t kon her phytopatho.logiseh la.bora- torium 1,Vii:lie Commelin Seho]te.n, war zijn,e inriehting b.e.treft, aan billijke, h,,ewcq n, og matige, e,ische.n vohlo,e.n. Door he4. ~,erhoo.o,d.~ budce.t werd ik in sta.a.t ge,steId, i~dor jaar de. wet.enschagpelijke~ hulpmi.dde,len en d,e b.ibliotheek tot gro.o,iere uitbreiding te bre,nge,n; er waren a,fzond,e.rlijlm laborat.oria voor mij en mijnen gd.sist,e,nt, een behomqijk inge.rieht museum, kame.rtjes voor d.e. biblioth.eek, ee.ne werk- kame,r voor lie,e,rlingen, e,e,ne donkere, kamer, enz. -- 36 Eehter bleef de inriehting c~en greet g(~brek aankleven : hot ontbreken van bijkans iedere gelegenhcid om besmet- ting~pro(;ven t~., nemeu. Hot t)lant~c.~nka~je, door den Heer Seholten aan hot laboratorium afgesta~m, mawr mqnvank(.~]ijk door hvm met mijne ve, rgunning ia gebruik ~houden w~nr zijne eigen plan~en, word ook op de.n duur niet o:ni~imd, ]m.ewvl ik op de noodzakelijkheid om een plantenkasjc hu' b~,sehikldng ~e hebben, herhaaldelijk woes. We] werde~t l;ogingen aangewend, om met behulp van e~ne a,fzondcrlijke Rijkssubsidie e,en nieuwe kas te bouwen in den aangr~n- zenden ruin van den Heer Seholten; maar doze poging+n mislukten. ],~en reel groo4er bezwaar hog dan hel, gc~mis van een pla.ni.enka~je, was hot algeheele gebrek "ran fem~in. W:el l)evond zieh aehter hot labora~oiium e~'n klein ]apje growl, een Amsterdamseh stad.stuintje; maar besehaduwd als hot wet;1 d,or tauten en hooge booliwn, was hot veer mijn (l()el w)lslagen cI~ge.~ehikt. Wcl werdeal door mij in ver,sehil.. lende Mreken des lands proefvclden aangele~d met 't. o~g op bepaa]de da'u' heerse.hende pluute.nziekten, en ken ik door de welwi[l.e.ndheid van e(~n paar Wageningsehe lee.raren over e~nig terrein van de. l~ijks ]toogere I_am(l-, Tuin, en :Bosch- bouwschool be~(.hil&en, -- maar dringende behoefte bled beslaan 'ran ('(,n stuk grt)nd in de onmiddellijkc nabijtmid van hot ]t~boratorium, hetwelk zonder tijdverlies op ieder uur va.n d~,n dag ken worden bezceht. HET PHITOPATHOLOGISCH LABORATORIUIII WILLIE COiilHELIH SCHOLTEli VAH 1895 TOF 1906, In dit gemis ken onmogelijl; worden voorzien, zoolang hot laboratorium in de Roomer u gevestigd bleef; want z clfs met opoffering van v~el gehl zou 4aar in de buurt geen terr~,in te krijgen zijn g~weost. Alleen door verplaatsing van de inriehting na~r W,~tcrgraa.f.~meer of naar e.ene der buiienbuurten vaal de gemt~ente Amsterdam zou men missehien terrein in de onmiddellijko nabijheid 37 vail h,et labo,ratorium hvbbe,n kunn,e,n krijgen; en daar zou- den e,en laboratorium, evne dlr'ecteurswoning e,n e,e,n/erreln voor proefneming zeJ~e,r niet mc,er aaln huur ge,koM hebben dan de twe,e perc,e,Nen z.nd,er terrein in d,e R~<'mer Vissch,er- stra.at. tHet groot.e ine:onvenient wm h.e,t, gemis wm t,e.rrein en plan f,enkas da.argdat.en, kon overigeals van af 1899 he~, phy- topaflMogisch l:abora.torium te Amsterdam, war zij,n,e, inrieh- tiHg b.etreft, aan billijke hoewcl matige, eisd~en vohlo,en. .~[e~ in:gang van 1 Dec'ember 1S99 was d.e ttc,er van Hail, candidaat in d.e plant-en dierkunde aan de Universitei~, van Amsterdanb tot adsi.s~.ent beno,emd, l)a.ar ,l,e,ze :n e.en.e, groole sta<l geboren cn gefogen was, wa,s <le ]lmdbouw ~,l zijne ver'schill.cmd(, v~,rt.akkingen lre,m gehe.e,1 vre,eend geble- yen ; en he.t sl)r:eek! wel van zelf dat :le. phyt,o.pa,tho]oog met. alleea met 1)oI,a,nis(,he e~t zoi~logisehe ke.nnis kan voi.st.a:an, ma.ar dat: hi] (ok met de t,>di der vers,qfillende land- en tuinbouwg.e,wassen ,q~ de hoogte mo.e.t zijn. ],;e:l jong bota. nitieu,s, di(~ gdle.e] w'e.emd .st.a at t.egeaover d.e, pra.ktijk, he.eft -- al is hij ~,ok uils,l.eken,'l in ziin yak als zoodanig thui,s-- jaren noodig, ,eer hi.i e.en go,e,(] ld~ytopalholoog is gew(,rden. Daa,r(mboven k,on m i.in a,d,sistent zieh n iet :]a(lelijk me{ de 1)orst op h~t,g'o,~,:n hem onlbr'a,k Ioele,ggen, daa,r hij hog m,et zijn do.ctoraal examen . in de maag" . zal.. ])aa.rna kwaan h,e,f bewerke,n van zijne dissertat,f,e, waa,rop hij in k[ei 1902 promoveerd,e. In di.en tusschent,ijd had hij e,en t,ijd lang in her baeleriologis~'h ]~aboratorium van D ~ ~Basenau te Am,st,erdam gewe:rld, e,n daarmt on,d,er leiding van I?ro~. Beye,rinck t,e, 1),e,lfl. Re,ed,s in 1903 ver'trok D" vaal IIall naar ~urina.me, waa,r hij op voordra.eh|, van Prof. Went tot , lnsp.e,eteur van den Imli,lbouw ~J was lwno.emd. Hij vertr.k dus op c.e." ti,i d, /,.on h;j .juist begon ge:schikt h~, wor&m, mij van een ge.de,elt,e, mijner ta,ak te, ontlasten. b 38 }Iet volgende jaar werd de tIee r A. W. I)~rost tot Land- bou~adsisLent a~m ho~ landbouwkundig laboratorimn in Suriname bencemd ; h ij vertrok daarheen in September 1904. HET PHITOPATHOLOGISCH LABORATORIUIII WILLIE COiilHELIH SCHOLTEli VAH 1895 TOF 1906, J)e Heer Drost was van de oprieht, ing v:m het phytopatho- 1.cg~sch labora.torlum W. C. S. af nan dez~e inrichting ver- bo nden geweest. : eerst als amanuensis, ~aler onder den titel , teehniseh beambte D. Uit de praktijk wm den tuinbouw voortgesproten, had hij zich ]angzamerhand meer welen- schappelijke kemfis eigen gemaakt ; en hij was nu veer her phytopa.thologisch laboratorium een zeer bruikbare, kracht geworden. Inderfljd werd geschreven dat her veer mij vleieM was, dat twee ann mijn laboratorium verbonden personen in beh~ekkingen a.an het nieuw opgerichte landbouwlabo- ratorium t,e Paramaribo werden benoemd; dit daargelaten, zal t, oeh moeten worden erkend, d~t hun vertrek, juist teen zij hunne voorbereidende school hadden doorloopen, oor- zaak was dat ik veer mijne ]nrichting slecht.s betrekkelijk weinig val: hunne hulp profiteerde. De I[ee r It. 3i. Qua~ljer, tot opvolger van D ~ van tfall bermemd, aanvaardde zijne a.dsistenfsbetrekldng in 190~, eel,st nadat hij zijn doet:n'a,al examen in pharmaeie ,'whter den rug had en even w;dr hij zijn praldisch apr~t,he- k(,rsexamen deed. Deze Heer ken zieh (h's dadelijk gehecl aan zijne tank wijden, en deed dat voorlaurend met grcoten ijver. Afkomstig uit eene kleine pleats, had hij reel op 't, platte land verkeerd en belang leeren stellen in al war het land- en t.uinbouwbedrijf aangaat. De lezers van bet , Tijdsehrift over Pla.nt,enziekten r hebben reeds met een paar zijner pmmevruehten kennis gemaakt en zullen spoedig a.ndere onderzoekingen van hem ]eeren kennen. In plants van den IIeer Drost verleende mij in de laatsto paar jaren de tIeer C. B. va.n der Zeyde ts Zaa'ada,m hulp al~ amanuen~sis. m 39 -- Ten slotte, zij her mij vergund met e,en enke,1 woord mijn jonge,n vriend, de.n Heer W. H. Obreen te gedenke.n, di.e eerst, e.en tijd ]ang als ijvorig leerling mijne eo,lleg.es volgde en als labo,ranf in he4 laboratorium werkie, en later-- zonder ooit ,'flh.ci,ee] a.an d,e inriehting ve.rbond.en fe zijn gewee,st -- zieh voor ha,at zeer verdi.enstelijk maakte, zo.o door he{ doen van sommigc onderzoekingen, als door hulp bij de bepro.e- ring van b.e,strijding~smi.dde.len, do.or hot verva.a,rdigen vaal photo.'s en door een begin f,e :maken me,t hot eal~a]ogiseerel~,. van d,e. bibliotheel~ der htrielfl,ing e,n her sa.m.~mste,llen van eene phytopathologis(:he bibliographie. Helaa.s ([,e,ze ijvo- rige, bereidwillige en bekwame jo:ngding weed t.e mid.den va,n zij n,e, werkza.aa~he,id door e,e:ne ern.st.ige, ziekte ov.e.rvalle,n, die hem ten grave sleepte. Zijne nagedaehtenis zal bij mij in dankbaar aandenken blijven. HET PHITOPATHOLOGISCH LABORATORIUIII WILLIE COiilHELIH SCHOLTEli VAH 1895 TOF 1906, -- Thans ga ik over tof de b,espreking vaa d,e werkza~am- he,(]en, ge,durmlde mijn Direetoraat van wege her phy~o- pa, thologiseh la,b.orah~rium ve.rri~'ht. Yolgens a~:t. 2 van de stieh~ingsacte, wordt dit labora- {orlum di,e,n,stba,ar gemaakt : (~ a,an he,t geven van inliehtingen aan d~ beoefenaars van &m landbouw, den luinbouw e:n d,e, houtte.elt, o,mfrent de oorzaken van ziekfe.n en besehadigingen vaat voo,r di~ vaklmn behmgrijke g.ewa,ssen, al~mede omtre,n{ de mi(hle.lelt Oln ,leze, ziekten en be,sehadiginge~l te voorkomen en fe be~sfrij den ; (c a,an d,e uitbroiding (ler k.e,nnis van plante,nzie]~ten en sehade,liike die,ren door weien,sehappelijk onde.rzoek; (( a,an de. belaaqgen van h.e,t cnd.erwijs van de. stude,nte,n in de plant- e,n dierkunde a.a,n :]e, UniversiIeit. van Am,ste*- dam, en ill 'f a]gemee.n van hot onderwijs in de phylopatho- logle ~.- -- 40 -- War be~reft de aanvrageu om inlichtingen omtreat pla.nt~enzieI2~en en schaddijkc dim, en e.n ocn.tre.nt &. mid.deleal let bestrijding, ]~an worden geconstateerd dat deee gedu- rr her tienjarig bestaan der inriehting vrij geregeld a.an- zienlijk zijn toegenomen. Dit kant eenigszias blijken uit her aantal in de versehil- lendc jaren ingekomen en uitgegane brieven. Dit bedroeg : Ingckomen. Uitgegane bricvcn in 1895 ? 376 1896 553 607 1897 659 756 1898 667 788 1899 753 927 1900 960 1192 1901 975 1156 1902 1169 1355 1903 878 1084 1904 1157 1359 1905 1098 1272 Ik wil daarbij her volgende doen opmcrken : 1 ~ Versehillcnde vragen om inliehtingen werden mij mondeling ged'aaa ; deze werden bij de uitgegane brieven op- gel.~.hl, nie.t bij d.e ingekemen brieven. Vo~)r ten deel is he~ d55raa, n toe i.e sehrijve.n, dat- blijk,ens bove.nsiaande tabel -- ie.de.r jaar her aaatal uitgegane brieven her aantal ingekomen brievan hie% onbelangrijk overtrof. 2 ~ In ddn brief we.rden herhaald, elijk ve.rsehillende vr,l- gen bealttwoord ;ann d.cn anderen kant eehter warener onder ~.le, ingekomen an uitgegane b rieven ook dezulke van admi- nistratieven aard; zoodat de boven aa.ngegeven ~.ijfers geen va, wk(',,,'i,/ overzi~'ht ge.ven van d~. uitg~.,br~.idheid van den inlichling.,:dienst in de ~'erschilJe.n.dc' jaren. Toch geven 2 ~ In ddn brief we.rden herhaald, elijk ve.rsehillende vr,l- gen bealttwoord ;ann d.cn anderen kant eehter warener onder ~.le, ingekomen an uitgegane b rieven ook dezulke van admi- nistratieven aard; zoodat de boven aa.ngegeven ~.ijfers geen va, wk(',,,'i,/ overzi~'ht ge.ven van d~. uitg~.,br~.idheid van den inlichling.,:dienst in de ~'erschilJe.n.dc' jaren. HET PHITOPATHOLOGISCH LABORATORIUIII WILLIE COiilHELIH SCHOLTEli VAH 1895 TOF 1906, Toch geven 41 deze ('ijfcrs duide,lijk genoe,g eeno belangrijke toe~eming a.an van dit ged.e,el~,e, de.r werl~zaamhede.n. 30 Bij he,t. jaar 1898 dient t,e wor&m opgemo~'kt, dat ik gedurende, dc maa,nde.n April, 5[ei en Juni, -- dus in e.en' tijd, wann.e,er gewo~nlijk de vragen om inlichtingen her tal- rijks( inkomen -- op reis naar Amerika was, waaM~or her laboratorium toen gesloten werd; zoodat her aanta.1 vragen om inlichtingen anders zeker aanmerkelijk grooter wa,s geweest dan nu her geval was. 40 De aanmerkdijke stijging van her a,an;tal ingekmnen en uifgega.n.e, brieven van a{ 1900 lig{, fen d,eel.e, in de, invoc- ring van den phytopathol.ogischen dienst. 5" I)at d.e. inlh~hfings;dienst, in 1903 min,der druk wa.s dan in he.t voorafga,a.nde, en in h.et volg.ende, jaar, wa.s gelie- gen ill vers~!hilleltd,e om.staatdighe.:!en. Yoore,erst i.n h.e,t felt, dat ,le we.e~'si~tvloeden ee,r'st zeer laa.t in 't voorjaar veroor- loofden, te zaaien; t,e.mvijl de zoo laat g=e,za.ai:le, g.ewas.seab e,e,mnaa.1 o.pgeko,mcn, zich me,esta! snd o,n~wikkeld,e~n. Te~l twe.ed.c, was de groot.e vochtigheid van d.en zmner oorzaak, flat zich b.elrekkei.ik w.einig inseldenpl.agen wm.rd,ede,n; tcr- wijl h.et weer w.el voehtig, ma.ar Met warm gc:no.eg was, om v.e.le s~ort,en van pava,sitaire zwammen ~ot st.e.rke, oaflwikke.- ling en ve.rmcerdering t,e bre,ngcn. Ten flerdc is ook zdler h.e,t ~'eit. dat D ~" wm I-la.ll w.eldra naar Suriname z, cm ver- trekken, .e,n dat hij in ve,rba:nd daarmee ottd.e.I~Sdieidt',n r.e,e:is begonn.e:n corr.espondentie~s .over be.pa.ald,e, p~alltenzidd,en Met voort.z.r van invloed op he.t aa.nfal ingeko,me,n en uifg,e- gano brieven gewee,st. Zo,ov.c.~q l~].ijkt wel, dat gedurend.e.d.e jaren van mijn Directora.a,l her a a.ntal vragen o,m inlqchtingen vaal den kant der praetici zo.t!r aanmerke,lijk is toogenomc'n. Vel.e pl'avti~'i zomh',n g~,w,g:,,hl al ],el n,~q'l,:wa.;mlig~; wal bij l,*'n "!; 't ge,bied van d,c sehad,clijke dieren e:n pla.ntenziekt, en 42 N voorkwam, ~]thans voor zoover het hun onbekend was; en her aanfal van hen, die vaa i,n]iehtinge,n van den kant van her phytopafhologisch laboratorium g~bruik maakten, nam gele,idelijk aa.nmerke]ijk tc,e. ~leer ell nmer begon her phyiopathologiseh laboratorium W;llie Commelin Seholten, althans voor d.e ontwikkelde land- bouwers en kweekers, en ook voor tie groote grondeige- n.aren, de geregelde, vraagbaak te word(m op 't ge, bie3 van plantenziekten en sohade]ijke dieren. Ik ho~)p dat her Instituut voor phyl.opathologie, dat thans onder mijne lehling die iaak zal vom~zetten, steed.s mevr in rela.tie me,t de praetiei moge komen. Va,n her. ideaa.1, dat in smnmige Staten tier Amerikaansehe I~publiek bereikt is, n.1. HET PHITOPATHOLOGISCH LABORATORIUIII WILLIE COiilHELIH SCHOLTEli VAH 1895 TOF 1906, t]at de Staatse.ntomoloog, t}e Staatsphytopatholoog of de deskundige van bet proefstation (experimeat station) met alle land- bouwers of ooft.telers in zijn gebied' ge.re~ld in retatie staat, -- van dat ideaal zullen wij hier te lande hog wel 1,angml tijd ver verwljdertl blijven ; en wel omflat _Xe(lerland, naast groote landbouwers en groote kweekers, een onnoeme- lijk aantal kMne boeren en kweekers left, temvijl in de meeste Staten va.n Amerika het groot grondbezit, ook voor den ooft.teler, regel is. Maar wanneer wij een.e verge.lijking in.stelIen tussehen he.t gebruik, (]at in Nederland in de laat- ste jaren door de praetiei werd gemaakt van her phytopatho- togiseh labosatorium en dat, hetwelk in andere landen van Europa van de ~ Auskunfststellen fr Pflanzensehntz ~ en dergelijke inriehtingen gema.~kt wordt, dan staat zekvr .Nederland in dezen mee bovenaan. En om een ove.rzieht te hvbben van wat bier te laade tegenwoordig dv phytopatht~- logie v~r den land- en tuinbouw beteekont, is he.t hie4 vold'tumde, dat men nota neemt va,a bet saiee0s toeinemende a.anta]: vragen om inliehtingen, d, io va.n de zijde de,. prat.t.M kwamen bij den Direebeur van lint phytopathologiseh labo- 43 m rato.,'ium te Amsterd,am, - ook vele ]~ijkshmdlmuwlem'arm~ vn llijkstuinbouwh,(,rar(m zijn op ,lit gebied met: vru('ht we,'l~zaam, to,'wiil zij i,, t,,'ijfolavh(igo govallvn mijne hulp inroepen, en mij verder gvregehl op de h(~gte houden "van wat ~r in hun ambts~ebied "root merkwmmli~zs op phytoln~- lhologisch gebied v,mrkomt. Stuns wt'rd dora' welenschal~l~.elijke, buiten de prak- ti,jk slaan(b, l~]alltkundigen (1(, meening uitgesproken, dat ]wt gewens('hl zmt zijn. &, werk:,;mmheid van den ])irv~'teur vl.lll het phyl,,palh(~log'isch iaboratorium, war aangaal bet vers(.hathm van inlichting(,ll aan pra~.tiei, It' b(~l)erlwn, l'h'n w(,lmlsdmppelijk g',,vo,'md persoon zml daa.rvom' eigenlijk (, {t, g,~e.:l ~ zi.in, ziu t iid te I:.ostbaar, daar hi.i diml b(,ier zou kunn('n lw.st,*d.en. ]I~,rhaahlelijl~ kmm,n tdkc, ns wt,er vra~'(,il onl/l.'t,:~t dt,- z~,lt',l~, zit~ktt, of h('tzr s('haddijke dier in; cn hv,t, ;s gt, c'lt :.lall~(qlalll(, ('l!. OC.ll(, vri.i onvruehtbare taak, allijd weer hei- zdf& t.e m~'t('n s(,hrijven; p.ers(mml, die wel.enschappelijk mind('r hong siaan, l<unnen dat werk best doen; de weten- sdml,p,q:ijk hooge," st~mmle p('rsonml moelen zi(.h met nle.pr zuiver w(~t.(msdmpl~,'lijl{ werk bezig houden. 11 I, d;!t sian,lpunl .s.taan inde.rdaad x-vrs,.hillen,le l)irve- leu.ren van lfllyt,qmlo]ogische l:~bm'aiol'ia. Zoo is bet-- of zoo ,'a.~ hvt althans e(,n l~ .i;~,'en g~,l~,d(m - - geslehl in h('t . lmb,,ral.oire de Imthologie vdgdt,ah, ~ ond('r d~ d{re~'ti(., 171111 (l('ll ovm'igens z('t,r v(,rdimlst~,lijken Prof. D" .l';d. Pril- lieux. HET PHITOPATHOLOGISCH LABORATORIUIII WILLIE COiilHELIH SCHOLTEli VAH 1895 TOF 1906, Prof..llug'o de Vries g'.:'(fi in ,lt,~,l I van h,,t , L:~nd- lmuwkundig Tijds(.hrift ~ (18.1.,, hl. 217 (,nz.) vene b.e..~('hri i- ring van dal lalmralm'ium. Op bl. 220 z('gt hij: , !Iier (,n ,laar zagr ik kis jes st, aan, waarin monsters van zit'ke kultuurpIant, en ing, ezonden waren. Voor zoover ]t('t ])('- ken,]~, zi&t(,,~ zi.in, win'den &~ze aan de leecl/,,l~. ~, ,'.'('~ , t.e.r ,leterminatie en zoo mogelijk ter beantw,;or,ling (1(,1' d,~,u" 44 don inzendcr ge~stelde vragen ~. Men g-ing aan her ]abom- torium van Pri]lieux blijkbaar vaaa de meening uit dat bij her geven van inliebtingen aaa praetici, de (lelernfina.lie, de bepaling van den aard en den naam van den parasiet, clio de ziekte veroorzaakt, de zaak is, waarop her eigenlijk aan- komt; en dat wat men dan aan (h,n praticus te schrijvvn heeft,, in de lrandbo~q~ell kan worden opgezoc.ht. don inzendcr ge~stelde vragen ~. Men g-ing aan her ]abom- torium van Pri]lieux blijkbaar vaaa de meening uit dat bij her geven van inliebtingen aaa praetici, de (lelernfina.lie, de bepaling van den aard en den naam van den parasiet, clio de ziekte veroorzaakt, de zaak is, waarop her eigenlijk aan- komt; en dat wat men dan aan (h,n praticus te schrijvvn heeft,, in de lrandbo~q~ell kan worden opgezoc.ht. Net is waar, dat nu en dan vrage.n omtrent d.ezel'fd,e ziekte inkomen, die op dezeIfde wijze kunnen wor~len beant- woord ; en her zou monnike.nwo:rk zijn, wannee,r de ])ireeteur of een wetensehappelijk gevormde adsisteat pe, rsoonlijk al de brieven a]s antwoonl daarop ging sehrijven. Een vroeger aan oen ander adras gesehreven brief kan dan gevoegelijk d,oor con' amanue.nsis of s~'hrijver wor~len get"Olneer~l, natuur- lijk evcntu~e] onder aanbrenging van de noodzakdijke ver- a.nde.ring~n. Maar dat gaat in zeer vele gevallen niet: wan- neer de bepaliag van den aard der ziekte of be.sehadiging is afgeloopen, kan op verre na niet altijd al het overigc in de handboeken wordcn opgezoeht: e.n het laten beantwoorden wm brievel~ over zi&ten, die vaker voorkwamen, kan dus lang niet ah.ijd aan leerlingen of ondergesehikte beambten wordeu overgtflat.en. Imme.rs war men den praetieus ter bestrijding va,n eene of amlere ziek/e of bc, sehadiging meet aanraden, hangt op verre na niet uit.sluitend af van den aard tier ziekte of wm de soort van parasiet, die. haar veroorzaakt. HET PHITOPATHOLOGISCH LABORATORIUIII WILLIE COiilHELIH SCHOLTEli VAH 1895 TOF 1906, War zou de ]aaldbouwer gezegd hal> ben, wanneer hem vanwege her phytopat.hologiseh labora- ratorium de road ware gegeven, 9oft te zaaien[ Ik geloof dat her geheim, waarom in Nederlaaqd meor saanoa~wea'king tussehen her, phytopathologiseh laboratorium en <le pm<.tioi }>estaat: d'm in de mc~ste andere ],anden, groo- 46 tendee]s juist d55rin schuilt, dat her geven van inliehtingen aan de mannen der p~,a.ktijk niet d,oor mij als een ]ast.lmSt we.rd aangezien, die. maar tijd cnt~ad~te aaa wete..nsehappe- lijk werk; maar dat juist (]it gedeelte va.n de werks.aaan- hede.n steeds al nommer s vaa her. progra~ma werd be- s('houwd (1). Ik weet zeer goed, (lat ik door mij van di(.~ a.dviezen maar walt vlug af te ma.ken of door die grootendee]s (1) D r L. Reh is in een zeer lezenswaardig opstel in . Zeitschrift f~tr wissensehaftliche Insektenbiologie 9 (1905. IIeft 7) er tegen opgekomen, dat in Duitschland altijd juist planthundigen aan 't hoofd van phytopa- thologisehe instituten worden geplaatst ; hij meent dat dierhundigcn daarop minstens evenveel aanspraak kunnen doen gelden. Zijn ideaal echter zou d~rin bestaan, dat men phytopathologische instituten stichtte, ~ an deren Spitze tin Landwirth steht, und an denen wissen- schaftlich selbstst~indige und einander gleichberechtigte Abteilungen die botanisehen, zoSlogischen, usw. Aufgaben ilbernehmen, so dass die ganzen Anstalten doch gemeinsam auf das eine grosse Ziel, die Fiirde- rung des far unseren Natioualwohlstaad so ntltzlichen Pflanzeaschutzes hinarbeiteten .. Hij beweert dat echter een dierkundige met evenveel of met meet reeht aan het hoofd van eea phytopathologisehInstituut kan staan als een plantkundige, cn gaat aldus voort: 9 Dass die ganze Disciplin dabei nicht schlechter fahrer wtirde, lehrt uns unter anderen das . Laboratorium Willie Commelin Scholten . in Amsterdam unter der Leitung des Zoologen Ritzema Bos, das far den Pflanzenschutz etwa soviel leistet wie sttmtliche en~sprechende deutsche Anstalten zusammen .. D r Reh kan hier natuurlijk niet her oog hebben op de wetenschappe- luke onderzoekingen, die aan de Duitsche inrichtingen en die, welke aan mijn laboratorium hebben plaats gehad ; bet spreekt toch van zelf, dat ~dn enkel, slechts aan matige eischen voldoend laboratorium zonder kassen en terreinen, waarin ik gedurende het grootste gedeelte van de tien jaren van zija bestaan~ 5falleen werkte 5f met geringe hulp, op weten- schappelijk gebied niet ken praesteeren wat talrijke, veer een gedeelte zeer goed ingerichte laboratoria met tal van wetenschappelijko perso- hen er aan verbonden, kunnen doen. HET PHITOPATHOLOGISCH LABORATORIUIII WILLIE COiilHELIH SCHOLTEli VAH 1895 TOF 1906, De ge~ardheid van den bodem, de ontwikkeling'speriode, waarin de plant verl~eert, de tijd van 't jaar, de vrueht- opvolging, de soort of variSteit van 't gewas, d,qt men teelt, en zoovele andere omsta.ndigheclen zijn van veel bet.eekenis met bet oog op den raad, dien men moe, t geve,n. Een vom'bevld. I~ermmsl~or{~Sn worden algcm(,~n mt't sueebs door bespuiting met B~m?~,auxsehe, pap beMl'(~den. Stel nu dat wil&e of Lalhyrus sylvestris Wagneri wordt 45-- ingezonden, aangeta.st, door Pe,.oz~ospora Viciae, en men s{elt r iuzending , ter Mdoening ,~ in handen van een lee,fling ~f een minder deskundig beambte, dan zal deze in negen~ig wm de tmnderd g~vallen adviseerml : be.spuit den akker met ]/ordeauxsehe pap; de landbouwer, die zoo'n advies krijgt, sehudt her hoofll, en komt misschien later niet mere" tenlg om advie, s; want de kwaal vetacoont zieh ~rst als 't gewas re.eds ho(~g is opg~s<'hoten, e.n dan is her onmogelijk z(>o'n l>eqmiting {e volvoea'm~,- daargela%zt hog dat men liever de pap niet brengt aver planten, die over niet al te langen tijd als veevoe.der zullen wordcn gebruikt. Zoo spoedig mogelijk afmaaien, -- zied.aar her advies, da.t iaa dezen ge.geven moest zijn; want sleehts bij uitzondering t, ree.dt &m in de tweed(, sn(~le her kwaad weber op. 't l~mvensta.a.nde is geen gefl'a.geerd geval ; bet heeft zich werkelijk ii~ mijne, praM, ijk v~orgedaa,n, d.~t een la,boran,t, in mijn laboralor;um den raad wihle geven, een flink opge- greet.1 wikkenveld met Bm'deauxsehe pap te be,spuiten. Een ander geval, dat zich eens voordoed, was her vol- gende. ~I{et, vitriolen ~ van her zaaizaad is bij taru'e een uit~te.kend midd,el om her opt,rede,n van bra,nd re, voo,rlmmen. Ec, n jongm<msl:h, di~ bij m U we.rkte, wilde ook ~dviseere.n, d~*, 9c~',*t~ a,htus t,e behamlelen, om her optreden vaaa ger, s,te- bra,nd tegen te gq,au, rl'o,en !.k hem att.~nt maa.kte, op her bozwaar, dot d,e gerstekorre,ls door hare kafjes mnslote,n blijven, en de bra,ndsporen zieh binnen de kafjes kuo.nen be.vinden, wilde hij in Mien ernst, d~ gels.t, pellen om ze daarna te vitrioh'n. (1) D r L. Reh is in een zeer lezenswaardig opstel in . Zeitschrift f~tr wissensehaftliche Insektenbiologie 9 (1905. IIeft 7) er tegen opgekomen, dat in Duitschland altijd juist planthundigen aan 't hoofd van phytopa- thologisehe instituten worden geplaatst ; hij meent dat dierhundigcn daarop minstens evenveel aanspraak kunnen doen gelden. Zijn ideaal echter zou d~rin bestaan, dat men phytopathologische instituten stichtte, ~ an deren Spitze tin Landwirth steht, und an denen wissen- schaftlich selbstst~indige und einander gleichberechtigte Abteilungen die botanisehen, zoSlogischen, usw. Aufgaben ilbernehmen, so dass die ganzen Anstalten doch gemeinsam auf das eine grosse Ziel, die Fiirde- rung des far unseren Natioualwohlstaad so ntltzlichen Pflanzeaschutzes hinarbeiteten .. steeds heb ingenomen als Directeur van her phytopathologisch labora- torium Willie Commelin Scholten, ook bij bevoegde beoordeelaars in bet buitenland waardeering vindt, niettegenstaande sommige geleer- den en napraters van dezen er mij wel eens een grief van maakten, dat ik niet meet . zuiver wetenschappelijk ,, werkte. HET PHITOPATHOLOGISCH LABORATORIUIII WILLIE COiilHELIH SCHOLTEli VAH 1895 TOF 1906, Waar hij er van spreekt dat mijne inrichting alleen , [i~r den Pflanzenschutz 9 etwa soviel leistet wie die samtlichen deutschen Anstalten zusammen, heeft hij zeker her oog ge- vestigd opwat het phytopathologiseh laboratorium Willie Commelin Scholten veer de:praktijk is geweest. En moge D r Reh bier ook al over- drijven~ niet zonder voldoening zie ik dat bet standpunt, 't welk ik - - ,17 - - aan soms n,og onervaran adsi,st,entan of aan leer]ingen oxer ~.~ lagen, meer tijd had overgehouden voor wet~nschappe]djk onde,rzoek, en a]licht meet naam had! gema,al<t in de, weten- sehai~pelijk,e, w,eretd ; ma,a,r ik weet o,ok, d'a,t ik o,p die wijse de phytop,a,t~mlogie niet bij de pract~ci he,t, b~rge,iweeht had doen verkrij gen, d a,t ik mind~r tot nut vaa de bodemkultuur i.n Nederla.nd had ge,werkt. En &i,'irom was her mij te doen, - - niet in de eerste plaats om naam te maken in de weten- schappelijke werehl. Trouwens her geven van inliehtingen aan praetici wordt in de st.atuten van bet phytopatTaolo,gisch laboratorium }V. C. S. her allereerst genoemd onder [le werkzaamheden van deze inriehting; en stellig ha,d o.ok de Ilegeering g.een aanleiding gewmden, in de laatste jaren de helft vau bet jaarlijksche budget voor hare rekening te nemen, wanneer de stichting niet in de eerste plaats w,or den Landbouw had gewerkt. -- I~eeds vddr 1895, maar la,t, er i.n s,te,e,ds toe~mraea~de mate, weed h.erha.ahle.lijk mijn advies do.or de, Re,geering ge,vraa~gd, ni,et a.tleen in zake phytopaiho~ogisehe, quae.st.ies, maar in 't algeme.en in allerlei zak.en, de, t.oegepa:s.t.e natuur- lijke hi,st~orie be.t.reffeade. In 1895 wa,s ik met. dea tte,er Bult,man afgewmrdigde de,r Nede,rlandsehe t~egeering naar eene eonfe,rentie t~e. Parijs, waar her. vraa,gst.uk van de, inter- 1rational.e. vo.ge.lbescherming we.rd b.esprokeaL In 1898 z~.nd mij d,e Rege,ering naqr d,e Yere,eaigde Staten vaa No~rd- Amerika, Oill e,oi}_ o,nderzoek in te 8telle,n a,angaande, de San Jos~ sehildluis en a.angaande, d,e be,t~eke~l~s, die. dit insekt voor oas land e,n in 't algemeen voor Europa. zou kunnen krijgen, tier door mij" ultgebraehte rapport w'r- schecn niet a.]l~n in de Staatscourant, maar werd ook op last der Reg,,eering in 't Duitseh vertaald. Dat mijn advies ook door her. Bes.tuur van ]let Land- bouwcomit6, door Besturen van land- en tu~nbouwgenoot- schappen en hare afdeelingen, d:oor den Directour der Ned. HET PHITOPATHOLOGISCH LABORATORIUIII WILLIE COiilHELIH SCHOLTEli VAH 1895 TOF 1906, tIeidem.aat,sehappij, door den Inspectenr van her Staatboseh- beheer, door de Rijkslandbouw- en Ilijkstuinbouwl.ceraren, door Directeuren van de Rijkslandbouwproefstations en van onderscheiden inrichtingen voor land- en tuinbouwonder- wijs herhaa.ldelijk werd gevraagd, spreekt ,,'el van zeLf. 0ok buitenlandsche vakgenoofen wonnen herhaaldelijk mijn advies in. Een aanbod van een aant.al landbouwers in Suriname, om ter plaatse een ond.erzoek in to stellen naar den aard der krullotcn in de cacao en naar de best.rijding der ziektc, werd door mij na rijp beraad afgewezen, daar ik mij dam'voor ~anger nit Nederland zou moeten verwijdel, c.n, dan met de belangen mijner bet.rekkingen hier te lande zou zijn in overeenste.mming to brengen. Herha.ahMijk trad ik bij verschillende tentoonstellin- gen als jurylid op voor de afdeelingen land- en tuinbouw- onderwijs (m -onderzoek. Bij gelegenheid vail d~n in 1904 en 1905 te Wageningen gehouden wef]strijd van pu]verisa- fern's fung~erde ik als voorzltter der jury. Her. nitg(~- braehf(~ vers]ag over dezen wedstrijd is te vinden in den XI ~" jaa~'gang van her ~ Tijdse.hrift over Plantenziekten ~, bl. 81-96. Ilapporteur was de heer S. Lako. In de tweede plants werd, overeenkomstig de staiaien, her phytopathologiseh labora~orium Willie Commelin Sehol- ten dienstbaar gemaa.kt m'm de vitbreidin.g der 1,.ennis van -- 4 9 ~ pla.tc~ziekte~t cn schadelijke diere~ door wctc~schappel~jk oml~ r~ocl,!. Op dit gebied heb ik mlnder ge,l,everd &~n ik gaarne zou hebbea ged,a,a~n. De inliehlingsdienst eischte do org~ans te re,el ti.jd om ]ang ~ehtere.en aan wetonsehapp,elijke on(ler- zoekin.o, en te kunne,n werke.n. Toeh zijn ve.rseheiden onderz.o,ekinge.n (~p phytopatho]o- g~s(.]l g~bied gMaan. Ik voor mij he,b da, a~rbij .c,eMer l~ooit uit het o.og verloren~ dat ik wefl~z~lam was hi he,t be4ang van de praktijk. Het kost e, en w et,ellsehappelijk pe,rsoon wel een~ e,e,nige, zelfopoffe.ring, om de e,e~ne of a,ndere kwe.stie, die zi, eh g,e.durende e,en o.nc].erzoek w)ordo,et en we,lke uit e,en zui- vet wc4e.nsehappe,lijk ~Jo.gpunt wel van bel,aag zou zijn verder te. vervolgo,n, to lat,en rust,o,~l, wanneer blijkt, dat daa.ru[~ voor (~e praktijk 1clefs verder te hqlen zou zijn. Overigens kunnen weer sores zaken, die uit een zuiver wetensehap- pelijk oogpunt van weinig belang zijn, voor de praktijk zeer belangrijk wezen. Of bijv. HET PHITOPATHOLOGISCH LABORATORIUIII WILLIE COiilHELIH SCHOLTEli VAH 1895 TOF 1906, van eene bepaalde sehihl- luissoort hog beweeglijke larven op de boomen voorkomen, wanneer deze in 't najaar de bladeren reeds hebben verloren of althans w'mneer de bladeren reeds opgehouden hebben te fungeeren, kan van gr~ot be]ang zijn uit een oeeonomiseh oogpunt; want de tijd, waarop men de boomen met een insecticide dient le beslmiten, wordi erdoor bepaald. Do versi,agen, jaar]ijks van v~ege, her phytop~tlmlogiseh 1,aboratorium uitgebraeht, geven een overzieht ()ok van de wetensehappelijke onderzoekingen, die daar werden verrieht. Daar zij eerst in her (c Landbouwkundig Tijdsehrift ~), later in bet ~ Tijdsehrift over Plantenziekten ~ werden gepubli- eeerd, behoeft bier daarop Met uitvo.erig t,e word~n t.erng'- gekomea. Ik wil tlier sle.ehts de b.e,lsngrijkste, publieaties even vermehlen. ttet ziju d~ volgenfle : 4 50 Ritzema Bos: over bestrijding van de dennenbastaardrupsen (Tijdschr. o. rl. z. ~, I, bl. 13-19; ~ Forstlich-natur- wissensch. Zeitschr. ~, 1895, IIe.ft 4). Ritzema Bos, over ([en, Amerika~mschen kakkerlak ~, scha- delijk in plantenkassen en de besIrijding wm dit insekt. (,, Tijdsehr. o. Pl.z. ~, II, bl. 22-28). Ritzenm Boa, over inspuiting va,rt bealzine in den bo~lem, als mi(Mel tege,n scha(lelijke iz~sek~en. (~Tijdschr. o, Pl.z. ~, II, bl. 28-43;~ 'l, ijdschr, o. Pl.z. D, VII, bl. 37;~ Zeit- schr. f(ir Pflanzenkrankh. ~, VIII, Ileft 1). 12itzema Bos, ove.r ]lotrytis Douglasii , sehadelijk aa.n kweek- de~tnen. (~ Tij,lschr. o. Pl.z. ~, IIi, bl. 6; ~( Forstl. nat ul~qssensc.h. Zeitsehr. ~, 1897, Heft 4). H. ,I. Lovink eal Ritze,ma ltos, ond,elwoekingen omtrent her ge,slach~ Reli,r~ia. ((( Tijclsehr. o. Pl.z. ~, III, bl. 8,3-134 ; ~eve.ns gepublieeerd a]~s uitgave, (le,r Nell. Hei, dema~tseh. en verd.er in cc CenLralb].,a,tt Fir Bacteriologic, u. Para- sit~nkuaade, ~, II, Abt. 150, 1903, n ~ 8). Ritzenaa B as, ove~" Botrytis Paeo~iae Oud., oorzaak waa ee~m ziekte bij pmen,en en Canva;]la.ri, a's. (~ Tijdschr. o. Pl.z. ~,lII, bl. 150-157; ~ Zeitsehr. fiir Pflanzen]~rank- heiten ~, VIII, Heft. 5). Ritzcma Bos, voortz~tting v,'m vroe,gere si.udie.s omtreaat aaltjeszi~ktc~, o. a. , Tij(.tsc.hr. o. Pl.z. ~, V, bl. 29, VI, bl. 46; X, bl. 110; XI, bl. 144; ~ Zeitsehr. {(ir Pfl,~nzenkrankh., XIII Ileft 4 ; XIV lIeft 3. ttandelin- Hen van h.et achtste Ned. Satuur- en Geneesk. Congres, gehouden ~ ]h)tterdam; 1902, hi. 180). ]litzema Bos, over eene bacterieziekte de,r syringen ((~ Tijd- so,hr, o. P].z. ~, V, bl. 177). llitzem,~ Bos, over kmfllo~en e.n heksenbezems in de Cacao- boomen in Sttriname. (~ Tijdsehr. o. :Pl.z. HET PHITOPATHOLOGISCH LABORATORIUIII WILLIE COiilHELIH SCHOLTEli VAH 1895 TOF 1906, ~, VI, hi. 65 ; Zeitsehr. f. Pfla~nzenkr,~'-~kh. ~, XI, Heft 1). -- 51 -- Van ][all, over bnclerieziekten in ~!~ ko,ol en l~ij muurbloe- men. (, Tijd.schr. o. l q.z. ~,, VI, bl. 1(i9). l{iizema Bos, cc Zur I~ebensgeschichte ties Muulwurfs ,,, Biol. ('.vntralblat.t 9 XVIII, n ~ 2. ltitzema Bos, .vet her vuur in de nareissen en zijn.e In,sir;j- ding (c, Tijdsdn'. o. lq.z. ,~, YII, bl. 12; ,, Zeilsd, r. fiir Pilanzo.nkranldleit,,n ,,, lid XIII, 2 lleft). van Hall, over eem, ziekie in de syl.ingon, vel om'zaald do.or l]olut:i.; c,l:/a,/.*. (,, Tijdschr. ~. lqantenz. ~,, VIII, hi. 142- l-t-i). Ide <m l{ilzonm Bos, over be.sfrijding van lu, re- en apt)e]- sdmrit. (,, 'l'ijdsdm o. Plantenz. ,, VIII, bl. 149-177). l{i4,zema. 13os, over het wegblijvo~ en omvallen der tulpen, vorom'zaakt &~or Botrytis p,rasitiea. (~c Tijdsehr. over Plantenz. ~, VIII, b]. 177-202; 9 Cenlralblait f/it lla~'- teriologie u. Parasite.nkunde ,,, II Abt.. Bd X, 190:i, n ~ 1-8). I{itzema. Bos, over her ontst~qn van giftatoffen in plant en- deelen, d.ie door parnsitisehe zwammen zijn aangetast, of door anderc o~rzaken zich nie.f nm'lnaal konden, onf- wikke]en. (,c .llygienisehe Bluden ~, 1901, n ~ 1, 2 en ;{). vim llal], l)iss0ria.tic, over baefcrie.&~ p]a,nie,nzi.ekie.n, 1902. wul IlalI, c,nqu61e. ,mt rent Ophiobolu.s hm'potrichu.s. (, Tijd- .~]u'. o. Planfvnz. ,, IX, l)]. 77-97). l{itz~,ma llos, ore.,' Monilia.ziekle.n de.r o.oftboomen. (~ Tijd- schr. o. Plan~enz. ,), IX, bl. 12.5-1,I7). ]litzema Bos, bijdrage tot de kennis van tie sehurftziekie van den aardappel. (Zie Itandelinge,n v:m her 9 ~ Xeder]. Natuur- en (~eneosk. Congres, gehouden fe's Graven- hage, 190:~; bl. 226). vaa~ Ilall, die Sant.t Joharutis krankheit (ler Erb,sen, verur- saeht yen FusaT'ium t'asi~dectum (, ]~eriehte der deut- schen botanischen Gesellschaft ~, 1908, Bd. XXI, Heft 1). -- 52 -- l~itzema Bos, ]~ankerstronken en va]lers bij de koo]',, veroor- za,akt door Phoma olcracea. (, Tijdsehr. o. Plante~z. ,, X, bl. 53-71, XI, bl. 106-118). Drost, over Pleurococcus vulgaris, a.ls e~dophytiseh ]eve.nde wier. (, Tijdsehr. o. P]a.ntenz. ~, X, bl. 71-73). Ritzema Bos, Onvruehtbaarheid bij zwarte bessen. (, Tijd- sehr. o. Pla,ntenz. 7), X, bh 137-145). Ritzema. Bos, over sehade d,oor huisj'esslakken. (, Tijdsehr. o. Plantenz )), X, bl. 145). Ritzema Bos, over de oorzake,n van slechte kiemba.arheid van in 1903 gew(mnen zaad. (co Tijdsehr. o. Pa.nte~z. ,, X, 5o bl. L~; XI, bh 124). Quaaje.r an Vfirtheim, onderzoekingea omtre,nt r:ookbeseha- diging re, Schiodam. (c~ Tijd, schr. HET PHITOPATHOLOGISCH LABORATORIUIII WILLIE COiilHELIH SCHOLTEli VAH 1895 TOF 1906, o. Plantenz. ~, XI, bl. 170). H. M. Quanjer, over Plutella cruci/erarum. ((( Tijdschrift voor enLomologie, deel XLIIX). H. M. Quanjer, over Plutella cruci/erarum. ((( Tijdschrift voor enLomologie, deel XLIIX). Aan de b.ovensta,ande opsomming van g edane onderzoe- kinge~ en in '~ lieht gegeven publieaties sluit zich aan eene opsomming van de volgealdo gedure~de 1895-1906 uitge- k H. M. Quanjer, over Plutella cruci/erarum. ((( Tijdschrift voor enLomologie, deel XLIIX). H. M. Quanjer, over Plutella cruci/erarum. ((( Tijdschrift voor enLomologie, deel XLIIX). Aan de b.ovensta,ande opsomming van g edane onderzoe- ki i ' li ht bli ti l it i h Aan de b.ovensta,ande opsomming van g edane onderzoe- kinge~ en in '~ lieht gegeven publieaties sluit zich aan eene opsomming van de volgealdo gedure~de 1895-1906 uitge- geve,n werken : Ritzema Bos, q Ziekten en Be,sehadigingen der Kultuur- g~wass.en )), 2 deepen. (Groninge,n bij J. B. Wolfe,s; 1897 e n 1898); 2 d~ d~*uk o~der den titel 9 Ziekten en Besehadigingen der Landbouwgewa.s~sen ~. 2 deelen. (Groningen, J. B. Wolters; 1902). Ritzema Bos, 9 Zi.ekten en Be schadJigingen dev oo~boo- men ,, 4 d,ee.]en. (Groningen, bij 5. t3. Wo]ters; 1905). Ritzema Bos, 9 Zi.ekten en Be schadJigingen dev oo~boo- men ,, 4 d,ee.]en. (Groningen, bij 5. t3. Wo]ters; 1905). Rite,zem,~ Bos, a Sehoolplate~l van voor den akke,rbo,uw seha- delijke dieren ~, met Handleiding. (Groningen, bij J. B. Wolter.s; 1905). Rite,zem,~ Bos, a Sehoolplate~l van voor den akke,rbo,uw seha- delijke dieren ~, met Handleiding. (Groningen, bij J. B. Wolter.s; 1905). Ritzema Bos, 9 Zoologie ~iir I,a~dwirte ,. (Berlin; Paul Parey), 2 ~ druk 1896, 30 druk 1900, 40 druk 1905. -- 53 -- Van de Engelsche uitgave van (lit werk,vertauld door Prof. Aiu~worth Davi~, vc.rscheen de 1" (lruk ill 1894, de 2 ~ in 1900. l~itz.ema Bt~s, ~ Schet.am uit het ;..even de.r Vogels ~, (Am- sler(lam, Va.n den ]h'oeeke, 1897.) Van de Engelsche uitgave van (lit werk,vertauld door Prof. Aiu~worth Davi~, vc.rscheen de 1" (lruk ill 1894, de 2 ~ in 1900. l~itz.ema Bt~s, ~ Schet.am uit het ;..even de.r Vogels ~, (Am- sler(lam, Va.n den ]h'oeeke, 1897.) In de derde l)laal,s moes|, volge.ns de statuten, het l)hy - topafllologis(.h labm'atoriunl Willie Commelin Scholteu wor(len (liensibaar genmakt , a.an (lc belal)gen van her, (mdcrwijs van tle stu(lenten in de I)lant.- eli dierkunde aan de l'niversiteii van 3.m,ier(]mn, en in "t algeme.en van het onderwijs in de phylol)athologie ~. Met. HET PHITOPATHOLOGISCH LABORATORIUIII WILLIE COiilHELIH SCHOLTEli VAH 1895 TOF 1906, uitzondering van twe~- jaren, waarin doer mij ge(m college werd gegeven, werden door mij in iede.ren em.'~us in t wee uren l>er week onderseheiden hoofdstukken uit de zie.k- tenleer (ler t)hmt.en belmndeld. Aanvankelijk wa.s (te phyit;- patho]ogie veto' nicmand een examenvak ; later werd zij dat veer hen, die het do(.toraal ex'unen in de 1)|ant- en (lierl~un(le wihIen afleggen. Ten ein.te (1(, examens niet te reel t(, verzwaroll, W('l'd c.ahter ge.woonlijk sleehts over een vrij 1x,p<.rli~ g'o,h'elte va,n de t)hyi.opathologie ge~xaminecr(l. Zoo k~os (I~ een h(~t m~(le, rwerp : galvormi.ng, - - eeu amler 3e zield.e.n, d.,,.or Ne.mai.oderL en Phyr t.e.~ree.g'ge.bn~.eht: --- ~,e.u d(.,.rde, de sehad.e.lijke zoogdiel'en en voge]s, --- e(,n vierde de [Tredineiql en Uslil'agine~n (roest- en bra.ndzwarn- men), -- weer andel,en k(.zen de I)|ani(~nziek~n ver<)orza- kende. Pyrenomyceten en ])iscomyeete.n; een koos de door zwmnmen ver(,orzaakh, ziek%n van (~onife.re.n on V;lll graan- gewassen : enz. Mijn<~ <'oll(~ge,~ werden in de verschillmlde, jaren door (x~n z~er ver.s<'hil]end o'etal studenten bezoeht; terwijl (Vt ge.tal (,en pa.ar jarpn ('('it 25-;I0 tal lx, droeg, waren er ook jaren van sleehts 5 hfn)rders; de mee.ste jaren bedrocg hun aantal (mgevoer (,en 12 tal. -- 54 De meeste hoorders waren studenten en candidaten ;n de plant- en die::kunde.; verder werden de colleges gevolgd door cenige pharmaceutcn, door en]:ele medic:i, alsmede door een p,'uar or~derwijzers en een' tuinbouwkundi~, die nict a.]s studenten waren ingesehrc:e.n. In aalrsluiting aan de colleges we,rden eenige malen in verschille,nde declen des laad~ i)hytopathologische excursion gemaakt. -- ttoewd over be]angs~elling van de zijde dcr stu.dcnten in 't geheel niet te k~agen viel, moet toch worden erkend, dat de colleges niet in alle, deelen aan hun doe1 l:on.den bca_ntwoorden. En gcc.n wonder l De studenten in de plant- en dierkun(le volgden de colleges om hunne na.tuurhisto- rische kenni~ t>e vermeer(tcren ; maa.r daar zij in 't algemeeu niet in de landbouwwetcnsehap thuis waren, kon de toepaz- sing van de natuurhis.torisehe studie op de bodemk:tltuur niet voldoende tot haar recht komen. De phytopathdogie is nu cenmaa] eenc toe.~Dqste wetenschap, wetker doel is, de t)roductiviteit vaa: den bodcm is vermeerderen door (le kwalen der kultuurplanten te bestrijden; zij baseert zich 66k op de plantkundc cn de dierkunde, ma.ar 56k op natuur- en seheikund.e, op de leer van de plantenteelt, en op de erva- ring der plantentclers. HET PHITOPATHOLOGISCH LABORATORIUIII WILLIE COiilHELIH SCHOLTEli VAH 1895 TOF 1906, De phytopathologie is gecn tak van de botanic, zooals vele plantkundigen schijnen tc mccncn, die zich voorstclI,en dat een bonanicus, wanneer hij e(m- voudig war extra, werk maakt van zwammen, zoo dadelijk phytopatholoog is geworden. Zij is evenmin een tak van de botanic, als zij een tak is van de zoologic, do physiea of de ch.emie. Die vakken zijn al te gadc.r grondleggea:d,> weterrschappen voor do phytopathologie, wdke zich zoowel op deze vakken baseert als op de praktisehe ervaring dcr kult, nurmannen. ~N'u zijn studcnten in de plant- en die.rkunde in de mecstc 55 -- gevallen te weinig met de praktijk van de verschillende takken van plantenteelt o p de hoogte, om de phytopatho- logie met vrucht te bestudeeren ; en voor de praktische resul- tat,en dezer to.egepa,ste wetenschap voelen zij dikwijls ook Met de wa.re be]angstelling. De studie de~' phytopatho,logie, knn a lleen tot ha ar 1~eeht komen aml ee,ne inrM~ting van Itooge,r Landbouw~o,nderwijs (Hoehsehule fiir Bodenkul~ur). Dit toegepaste yak te d,oce,e- ren aan eerie Unive~siteit,waaraan geon ]!and b,ouwin,s~dtuut is verbonden, komt ong.eveer op 't zelfde neer als : een college in ophthahnologi.e, te g.e.ven aan e,ea.e. IYniver'sit~eit zonder medisehe faeulteit. De Pro~essor voor dat v, ak zou voor lie stud,ent.e,n, die hoo.fdzakelijk de zoiilo.gie en de verg,elijkende physiologic, tot stndievak hebhea geko.ze.n, eea a.antal col- l.eges kun.n.en geven over de a.na,tomie en physio.l.ogie van her oog, waarva.n zij heel war nut zouden kunnen hebben; e, ll ook de studenten in de. physiea z,ouden met nut ao,odanig college van den ophthalmoloog kunnen volgen ;--maar noeh de. zoSlogea~ no.eh de physiei zouden door 't vo.lge.n va.n zo,o- da.nig college ophtha,lmologen wordea. Om o,phthMlnoloog te word.en, dient men eers,t mede,eljne;n te studee,ren ; daarna ka.n me.n zivh in e,ene b,epaaht,e riehting speeialiseeren. Om phyt,opatho.loog te wor3en, dient men vooraf pl:antenteelt te hebben bestude,erd, of althans op de ee.ne o.f a.ndere ma,nie.r zieh de kennis van de teelt wm de 'kultuurgewassen te hebben eige,n gemaaJd. De,n b.ota.nieu,s, die phytopathologie wil studeeren, ontbreeld ook meestal te veel de belangsIelling in de voordeelen, welke dit. yak den praeiieus trae'ht te ver- sehaffen; bij hem is de wetensehappelijke sfudie nommer 66n, en belangstelling wmr de praktijk heeft hij maar' al te vaak alleen zoo lang hij denkt, uit de ervaringen der prak- tijk aanknoopingspunten te vinden, die vtmr zijne studie van belang zijn. HET PHITOPATHOLOGISCH LABORATORIUIII WILLIE COiilHELIH SCHOLTEli VAH 1895 TOF 1906, -- 56 -- Ik heb dan ook meer ea meet mijne, colleges aan do Amsterdamsehe U.niversiteit, bij allo betlaagst~lling, die door HI{. ~.~oucle~nfen wer.d betoond, leeren besch, ouweal a~s eerie aanvulling bij hunne aa.tuurhisl,ori~che studia, -- ~liet al.s e~.ne opleiding l,ot phytopathologea; da.arvoor moe.~ ik de U~iversiteiten, zooals zij bij (ms zijn in~,,'ieht, ong'c- sehikl, a,ehten. Dat neemt natuurlijk niet weg, da.t zieh tamer de student, en aan eene Universiteitt personem ktmne~l bevinden, die door hunnen natuurlijken a.aaHeg, door hmmo belangst.elling in, en door de door h~n opgedano kelmis v~m (le versehilleade takken der t)laatenteell, wel dege.lijk geschiktt zijn, om goede phytopathologen te wof den. -- ~%devl, mijn veri.rek n.aar Amsterdam had ik aan do toenma]igo Hoog~,re Lind- en ]3oschbouwschoot t(~ Wagc- ningen her onderwijs in de ken_his der schudelijko dieren behouden, en ]afer, toen de wetenschappelijke cursus aan de Rijksfuinbouwsehool werd ingerieht, ham ik aaa die afdoc- ling her onderwijs in de gehede phytol~thologie op m;j. Dit, onderwijs bleef ik g,e.ven gedurende mijn tie.njarig ver- blijf to Amsterdam. -- Ten slotfo meende ik, dat her ook de taak van he~ phyl,opathologiseh ]a.bo~,n.torium behoorde, de kennis der plantenziekfen en schadelijkc dieren o.nder ]andbouwers, kweekers, ooft- en groeafe.telers en boschbouwcrs Iv verbre;- den, en de be]aagste]ling in de phytopathologie bij de prac- 'ciei t,e wekke,n. En ik lmb de e~'varing r dnt her le.vende woord in deaen reel vermag. Da.t de phytx~patho- logie m.eer e.n m e.er bij de praet~ei hier {e. laa,le bet, burger- ,~.5t hee.ft verk~gen, moet .,eer zeker 66k voor eea decl worden toegesehreven aan de voordrachten, die deor her personeel der stichting horhaa.lde]ijk in onderseheiden dee- len dcs lands werden gehou&m. u ble:'k het. heel nuttig - - 5 7 n te zljn, wann eer na afloop der ]ezing de gelegenhe~d werd opengesteld, d.en spreker om inlichtingen te vrageu omtrent phytopathologische kwesties. In stre ken, vaa waar vroeger nooit inze,ndingen kwamen, werden dikwijls na eene gehoud.en l ezing ve,rscheide,n trouwe corre,spoaden/en van bet phytopathologisch laboratorium aangeworven. Gedurende bet tijdperk van Sept. HET PHITOPATHOLOGISCH LABORATORIUIII WILLIE COiilHELIH SCHOLTEli VAH 1895 TOF 1906, 1895 t.ot Januari 1906 werden door mij voordra.chten gehoud,en te Am~sterdam (3 maal), tIaarl,em, Zaandam (2 maal), in den Bangerd, te Levuw0,rden, Groningen (2 ma,al), Oldehove, Warffum, Loppersum, Appingedam, Beerta, Winschoten, Seheemda, Zuidbro,eL Zuidlare.n, Nieuw Buinen, Gm~sse], (Iro.en]o, Wint~erswijk, u Ze]hem, Brummen, Zutphen, Yo,rst, Twello, Apehloorn, Nijkerk, Wageningen, Arnhe,m, Nijmege:n, Tiel, Buren, Cul,~.mborg, ]lijsenburg, Z,eist, Utre.eht, Ba,arn, Hilversum, Bussum, AaJ~sme.e,r (2 maul), Bre.uke.l.e.u, Leid.en, Bo,skoop, 's-Oravenhage, Rotterdam, Axel en Tilburg. -- Do,or D" vaa Ha].l werde~n voordrachten gehoude,n te Am.sterd'lm, Baarn, tIeinkenszand en Kruinin- gen ; door A. W. Drost te Ba.arn en te Bus.sum. D.e vo.ordra.chten werden d,oo.rgaans gehond~e.n bf met st,eun van de Nedea']and, sche phytopat.hologische Ve,reeni- gMg, voor we]ke ook D r He.[nsius e.enige ]ezingen hie.ld, bf met st~un van de e.ene of aadere land- of tuinbouwmaat- schappij. -- Ten .sloit.e moet.en al~ een,e, hoo,gst, be~angrijke werk-. zaamheid va,n her phytopathologisch labora,torium worden be.schouwd de be,zoeken, do.or he.t t)ers, on.eel afgelegd bij d,e hutdbouwers, kwe,ekers, ooftt.e,lers, enz. ]),aar, o,p bet ar- beid,ste,'rein der onderseh.ei&n praetiei, is ge]egenhehl te (~ver niet, alle.ea om inlichtinge~ {,e v.e,rstre,kken omtrmd de daar voorkomemle, ziekt.en en beschadigingen en mnirent hare be,strijding, maar .er is ook ge]egenheid, om kc~nni:, to 58 -- maken met. ziekten en bcsehMiging~.n, (lie den bezoeker tot dttsver onbekend waren. Ook doer men daar reel speeiale ]/ennis op omtrent de ku]tuur der gewasscn onder verschil- lende omstand.,igheden : een eersie vereischte voor hem, die met vrueht als phyt()patholoog wil werkzaam zijn. Yan d(.' gelegenheid om praetiei te beT, oeken en de voorl~omende zk'ktcn en besehadigingen in l.oeo waar te nemen, werd dan ook veelvuhlig gebruik gemaakt. Wanneer ik aan he~ einde va.n mijne werkzaamheid a]s Direeteltr van her phyiopa.thologisch la.boratorium Willie Commelin Scholten hog eens e,en blik sla. op (le tien jaren, die achier mij lighten, dan kan ik met eenig~ vohl~ening getuigen dat her uitgezaai(le za.ad wortel hecft geseholen. ])e phytopathologie heeft zieh eene plaais wet~n te veroveren bij hen, die zi(.'h in h'ede.rland met (te bo(lemkul~uur bezig hou(len. De opriehting van bet: Rijks Insiituuf voor phyfo- pt~t.hologie, verbonden aan (le Rijks Hoogere Land-, Tuin- en Bcsehbouwsehool te Wageningen, zal mij in staat stellen, omler gunstiger omsfau.dighe,.lea te werken dan tot (lusver. HET PHITOPATHOLOGISCH LABORATORIUIII WILLIE COiilHELIH SCHOLTEli VAH 1895 TOF 1906, Ik hoop dat her mij gege.ven moge zijn, aan deze nieuwe inriehting nog eenige jaren in her belang wm de v~3erhl.nd- sehe bo(lemku}{uur we.rkzaam te zijn, zoowel (loor her geven va.n adviezen en d~)or her geven van omlel~vijs in engeren en ruimeren kring, alsook door wetensehappelijk onderzoek. pp j J. Rn'z~.:~a., Bos. Wageningen, Maart. 1906. Wageningen, Maart. 1906.
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Bioremediation efficacy—comparison of nutrient removal from an anaerobic digest waste-based medium by an algal consortium before and after cryopreservation
Journal of applied phycology
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Cronfa - Swansea University Open Access Repository This is an author produced version of a paper published in: Journal of Applied Phycology Cronfa URL for this paper: http://cronfa.swan.ac.uk/Record/cronfa36103 p Silkina, A., Nelson, G., Bayliss, C., Pooley, C. & Day, J. (2017). Bioremediation efficacy—comparison of nutrient removal from an anaerobic digest waste-based medium by an algal consortium before and after cryopreservation. Journal of Applied Phycology, 29(3), 1331-1341. http://dx.doi.org/10.1007/s10811-017-1066-x * Alla Silkina a.silkina@swansea.ac.uk; alla.silkina@gmail.com Paper: p Silkina, A., Nelson, G., Bayliss, C., Pooley, C. & Day, J. (2017). Bioremediation efficacy—comparison of nutrient removal from an anaerobic digest waste-based medium by an algal consortium before and after cryopreservation. Journal of Applied Phycology, 29(3), 1331-1341. http://dx.doi.org/10.1007/s10811-017-1066-x Silkina, A., Nelson, G., Bayliss, C., Pooley, C. & Day, J. (2017). Bioremediation efficacy—comparison of nutrient removal from an anaerobic digest waste-based medium by an algal consortium before and after cryopreservation. Journal of Applied Phycology, 29(3), 1331-1341. http://dx.doi.org/10.1007/s10811-017-1066-x This item is brought to you by Swansea University. Any person downloading material is agreeing to abide by the terms of the repository licence. Copies of full text items may be used or reproduced in any format or medium, without prior permission for personal research or study, educational or non-commercial purposes only. The copyright for any work remains with the original author unless otherwise specified. The full-text must not be sold in any format or medium without the formal permission of the copyright holder. Permission for multiple reproductions should be obtained from the original author. Authors are personally responsible for adhering to copyright and publisher restrictions when uploading content to the repository. http://www.swansea.ac.uk/library/researchsupport/ris-support/ J Appl Phycol (2017) 29:1331–1341 DOI 10.1007/s10811-017-1066-x Bioremediation efficacy—comparison of nutrient removal from an anaerobic digest waste-based medium by an algal consortium before and after cryopreservation Alla Silkina1 & Graham D. Nelson1 & Catherine E. Bayliss1 & Craig L. Pooley1 & John G. Day2 Received: 28 September 2016 /Revised and accepted: 17 January 2017 /Published online: 5 February 2017 # The Author(s) 2017. This article is published with open access at Springerlink.com Abstract An algal consortium was isolated from an integrat- ed steelmaking site at TATA Steel Strip Products Ltd. in Port Talbot, UK, and its bioremediation capacity tested. Excellent Bbioremediation^ was observed when the mixed culture was Bapplied^ to diluted effluent from an enhanced anaerobic di- gestion plant at Dŵr Cymru Welsh Water at Port Talbot, UK. After 5 days of cultivation in a 600-L photobioreactor, 99% of the total nitrogen (initial level, 4500 μmol L−1) and total phos- phorus (initial level, 690.4 μmol L−1) were removed from the waste stream. The consortium was deposited in the Culture Collection of Algae and Protozoa (CCAP), an international depository authority for microalgal patents, as CCAP 293/1. This material has been successfully cryopreserved using a two-step cryopreservation protocol with dimethyl sulphoxide (5% v/v) used as a cryoprotectant. On recovery of samples after 3 months storage at −196 °C, the specific bioremediation activity of the revived consortium was tested. The capacity of the revived culture to bioremediate effluent was not signifi- cantly different (p < 0.05) from a non-cryopreserved control, with 99% of total nitrogen and phosphorus remediated by day 4. Although non-axenic algal cultures have previously been cryopreserved, this is the first report of the successful cryo- preservation of mixed algal consortium, with validation of its ability to bioremediate after thawing comparing non- cryopreserved cultures with a revived post-thaw algal consortium. The study also highlights the need to ensure the long-term security and the requirement to validate the func- tionality of conserved inocula with biotechnological/ bioremediation potential. Keywords Alga . Algal consortium . Bioremediation . Cryopreservation Keywords Alga . Algal consortium . Bioremediation . Cryopreservation 2 The Culture Collection for Algae and Protozoa, Scottish Association for Marine Science, Scottish Marine Institute, Oban PA37 1QA, UK 1 Centre for Sustainable Aquatic Research (CSAR), Swansea University, Swansea SA2 8PP, UK Introduction There is a long tradition of bioremediation of waste streams by microalgae with early work undertaken by Oswald and col- leagues in the 1950s (Oswald et al. 1953a, b, 1957b) leading to the subsequent widespread use of high rate algal ponds (HRAPs) for effluent treatment in many countries. In addition to the capacity of algae in HRAPs to utilise nutrients, includ- ing dissolved nitrates and phosphates, some taxa can degrade toxicants such as high molecular weight polycyclic aromatic hydrocarbons (Juhasz and Naidu 2000). Furthermore, algae can adsorb heavy metals and other toxicants in aquatic envi- ronments (Gadd 2009). Current technology for algal wastewa- ter treatment uses HRAPs; however, low algal productivity (generally ∼10 t ha−1 year−1) and requirement for expensive processes for algal harvest limit the commercial exploitation of algal biomass (Craggs et al. 2011). With the development of novel technologies, this process has become more efficient and cost effective, for example the use of shallow, paddlewheel-mixed, HRAPs can result in productivities >30 t ha−1year−1 and better consistency of nutrient removal (Craggs et al. 2011). Furthermore, considerable amounts of research have been focused on the development of photobioreactors (PBRs) over the past two decades and a wide range of different designs and configurations have been built and tested around the world (Pulz 2001; Singh and Sharma 1332 J Appl Phycol (2017) 29:1331–1341 2012). Successful PBR cultivation systems, with high bio- mass productivity, have been demonstrated in high latitudes (Fuentes-Gruenewald et al. 2015). These have used semi- continuous cultivation approaches with resultant high nutrient removal rates employing Porphyridium purpureum and Scenedsmus sp. (Ruiz-Marin et al. 2010). to the algal uptake, phosphorus precipitation enhances the remediation process (Laliberte et al. 1997). A further advan- tage is that a mixed consortium has better ability to uptake carbon, as oxygen evolution by the algae facilitates aerobic bacterial growth and in addition many of the algae involved can directly sequester carbon via mixotrophic, or heterotro- phic, growth (Oswald et al. 1957a; Day and Turner 1992). The waste material from an anaerobic digestion (AD) plant was used in this study. This effluent contains high concentra- tions of nitrogen (in the form of ammonia) and phosphorus and may cause environmental problems if discharged without treatment (Li et al. 2011). Introduction The reduction of nutrient levels in effluent is required both to avoid environmental damage on discharge and to conform to the local and domestic legal re- quirements (Welsh Government DfEFaRA 2014). These re- quirements can be performed by microalgae because of their capacity to use nitrogen and phosphorus in their metabolism (Pittman et al. 2011). However, the efficiency of the algal bioremediation process primarily depends on the algal strain selected or the constituents of a microbial consortium in- volved. To date, the majority of studies that have focused on remediation have utilised uni-algal cultures (Wang et al. 2010; Lizzul et al. 2014; Praveenkumar et al. 2014; Whitton et al. 2016; Schulze et al. 2017). The use of algal consortia has been less extensively studied, although a number of recent reports have indicated that this strategy can outperform pure culture applications (Dalrymple et al. 2013; Samorì et al. 2013). The maintenance of a functionally stable, reproducible mas- ter stock culture, in this case, an algal consortium, is a pre- requisite for sustainable remediation, as it is in any other bio- technological process employing microorganisms. Furthermore, the ex situ maintenance and deposition of the microorganism(s) in an international depository authority (IDA) under the terms of the Budapest Treaty (Anonymous 1977) are absolute require- ments on submission of a patent. This raises specific challenges as IDAs are contracted to maintain the organisms, without loss of their desired patent-related characteristics for in excess of 15 years. Whilst for some algae, serial transfer may suffice and stability of metabolite production may be maintained, as in the case of pigment mutants of Parachlorella kesslerii (Müller et al. 2007), for other algal taxa, it may result in loss of key characters/ capabilities (Day and Fleck 2015). Serial transfer cannot abso- lutely guarantee retention of key characteristics, and although there are no published data on the retention or loss of bioreme- diation capability of algal consortia that have been held under laboratory conditions for extensive periods, alternative conser- vation strategies that do not require maintaining growing cul- tures may be optimal. In addition to the possibility of loss or reduction in efficacy of the characteristics relevant to the patent application, serial transfer by its nature has the potential to result in human-error-induced mistakes such as mislabelling or con- tamination on transfer. Introduction Therefore, in addition to issues associated with functional stability, alternative long-term preservation methods that minimise handling/manipulation are needed to guarantee that materials remain Bfit for purpose^. Cryopreservation, storage at ultra-low temperatures (normally −80 °C for prokaryotes and <−120 °C for eukaryotes), is widely accepted as the optimal method for the conservation of patented strains, although for many bacterial taxa, freeze-drying (lyophilisation) may also be applicable (Day and Stacey 2008). G ll l l lt h ld i h l b t i l pp ( y p ) A key aspect of this study has been the use of a mixed algal bacterial consortium from a polluted ecosystem, which was the basis of a patent submission (Silkina and Nelson 2014). The stability and sustainability of mixed cultures has previ- ously been reported (Kumar and Goyal 2009; Van Den Hende 2014; Van Den Hende et al. 2014a, 2014b, 2015, 2016). Furthermore, isolated mixed consortia from polluted ecosys- tems have already adapted to survive on the waste stream as the natural selection process has already been undertaken. Thus, there is a high probability that the consortium will be more stable than an artificially formulated algal/bacterial mix- ture. Additionally, due to the high level of tolerance of pollut- ants, it is probable that mixed consortia should acclimate more quickly than a monoculture culture. Studied mixed consortia composed of tolerant species, most commonly chlorophytes, have previously been reported for their capacity to remediate waste water (Khan et al. 2008; Ruiz-Marin et al. 2010; Wang et al.2010). In these studies, the mixed consortia had higher rates of uptake of nitrogen and phosphorous, as the different species could utilise different uptake mechanisms. Algae are known to have mechanisms for the uptake of various forms of nitrogen, namely, ammonia, nitrate and amino acids; these are highly variable within the algal community and individual algal strains may have differing nutrient uptake capacities (Olguın 2003; Barsanti and Gualtieri 2006; Cai et al. 2013). The growth of the mixed consortia results in a rapid increase in pH of the growing culture. Under this condition, in addition Generally, algal cultures held in research laboratories, cul- ture collections or by commercial organisations for biotech- nological applications are maintained as uni-algal (monocultures) but not necessarily axenic cultures (Lorenz et al. 2005). Furthermore, the commensal bacteria may be vital to maintaining healthy functional cultures (Prakash et al. 2011; Amaral et al. 2013). Algal consortium and cultivation The ACCOMPLISH algal consortium was isolated by taking water samples from an integrated steelmaking site in spring 2012 (Port Talbot, UK) and cultivating these on a defined freshwater algal medium based on the nutrient levels employed in f/2 medium (Guillard 1975). The most robust consortium, i.e. where no obvious change in the algal mix was observed on successive transfers, has been used in a bio- remediation study using nutrient media based on the waste stream from an enhanced anaerobic digestion plant (Port Talbot, UK) (Silkina et al. 2015). Since February 2012 this consortium has been maintained in the Bioscience Department microalgal culture collection (Swansea) in a freshwater algal medium based on the f/2 nutrient profile (Guillard 1975) under 18 °C, 100 μmol photons m−2 s−1 with 16:8 light/dark cycle. The material was deposited in the Culture Collection of Algae and Protozoa as a patent deposit and allocated the unique identifier CCAP 293/1 (note: patent strains are not in the public domain and cannot be directly obtained from the CCAP). A bench scale trial was conducted using 2-L flasks exter- nally illuminated on one side by a twin florescent tube (Natural daylight Osram tube). Each flask was sealed using a nitrile rubber bung with two separate holes drilled for glass tubes to be inserted, one for aeration and sampling and the other for venting the exhaust gas. The room temperature was maintained at 18.0 ± 3 °C. Lighting from the florescent tubes was provided by an 18:6 light cycle at 200 μmol photons m−2 s−1 with sam- ples taken at the end of the light cycle. The flask was contin- uously sparged with ambient air at 0.1 vvm, with the addition of 0.03% (v/v) CO2 during the light cycle. The pH was main- tained by the addition of 10 mg L−1 sodium bicarbonate. Daily samples (15 mL) were aseptically taken from the cultures, their pH immediately measured, then cell concentra- tion and biovolume were assessed by Coulter Counter (Multisizer 4). Samples for water chemistry analysis were tak- en every 24 h and centrifuged for 15 min at 3000×g. Supernatant samples were then passed through GF/F Whatman filters and stored frozen at −20 °C for 1 week prior to analysis. The predominant algal species in the consortium was iden- tified by Banco Espanol de Algaes (BEA), Grand Canarias, Spain, as Franceia amphitricha. Introduction Cryopreservation has been widely employed to maintain algal cultures; however, for the majority of the protocols employed, axenicity has been the key to suc- cessful recovery of a healthy algal culture (Taylor and Fletcher 1998; Day and Fleck 2015). Where cryopreservation has been applied to non-axenic algal cultures, in some cases, additional procedural steps may be required to reduce available carbon J Appl Phycol (2017) 29:1331–1341 1333 Table 1 Taxonomic designation of algal taxa in CCAP 293/1 consortium Species Family Class Franceia amphitricha Oocystaceae Trebouxiophyceae Scenedesmus sp. Scenedesmaceae Chlorophyceae Chlorella sp. Chlorellaceae Trebouxiophyceae Chlamydomonas sp. Chlamydomonadaceae Chlorophyceae Desmodesmus sp. Scenedesmaceae Chlorophyceae Table 1 Taxonomic designation of algal taxa in CCAP 293/1 consortium released as a result of cell lysis that may result in a bacterial Bbloom^ and subsequent algal death (Heesch et al. 2012; Amaral et al. 2013). This study explored a scenario where there was a functionally stable, mixed, algal-bacterial flora. The objective was to demonstrate that cryopreservation could be used to conserve this undefined algal consortium and to validate that the application of a standard cryopreservation approach could be employed to ensure functional stability of the consortium. released as a result of cell lysis that may result in a bacterial Bbloom^ and subsequent algal death (Heesch et al. 2012; Amaral et al. 2013). This study explored a scenario where there was a functionally stable, mixed, algal-bacterial flora. The objective was to demonstrate that cryopreservation could be used to conserve this undefined algal consortium and to validate that the application of a standard cryopreservation approach could be employed to ensure functional stability of the consortium. Material and methods assess optical density, a direct light measurement was selected in the NIR (near-infrared) spectrum, at a wavelength of 750 nm, so that colour influences would not affect the optical density values. The supernatant was then autoclaved at 121 °C for 20 min and, after cooling, stored in hermetically sealed containers at 4 °C for no longer than 1 month. Dilutions of this material to final concentrations of 1, 5 and 10% (v/v) were made to test their suitability in media formulations. Algal consortium and cultivation Other algal taxa present in the consortium included Scenedesmus sp., Chlorella sp., Chlamydomonas sp. and Desmodesmus sp. (Table 1). Bioremediation assessment Scale-up cultivation experiments were performed using two 600-L capacity horizontal tubular photobioreactors (BioFence, from Varicon Aqua Solution manufacturing) located in a heat- ed greenhouse, Swansea, UK. The duration of experiment was a 12-day consecutive period. The abiotic conditions were tem- perature 18–25 °C and 2% CO2 injection, which was regulated by pH measurements (i.e. CO2 addition when the medium was above pH 7.5). The natural (greenhouse) light conditions were variable over the experimental period ranging from 300 to 1000 μmol photons m−2 s−1, with a light/dark cycle of 16:8 h. During the cultivation period, waste-based medium [10% (v/v), 100 mL of prepared waste solution for 1 L of culture] was tested against a control f/2 medium. The control medium employed was a freshwater algal medium based on the nutrient levels employed in f/2 medium (Guillard 1975). The waste-base nutrient medium was prepared using Dwr Cymru Welsh Water’s (DCWW) anaerobic digested (AD) effluent sampled directly after the digester gravity belt filters. Fresh samples of DCWW’s AD waste effluent were stored in a fridge at 4 °C before being treated. The samples had high levels of turbidity and were passed through a 100-μm bag filter and then filtered through a 0.2-μm hollow fibre cartridge (GE Healthcare, USA) to improve the effluent’s clarity. To 1334 J Appl Phycol (2017) 29:1331–1341 Cryopreservation of the algal consortium Cryopreservation was performed according to Day and DeVille (1995). Dimethyl sulphoxide (DMSO) (Sigma- Aldrich Ltd., UK) was filter sterilised in a sterile f/2 freshwater algal medium to a final concentration of 10% (v/v) using a 0.20-μm sterile syringe filter (Iwaki, Japan). An aliquot (10 mL) of the 10% (v/v) DMSO solution was aseptically added to 10 mL of an early stationary phase culture of the algal consortium CCAP 293/1 in a sterilised Universal bottle (25 mL). The Universal was inverted several times to ensure complete mixing and the algal culture in 5% (v/v). DMSO was dispensed in 1.0 mL aliquots into cryovials (Greiner Bio-One GmbH, Germany). These were then incubated at room tem- perature (∼20 °C) for 20 min prior to cryopreservation to Cell growth Every 24 h, cell concentration, cell size and biovolume mea- surements were performed by Coulter Counter Multisizer 4, Beckman, USA, to quantify culture growth as described by (Mayers et al. 2013). After 24 h, and again after 3 months, storage triplicate samples were transferred in LN2 from the cryostorage facility to the lab. They were then thawed by direct immersion in a preheated water bath at 40 °C and were removed as soon as all visible ice had melted. Immediately after thawing, the samples were aseptically inoculated into tissue culture flasks contain- ing 20 mL of the sterile f/2 freshwater medium. The flasks were wrapped in aluminium foil to prevent the possibility of light-induced stresses/metabolic uncoupling and incubated at 20 °C in the dark for 36 h before removal of the aluminium. For culture regrowth, samples were incubated under a 12:12 h light/dark regime, irradiance ∼30 μmol photons m−2 s−1 PAR for 3 weeks and periodically visually assessed and examined by phase contrast microscopy to confirm regrowth. The cul- tures (after cryopreservation) derived from samples which had been stored for 3 months were then dispatched by post to Centre for Sustainable Aquatic Research (CSAR), Swansea, UK, to undertake post-preservation functional/bioremediation stability assessment. The growth rate was calculated based on biovolume using the formula provided by Levasseur et al. (1993): Growth rate : K0 ¼ Ln N 2=N 1 ð Þ= t2−t1 ð Þ where N1 and N2 = biovolume (measured by Coulter counter) at time 1 (t1) and time 2 (t2), respectively. Sample analyses enable the cryoprotectant to enter the cells. The cryovials were then transferred to a controlled rate cooler (Kryo 360 3.3, Planer plc, UK). The samples were cooled at −1 °C min−1 between 20 and −40 °C, with auto-ice nucleation at −5 °C, and then held for a further 15 min at −40 °C. The cryovials were then rapidly removed from the cooler unit, plunged into liquid nitrogen (LN2) and transferred to the CCAP cryobank for storage in liquid phase liquid nitrogen (−196 °C). Statistical analysis Statistical analysis of data was conducted using R software, using two- or three-factor analysis of variance allowing us to assess the differences between each treatment (i.e. medium; strain) and the data evolution in time. The analysis was con- ducted on the following variables: cell density, cell size and biovolume. When a significant difference was found, a post hoc Tukey test was used at a confidence level of 95%. The statistical interaction between factors was also assessed by the Spearman correlation test. Remediation N and P uptake The nutrient levels in the algal media were analysed every other day using an automated segmented flow analyser (AA3, Bran Luebbe, Germany). The automated procedure for the determination of nitrate (NO3) and nitrite (NO2) uses the process whereby nitrate is reduced to nitrite by a copper- cadmium reductor column (Armstrong et al. 1967). The nitrite then reacts with sulfanilamide under acidic conditions to form a diazo compound. This compound then couples with N-1- naphthylethylene diamine dihydrochloride to form a purple azo dye. Nitrate/nitrite analysis was performed, which along with ammonia, measured by the Berthelot reaction, in which a blue-green coloured complex is formed that is measured at 660 nm, gave total nitrogen (TN). The automated procedure for the determination of ortho-phosphate is based on the col- orimetric method in which a blue colour is formed by the reaction of ortho-phosphate, molybdate ion and antimony ion followed by reduction with ascorbic acid at a pH < 1. The reduced blue phospho-molybdenum complex is read at 880 nm. Bioremediation using the algal consortium in a 2- and 600-L photobioreactor c Bioremediation—re- moval of phosphorous (error bars indicate the standard deviation, the number of independent replicates = 3) J Appl Phycol (2017) 29:1331–1341 1335 1 10 1 2 3 4 5 6 7 8 Cell concentration (x106 cell mL-1) Time (day) 0 1000 2000 3000 4000 5000 6000 7000 0 1 2 3 4 5 6 7 8 Concentration (Total N µmol L-1) Time (day) 0 5 10 15 20 25 30 35 40 0 1 2 3 4 5 6 7 8 Concentration (Total P µmol L-1) Time (day) (c) (b) (a) 1335 1 10 1 2 3 4 5 6 7 8 Cell concentration (x106 cell mL-1) Time (day) 0 1000 2000 3000 4000 5000 6000 7000 0 1 2 3 4 5 6 7 8 Concentration (Total N µmol L-1) Time (day) 0 5 10 15 20 25 30 35 40 0 1 2 3 4 5 6 7 8 Concentration (Total P µmol L-1) Time (day) (c) (b) (a) 1335 1 10 1 2 3 4 5 6 7 8 Cell concentration (x106 cell mL-1) Time (day) 0 1000 2000 3000 4000 5000 6000 7000 0 1 2 3 4 5 6 7 8 Concentration (Total N µmol L-1) Ti (d ) (b) (a) 1 10 1 2 3 4 5 6 7 8 Cell concentration (x106 cell mL-1) (a) (a) 1 2 3 4 5 6 7 8 Time (day) 0 1000 2000 3000 4000 5000 6000 7000 0 1 2 3 4 5 6 7 8 Concentration (Total N µmol L-1) Time (day) 0 5 10 15 20 25 30 35 40 0 1 2 3 4 5 6 7 8 Concentration (Total P µmol L-1) Time (day) (c) (b) (b) 0 1000 0 1 2 3 4 5 6 7 8 Conce Time (day) 0 5 10 15 20 25 30 35 40 0 1 2 3 4 5 6 7 8 Concentration (Total P µmol L-1) Time (day) (c) 0.35 day−1 in freshwater f/2; however, the duration of the exponential phase was 3 days in the cultures grown on waste nutrients and 5 days in f/2. The growth observed in media supplemented with 1 and 5% of waste effluent was lower, and statistical analysis indicated that the medium formulation used had an impact of the growth estimated by cell density (p < 0.05) (Fig. 1a) (Table 2). Fig. 1 Growth and nutrient uptake of the ACCOMPLISH algal consortium in 250-mL flask under f/2 (black circle), 1% (white circle), 5% (black square) and 10% (white square) of effluent nutrients. a Cell density. b Bioremediation—removal of ni- trogen. c Bioremediation—re- moval of phosphorous (error bars indicate the standard deviation, the number of independent replicates = 3) Bioremediation using the algal consortium in a 2- and 600-L photobioreactor The algal consortium was successfully grown on media sup- plemented with Bwaste nutrients^ at 1, 5 and 10% (v/v) and control f/2 (Fig. 1a). Adaptation to the new nutrient regime was observed over the first 2 days under all waste dilutions tested and the best growth observed was in a medium supple- mented with 10% waste effluent. The exponential growth rate in the 10% growth medium was 0.4 day−1, in comparison to J Appl Phycol (2017) 29:1331–1341 1335 1 10 1 2 3 4 5 6 7 8 Cell concentration (x106 cell mL-1) Time (day) 0 1000 2000 3000 4000 5000 6000 7000 0 1 2 3 4 5 6 7 8 Concentration (Total N µmol L-1) Time (day) 0 5 10 15 20 25 30 35 40 0 1 2 3 4 5 6 7 8 Concentration (Total P µmol L-1) Time (day) (c) (b) (a) Fig. 1 Growth and nutrient uptake of the ACCOMPLISH algal consortium in 250-mL flask under f/2 (black circle), 1% (white circle), 5% (black square) and 10% (white square) of effluent nutrients. a Cell density. b Bioremediation—removal of ni- trogen. Bioremediation using the algal consortium in a 2- and 600-L photobioreactor The interaction between both factors was significant for both the cell density and the biovolume (p < 0.05), demonstrating that there was a signifi- cant difference in growth between both media; however, that difference was not the same over the time course of the exper- iment. Indeed, the cell density results indicated that there were no apparent differences between the two media at the begin- ning of the experiment, but there were differences after a few days of incubation. Furthermore, the post hoc Tukey test in- dicated that employing the control medium resulted in higher cell density and biovolume. Finally, the correlation test showed that the three variables (cell density, cell size and biovolume) were correlated, demonstrating that under all con- ditions tested that when a high cell density was observed, cells were larger and total biovolume higher (Table 2). The nutrient uptake by the algal consortium is shown in Fig. 1b, c. Nitrogen was gradually removed from the media by the algal cultures under all the experimental conditions tested. Phosphate uptake was more rapid and by day 4, all the phos- phorus in the media had been assimilated by the algal cultures under all nutrient conditions tested. The waste medium used had an impact on the nitrogen uptake rate (p < 0.05) and the results of post hoc tests (Fig. 1). The interaction between the concentration of both factors was significant for the cell den- sity and the biovolume (p < 0.05) indicating that there was a significant difference in performance between media; J Appl Phycol (2017) 29:1331–1341 1336 Table 2 Biovolume, cell concentration and cell size of algal consortium growing on different nutrient conditions and scale (± indicates the standard deviation, the number of independent replicates = 3) Treatment Biovolume (× 108 μm3 mL−1) Cell concentration (× 106 cell mL−1) Cell size (μm) 1% effluent (250 mL) 2.3 ± 0.3 2.7 ± 0.3 5.2 5% effluent (250 mL) 5.2 ± 0.5 3.6 ± 0.3 5.5 10% effluent (250 mL) 2.8 ± 0.1 7.7 ± 0.1 5.5 Control (f/2) (250 mL) 1.8 ± 0.2 5.5 ± 0.4 5.4 10% effluent (600 L) 4.8 ± 0.5 3.7 ± 0.1 7.7 Control (f/2) (600 L) 5.8 ± 0.5 6.5 ± 0.3 7.1 The algal consortium grown on waste medium had slow phosphorus uptake during the first 3 days; after that, the phosphorus uptake accelerated. Bioremediation using the algal consortium in a 2- and 600-L photobioreactor By day 6, all the phosphorus had been removed by the algal consortium showing rapid waste remediation, comparable to the control (freshwater f/2) treatment. however, that difference was not related to the time of sam- pling. The cell density results indicated that there were no apparent differences between culture nutrient uptake in the control and experimental culture media at the beginning of the experiment, but differences were observed after a few days of incubation, due to the adaptation to the media. The algal consortium was successfully grown in PBR remediating a waste-based medium (Fig. 2a), (Table 2). The lag phase, adaptation to the reactor and nutrient conditions, was observed for the first 2 days for both control and waste nutrient conditions; the growth rate in this period was 0.1 day−1. The subsequent exponential phase lasted 4 days with a growth rate of 0.35 day−1 for the culture grown on AD waste nutrients. This was significantly different (p > 0.005) from the control (f/2) medium where the culture grew at a slower rate and entered stationary phase after 5– 6 days of cultivation. The highest final cell density (3 × 107 cells mL−1) was observed in the waste water-grown culture (Fig. 2a) (Table 2). Statistical analysis demonstrated that both the time of experiment and the medium used influence the three studied variables: cell density, cell size and biovolume (p < 0.05). Furthermore, the interaction between both factors was significant for the cell density and the biovolume (p < 0.05) indicating that there was a significant difference in culture growth between both media (Table 2); however, that difference was not dependent on the time of sampling. Indeed, the cell density data indicated that there were no apparent dif- ferences between adaptations in the two different media for- mulations at the beginning of the experiment, but there were differences after a few days of incubation. Furthermore, the post hoc Tukey test demonstrated that using the waste medium resulted in higher cell density and biovolume. Finally, the cor- relation test indicated that the three variables were correlated, demonstrating that at higher cell density, there were also bigger cells and thus greater total biovolume (Table 2). Validation of bioremediation capacity of the cryopreserved algal consortium On thawing of samples after 24 h and 3 weeks storage under liquid nitrogen, relatively rapid recovery and regrowth of the consortium were observed in all replicate cultures. Within 2– 3 weeks, the cultures were comparable in density and appear- ance to the culture prior to cryopreservation. No overgrowth of the algal culture by the commensal bacteria was observed, and on microscopy, all replicates appeared to be effectively identical with respect to algal morphotypes present and were indistinguishable by light microscopy from the non- cryopreserved control samples that had been maintained in parallel under the standard cultivation regime. On testing the growth of the two cultures, the non- cryopreserved algal consortium and the algal consortium after cryopreservation, over 13 days, good growth levels were ob- served (Fig. 3). No significant difference (p < 0.05) was ob- served between growth rates of the algal consortium before and after cryopreservation, which were 0.6 and 0.7 day−1, respectively (Fig. 3a). The nutrient remediation functionality (N and P uptake rate) was very similar in comparison with the previous experiment, and by day 4, 99% of the total nitrogen and total phosphorus were remediated by both of the cultures studied (Fig. 3b, c). Table 2 Biovolume, cell concentration and cell size of algal consortium growing on different nutrient conditions and scale (± indicates the standard deviation, the number of independent replicates = 3) Discussion c Bioremediation—re- moval of phosphorous (error bars indicate the standard deviation, the number of independent replicates = 3) J Appl Phycol (2017) 29:1331 1341 1337 1 10 100 0 1 2 3 4 5 6 7 8 9 10 11 Cell concentration (106 cell mL-1) Time (day) 0 1500 3000 4500 6000 0 1 2 3 4 5 6 7 8 9 10 11 Concentration (Total N µmol L-1) Time (day) (a) (b) 0 20 40 60 80 100 120 140 160 180 0 1 2 3 4 5 6 7 8 9 10 11 Concentration (Total P µmol L-1) Time (day) (c) 1 10 100 0 1 2 3 4 5 6 7 8 9 10 11 Cell concentration (106 cell mL-1) Time (day) 0 1500 3000 4500 6000 0 1 2 3 4 5 6 7 8 9 10 11 Concentration (Total N µmol L-1) Time (day) (a) (b) 0 20 40 60 80 100 120 140 160 180 0 1 2 3 4 5 6 7 8 9 10 11 Concentration (Total P µmol L-1) Time (day) (c) 1 10 100 0 1 2 3 4 5 6 7 8 9 10 11 Cell concentration (106 cell mL-1) Time (day) (a) 1 10 100 0 1 2 3 4 5 6 7 8 9 10 11 Cell concentration (106 cell mL-1) Time (day) 0 1500 3000 4500 6000 0 1 2 3 4 5 6 7 8 9 10 11 Concentration (Total N µmol L-1) Time (day) (a) (b) Fig. 2 Growth and nutrient uptake of the ACCOMPLISH algal consortium in a 600-L PBR under (black diamond) f/2 and (white diamond) 10% of effluent nutrients. a Cell density. b Bioremediation—removal of ni- trogen. c Bioremediation—re- moval of phosphorous (error bars indicate the standard deviation, the number of independent replicates = 3) (a) 0 1 2 3 4 5 6 7 8 9 10 11 Time (day) 0 1500 3000 4500 6000 0 1 2 3 4 5 6 7 8 9 10 11 Concentration (Total N µmol L-1) Time (day) (b) Time (day) Concentration (Total N µmol L-1) (b) ( y) 0 20 40 60 80 100 120 140 160 180 0 1 2 3 4 5 6 7 8 9 10 11 Concentration (Total P µmol L-1) Time (day) (c) consortium, where the dominant groups of algal species belong to either the Chlorophyceae or Trebouxiophyceae. Discussion The nitrogen uptake observed was not significantly dif- ferent (p < 0.05) between waste and control (f/2) conditions (Fig. 2). By day 6, total nitrogen was remediated, i.e. re- moved from media by the algal consortium (significantly different (p < 0.05) than f/2 medium grown consortium). The phosphorus uptake for control and waste-based media growth conditions were significantly different (p < 0.05). Conventional wastewater treatment technologies (e.g. anaero- bic digestion) still have technical-economic limitations, mainly caused by their high energy requirement and relatively poor nutrient removal (de Godos et al. 2010). The development of algal technology for bioremediation applications has the poten- tial to overcome these constraints as they can provide a system 1337 J Appl Phycol (2017) 29:1331–1341 1 10 100 0 1 2 3 4 5 6 7 8 9 10 11 Cell concentration (106 cell mL-1) Time (day) 0 1500 3000 4500 6000 0 1 2 3 4 5 6 7 8 9 10 11 Concentration (Total N µmol L-1) Time (day) (a) (b) 0 20 40 60 80 100 120 140 160 180 0 1 2 3 4 5 6 7 8 9 10 11 Concentration (Total P µmol L-1) Time (day) (c) Fig. 2 Growth and nutrient uptake of the ACCOMPLISH algal consortium in a 600-L PBR under (black diamond) f/2 and (white diamond) 10% of effluent nutrients. a Cell density. b Bioremediation—removal of ni- trogen. Discussion Taxa belong- ing to these classes, particularly Chlorella and Scenedesmus, have been demonstrated to have high bioremediation potential (Chinnasamy et al. 2010a; 2012 Su et al.; Shene et al. 2016). However, F. amphitricha has not previously been reported in bioremediation studies (Tsarenko and John 2011). capable of high levels of nutrient removal, with the capacity to remediate a wide spectrum of waste nutrients by an environ- mentally friendly alternative to conventional methods (Boelee et al. 2011; Posadas et al. 2013; Polishchuk et al. 2015). The advantages of the use of mixed microalgal consortium have been the focus of recent research (Kerckhof et al. 2014; Mahapatra et al. 2014; Lahel et al. 2016). Furthermore, similar to the results of this study, high levels (above 90%) of remedi- ation of inorganic nutrients have been reported in studies using both mixed natural or artificially formulated algal consortia (Fergola et al. 2007; Chinnasamy et al. 2010b; Renuka et al. 2013) . In this study, we have used a natural algal mixed The use of municipal waste nutrient source was one of the objectives of this study and the remediation of 4500 μmol L−1 (64 mg L−1) of N and 690 μmol L−1 (21 mg L−1) of P by a reduction of 99% was achieved using the algal consortium. Other research studies (Woertz et al. 1338 J Appl Phycol (2017) 29:1331–1341 1 10 100 1000 0 1 2 3 4 5 6 7 8 9 10 11 12 13 14 Cell concentration(106 cell mL-1) Time (day) 0 100 200 300 400 500 600 700 0 1 2 3 4 5 6 7 8 9 10 11 12 13 14 Concentration (Total N µmol L-1) Time (day) (b) (a) 30 40 50 60 70 80 ation (Total P µmol L-1) (c) 1 10 100 1000 0 1 2 3 4 5 6 7 8 9 10 11 12 13 14 Cell concentration(106 cell mL-1) Time (day) (a) (a) (a) Cell concentration(106 cell mL-1) asamy et al. 2010b; Silva-Benavides and 2; Su et al. 2012) have reported comparable remediation capacities of 41–100% f for phosphorus. Discussion However, there are few reports on the retention of functionality of cryopre- served microbial consortia for environmental remediation in- cluding Augustynowicz et al. (2008) who demonstrated the capacity of a revived cryopreserved mixed bacterial commu- nity to degrade petroleum-derived environmental contaminants and Kerckhof et al. (2014) who successfully optimised the conservation of a methanotrophic co-culture (MOB), with potential for mitigation of greenhouse gas emis- sions, environmental pollutant removal and bioplastics pro- duction, as well as an oxygen-limited autotrophic nitrification/denitrification (OLAND) biofilm, with enhanced economic and ecological benefits for wastewater treatment. These consortia both retained good levels of functionality, although the preservation of the community structure (as de- termined by 16S rRNA gene sequencing) was incomplete. In our study community structure, beyond the presence of the key algal taxa, this was not determined, as functionality was the primary requirement. this is achievable at pilot-scale, rather than lab-scale (0.25– 5 L) volume. The cultivation of the algal consortium with waste nutrient removal in a 600-L PBR in greenhouse reflected real environmental conditions (variable light, pH, nutrients availability, temperature) similar in any po- tential industrial site, rather than in laboratory. All these parameters influence the consortium behaviour and biore- mediation capacity; however, although the remediation process in PBR was slightly longer than in the lab-scale experiment, as has previously been reported, the culture was more stable (Bordel et al. 2009). Overall, the scalabil- ity, nutrient removal capability and cell density of the algae produced demonstrated the potential of an integrated sys- tem capable of both bioremediation and the production of algal biomass. In the biotechnological application of algae in phyco-reme- diation, as in any other industrial application, stability of the alga(e) and/or the mixed consortium is a pre-requisite for en- suring sustainable results/production. In case of successful waste remediation by algae, the main challenges are to main- tain the long-term effectiveness and long-term homeostasis of the mixed consortium. (Gonçalves et al. 2016). The use of cryopreserved master stock cultures provides insurance for the sustainability of production. Furthermore, it provides prac- titioners with an option to ensure consistency in batch-based processes, where inoculum build-up may be initiated for each batch from the master stock culture. Discussion However, this study h 1 10 100 0 1 2 3 4 5 6 7 8 9 10 11 12 13 14 Cell concentration(106 cell mL-1) Time (day) 0 100 200 300 400 500 600 700 0 1 2 3 4 5 6 7 8 9 10 11 12 13 14 Concentration (Total N µmol L-1) Time (day) (b) (a) 0 10 20 30 40 50 60 70 80 0 1 2 3 4 5 6 7 8 9 10 11 12 13 14 Concentration (Total P µmol L-1) Time (day) (c) Time (day) 0 100 200 300 400 500 600 700 0 1 2 3 4 5 6 7 8 9 10 11 12 13 14 Concentration (Total N µmol L-1) Time (day) (b) Concentration (Total N µmol L-1) (b) ( y) 0 10 20 30 40 50 60 70 80 0 1 2 3 4 5 6 7 8 9 10 11 12 13 14 Concentration (Total P µmol L-1) Time (day) (c) (c) 2009; Chinnasamy et al. 2010b; Silva-Benavides and Torzillo 2012; Su et al. 2012) have reported comparable 2009; Chinnasamy et al. 2010b; Silva-Benavides and Torzillo 2012; Su et al. 2012) have reported comparable remediation capacities of 41–100% for N and 12–100% for phosphorus. However, this study has demonstrated that 1339 J Appl Phycol (2017) 29:1331–1341 Fig. 3 Growth and nutrient uptake for the ACCOMPLISH algal consortium before and after cryopreservation in flask (250 mL) system; black diamond—ACCOMPLISH mixed consortium before cryopreservation, white diamond—ACCOMPLISH mixed consortium after cryopreservation. a Cell density. b Bioremediation—removal of nitrogen (flask system). c Bioremediation—removal of phosphorous (error bars indicate the standard deviation, the number of independent replicates = 3) R capacity post-thaw of Porphyridium cruentum to produce ze- axanthin and beta-carotene and for the cyanobacterium Planktothrix to produce a cytotoxin. In addition, Nakanishi et al. (2012) have demonstrated that the chlorophyll content of Nannochloropsis oculata ST-4 and Tetraselmis tetrathele T-501 was not significantly changed after 15 years of cryostorage. More recently, Hipkin et al. (2014) reported the successful cryopreservation of the transgenic diatom Thalassiosira pseudonana CCAP 1085/23, which overexpressed a GFP-tagged nuclear localised protein, pre- and post-cryopreservation as a proxy for a biotechnological product. To date, there have been no previous reports on the cryopreservation of a mixed algal consortium. Discussion Cryopreservation is also an important tool for the Biological Resource Centre (BRC) community to hold patented strains, minimising risks associ- ated with alternative procedures such as serial transfer and facilitating the exploitation of non-traditional biological re- sources (Stacey and Day 2014). Under the patent procedure, the strain deposited should retain the trait/capacity of commer- cial relevance. As part of the procedure, on the request of the depositor/owner of the strain, the cryopreserved samples may be revived, dispatched and re-tested for their biotechnological capabilities. There are numerous reports on the conservation of algae by cryopreservation for biotechnological or aquacul- ture use, highlighting the importance of minimising risks as- sociated with losing, contaminating or the potential loss of traits by genetic drift (Cañavate and Lubińn 1995; Day et al. 2005; Rhodes et al. 2006). However, despite the importance of having evidence based on the retention of biotechnological potential, there are few commercially relevant reports in the literature. Hédoin et al. (2006) demonstrated the retention in In this study, we have confirmed the integrity of mixed consortium after the cryopreservation and demonstrated its capacity to bioremediate effluent effectively. This has signifi- cant implications to the algal biotechnology sector as a whole, where in reality, mixed/non-axenic cultivation will be a pre- requisite for future economic success. In addition, this paper provides a successful model of a procedure that has allowed the protection of intellectual property (IP) associated with a mixed consortium. This is in the vanguard of the BRC com- munities’ attempts to service the challenges associated with rapid developments in algal/protistan biotechnology. In conclusion, microalgae, with their photoautotrophic ca- pabilities, are able to uptake the waste nutrients using solar energy and carbon dioxide and can thus convert these nutri- ents to valuable biomass. This capability will, in the opinion of the authors, be a major component in a more integrated bioeconomy that will help to manage pollution worldwide, resulting from expansion of the global population and indus- trial activities. Underpinning these capabilities by guarantee- ing the functional stability of conserved consortia is a key component in ensuring sustainability and long-term biotech- nological exploitability. 1340 J Appl Phycol (2017) 29:1331–1341 Acknowledgements The authors are grateful for the underpinning sup- port of colleagues at CSAR (Swansea) and SAMS (Oban). Alla Silkina, Graham G. Nelson, Catherine E. Bayliss and C. L. References Gadd GM (2009) Biosorption: critical review of scientific rationale, en- vironmental importance and significance for pollution treatment. J Chem Technol Biotechnol 84:13–28 Amaral R, Pereira JC, Pais AACC, Santos LMA (2013) Is axenicity cru- cial to cryopreserve microalgae? Cryobiology 67:312–320 Anonymous (1977) Budapest Treaty on the international recognition of the deposit of microorganisms for the purposes of patent procedure. Regulations BT, Geneva Gonçalves AL, Pires JCM, Simões M (2016) A review on the use of microalgal consortia for wastewater treatment. 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Pol J Environ Stud 17:655–664 Hédoin H, Pearson J, Day JG, Philip D, Young AJ, Hall TJ (2006) Porphyridium cruentum A-408 and Planktothrix A-404 retain their capacity to produce biotechnologically exploitable metabolites after cryopreservation. J Appl Phycol 18:1–7 Barsanti L, Gualtieri P (2006) Algae: anatomy, biochemistry, and biotech- nology. Taylor & Francis, Boca Raton Hipkin R, Day JG, Rad-Menéndez C, Mock T (2014) The first evidence for genotypic stability in a cryopreserved transgenic diatom. J Appl Phycol 26:65–71 Boelee NC, Temmink H, Janssen M, Buisman CJN, Wijffels RH (2011) Nitrogen and phosphorus removal from municipal wastewater efflu- ent using microalgal biofilms. Water Res 45:5925–5933 Juhasz AL, Naidu R (2000) Bioremediation of high molecular weight polycyclic aromatic hydrocarbons: a review of the microbial degra- dation of benzo[a]pyrene. 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Supplementary Figure from Upfront Biology-Guided Therapy in Diffuse Intrinsic Pontine Glioma: Therapeutic, Molecular, and Biomarker Outcomes from PNOC003
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A p = 9.6e-1 0.00 0.25 0.50 0.75 1.00 0 10 20 30 40 50 Time Overall Survival CBTN (H3K27-altered DMG) PNOC003 (H3K27-altered DIPG) 22 11 4 1 1 1 28 19 4 0 0 0 PNOC003 CBTN 0 10 20 30 40 50 Time Number at risk PNOC003+CBTN A p = 9.6e-1 0.00 0.25 0.50 0.75 1.00 0 10 20 30 40 50 Time Overall Survival CBTN (H3K27-altered DMG) PNOC003 (H3K27-altered DIPG) 22 11 4 1 1 1 28 19 4 0 0 0 PNOC003 CBTN 0 10 20 30 40 50 Time Number at risk PNOC003+CBTN TP53 mut wt (N=35) (N=15) 2.3 reference (1 08 - 5 1) 0.032 * E p = 5.2e-4 0.00 0.25 0.50 0.75 1.00 0 10 20 30 40 50 Time Overall Survival TP53mut;10del TP53mut;10wt TP53wt;10wt 14 4 1 0 0 0 20 14 1 0 0 0 14 11 6 1 1 1 TP53mut;10wt TP53mut;10wt TP53mut;10del 0 10 20 30 40 50 Time Number at risk PNOC003+CBTN p = 2.4e-2 0.00 0.25 0.50 0.75 1.00 0 10 20 30 40 50 Time Overall Survival 15 6 1 0 0 0 7 5 3 1 1 1 TP53wt TP53mut 0 10 20 30 40 50 Time Number at risk TP53mut TP53wt CBTN p = 9.6e-1 0.00 0.25 0.50 0.75 1.00 0 10 20 30 40 50 Time Overall Survival CBTN (H3K27-altered DMG) PNOC003 (H3K27-altered DIPG) 22 11 4 1 1 1 28 19 4 0 0 0 NOC003 CBTN 0 10 20 30 40 50 Time Number at risk PNOC003+CBTN 4 5 D p = 1.3e-1 0.00 0.25 0.50 0.75 1.00 0 10 20 30 40 50 Time Overall Survival 10del 10wt 8 3 1 0 0 0 13 8 3 1 1 1 10wt 10del 0 10 20 30 40 50 Time Number at risk CBTN F Supplemental fgure 1. 10del Supplemental fgure 1. D p = 1.3e-1 0.00 0.25 0.50 0.75 1.00 0 10 20 30 40 50 Time Overall Survival 10del 10wt 8 3 1 0 0 0 13 8 3 1 1 1 10wt 10del 0 10 20 30 40 50 Time Number at risk CBTN Supplemental fgure 1. A p = 9.6e-1 0.00 0.25 0.50 0.75 1.00 0 10 20 30 40 50 Time Overall Survival CBTN (H3K27-altered DMG) PNOC003 (H3K27-altered DIPG) 22 11 4 1 1 1 28 19 4 0 0 0 PNOC003 CBTN 0 10 20 30 40 50 Time Number at risk PNOC003+CBTN D A COHORT CHR10 TP53 PNOC003 CBTN del wt mut wt (N=28) (N=22) (N=15) (N=34) (N=35) (N=15) 1.2 reference 2.3 reference 2.3 reference (0.64 - 2.2) (1.13 - 4.8) (1.08 - 5.1) 0.57 0.022 * 0.032 * # Events: 49; Global p-value (Log-Rank): 0.0022 AIC: 280.5; Concordance Index: 0.65 Hazards ratio E p = 5.2e-4 0.00 0.25 0.50 0.75 1.00 0 10 20 30 40 50 Time Overall Survival TP53mut;10del TP53mut;10wt TP53wt;10wt 14 4 1 0 0 0 20 14 1 0 0 0 14 11 6 1 1 1 TP53mut;10wt TP53mut;10wt TP53mut;10del 0 10 20 30 40 50 Time Number at risk PNOC003+CBTN -5 p = 1.3e-1 0.00 0.25 0.50 0 10 20 30 40 50 Time Overall Survival 8 3 1 0 0 0 13 8 3 1 1 1 10wt 10del 0 10 20 30 40 50 Time Number at risk F COHORT CHR10 TP53 PNOC003 CBTN del wt mut wt (N=28) (N=22) (N=15) (N=34) (N=35) (N=15) 1.2 reference 2.3 reference 2.3 reference (0.64 - 2.2) (1.13 - 4.8) (1.08 - 5.1) 0.57 0.022 * 0.032 * # Events: 49; Global p-value (Log-Rank): 0.0022 AIC: 280.5; Concordance Index: 0.65 Hazards ratio B C E p = 5.2e-4 0.00 0.25 0.50 0.75 1.00 0 10 20 30 40 50 Time Overall Survival TP53mut;10del TP53mut;10wt TP53wt;10wt 14 4 1 0 0 0 20 14 1 0 0 0 14 11 6 1 1 1 TP53mut;10wt TP53mut;10wt TP53mut;10del 0 10 20 30 40 50 Time Number at risk PNOC003+CBTN p = 2.4e-2 0.00 0.25 0.50 0.75 1.00 0 10 20 30 40 50 Time Overall Survival 15 6 1 0 0 0 7 5 3 1 1 1 TP53wt TP53mut 0 10 20 30 40 50 Time Number at risk TP53mut TP53wt CBTN p = 9.6e-1 0.00 0.25 0.50 0 10 20 30 40 50 Time Overall Surv 22 11 4 1 1 1 28 19 4 0 0 0 PNOC003 CBTN 0 10 20 30 40 50 Time Number at risk -log10 P Overall Survival 0 1 2 3 4 5 -10 -16 -12 -17 -13 -11 +1 -18 -14 -5 -19 p = 1.3e-1 0.00 0.25 0.50 0 10 20 30 40 50 Time Overall Survi 8 3 1 0 0 0 13 8 3 1 1 1 10wt 10del 0 10 20 30 40 50 Time Number at risk F B p = 2.4e-2 0.00 0.25 0.50 0.75 1.00 0 10 20 30 40 50 Time Overall Survival 15 6 1 0 0 0 7 5 3 1 1 1 TP53wt TP53mut 0 10 20 30 40 50 Time Number at risk TP53mut TP53wt CBTN E p = 5.2e-4 0.00 0.25 0.50 0.75 1.00 0 10 20 30 40 50 Time Overall Survival TP53mut;10del TP53mut;10wt TP53wt;10wt 14 4 1 0 0 0 20 14 1 0 0 0 14 11 6 1 1 1 TP53mut;10wt TP53mut;10wt TP53mut;10del 0 10 20 30 40 50 Number at risk PNOC003+CBTN B E C -log10 P Overall Survival 0 1 2 3 4 5 -10 -16 -12 -17 -13 -11 +1 -18 -14 -5 -19 C COHORT CHR10 TP53 PNOC003 CBTN del wt mut wt (N=28) (N=22) (N=15) (N=34) (N=35) (N=15) 1.2 reference 2.3 reference 2.3 reference (0.64 - 2.2) (1.13 - 4.8) (1.08 - 5.1) 0.57 0.022 * 0.032 * # Events: 49; Global p-value (Log-Rank): 0.0022 AIC: 280.5; Concordance Index: 0.65 Time F F # Events: 49; Global p-value (Log-Rank): 0.0022 AIC: 280.5; Concordance Index: 0.65 Hazards ratio
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Inactivated ostreid herpesvirus-1 induces an innate immune response in the Pacific oyster, Crassostrea gigas, hemocytes
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Brief Research Report 28 April 2023 DOI 10.3389/fimmu.2023.1161145 TYPE PUBLISHED OPEN ACCESS EDITED BY Daniela Melillo, National Research Council (CNR), Italy REVIEWED BY Alejandro Romero, Spanish National Research Council (CSIC), Spain Junfa Yuan, Huazhong Agricultural University, China *CORRESPONDENCE Lizenn Delisle lizenn.delisle@Cawthron.org.nz Anne Rolton anne.vignier@cawthron.org.nz 07 February 2023 ACCEPTED 18 April 2023 PUBLISHED 28 April 2023 RECEIVED CITATION Delisle L, Rolton A and Vignier J (2023) Inactivated ostreid herpesvirus-1 induces an innate immune response in the Pacific oyster, Crassostrea gigas, hemocytes. Front. Immunol. 14:1161145. doi: 10.3389/fimmu.2023.1161145 COPYRIGHT © 2023 Delisle, Rolton and Vignier. 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. Inactivated ostreid herpesvirus-1 induces an innate immune response in the Pacific oyster, Crassostrea gigas, hemocytes Lizenn Delisle 1*, Anne Rolton 1* and Julien Vignier 2 1 Biosecurity Group, Cawthron Institute, Nelson, New Zealand, 2 Aquaculture Group, Cawthron Institute, Nelson, New Zealand Infectious diseases are a major constraint to the expansion of shellfish production worldwide. Pacific oyster mortality syndrome (POMS), a polymicrobial disease triggered by the Ostreid herpesvirus-1 (OsHV-1), has devastated the global Pacific oyster (Crassostrea gigas) aquaculture industry. Recent ground-breaking research revealed that C. gigas possess an immune memory, capable of adaption, which improves the immune response upon a second exposure to a pathogen. This paradigm shift opens the door for developing ‘vaccines’ to improve shellfish survival during disease outbreaks. In the present study, we developed an in-vitro assay using hemocytes – the main effectors of the C. gigas immune system – collected from juvenile oysters susceptible to OsHV-1. The potency of multiple antigen preparations (e.g., chemically and physically inactivated OsHV-1, viral DNA, and protein extracts) to stimulate an immune response in hemocytes was evaluated using flow cytometry and droplet digital PCR to measure immune-related subcellular functions and gene expression, respectively. The immune response to the different antigens was benchmarked against that of hemocytes treated with Poly (I:C). We identified 10 antigen preparations capable of inducing immune stimulation in hemocytes (ROS production and positively expressed immunerelated genes) after 1 h of exposure, without causing cytotoxicity. These findings are significant, as they evidence the potential for priming the innate immunity of oysters using viral antigens, which may enable cost-effective therapeutic treatment to mitigate OsHV-1/POMS. Further testing of these antigen preparations using an in-vivo infection model is essential to validate promising candidate pseudo-vaccines. KEYWORDS immune priming, Ostreid herpesvirus-1, pseudo-vaccination, innate immune memory, flow cytometry, reactive oxygen species, droplet digital PCR Frontiers in Immunology 01 frontiersin.org Delisle et al. 10.3389/fimmu.2023.1161145 OsHV-1 infection in natura. More recently, Fallet et al. (36) showed that early life exposure of C. gigas to ‘microorganisms’ provided inter-generational protection against recurring OsHV-1 infections, indicating a potential trained immunity via epigenetic modifications. In bivalves, hemocytes play a central role in immunity, identifying and destroying pathogens through phagocytosis, oxidative stress, apoptosis, and autophagy – functions that can be characterized using flow cytometry and molecular analyses (37–43). Transcriptome analyses in oysters primed with Poly (I:C) have identified several pattern recognition receptors (PRRs) involved in antiviral signaling. These include, for example, retinoic acid-inducible-gene-I and Toll-like-receptors homologs of the Jak-Stat pathway, stimulator of interferon genes, interferon regulatory factors, and many IFN-stimulated genes (i.e., Viperin or ADAR), which are all implicated in the detection of virus and antiviral functions (44–48). We aim to evaluate the potency of inactivated OsHV-1 preparations (antigens) to elicit an antiviral response in C. gigas hemocytes. This research is undertaken in the context of developing new strategies of immune priming to improve oyster resilience to POMS. 1 Introduction The occurrence of mass mortality events and the emergence of infectious diseases affecting marine organisms have increased dramatically in recent years, exacerbated by a changing environment (1–3). These outbreaks can have disastrous consequences on biodiversity and cause rapid population declines, particularly in cultured livestock (4, 5). Diseases are the major limiting factor for the expansion of the aquaculture industry, with losses attributed to infectious microbial diseases alone exceeding US $ 6 billion per annum (6). One striking example is Pacific oyster mortality syndrome (POMS), which is associated with the detection of the Ostreid Herpes virus-1 (OsHV-1) and its variants. Over the last 15 years, OsHV-1 has decimated Pacific oysters, Crassostrea gigas, worldwide (7; see for review 8–10). The virus induces an immune-compromised state in infected oysters, which evolves toward subsequent bacteremia by opportunistic bacterial pathogens, leading to mortality rates of up to 100% in juveniles (11). Recorded for the first time in France in 2008, OsHV-1 µvar rapidly spread along the European coastline (9, 10, 12), and closely related variants of the virus were further detected during mortality events in Australia (13), New Zealand (14), Korea (15), and more recently in California (16). The inability to contain the rapid spread of the virus combined with an absence of therapeutic treatments resulted in huge losses of aquaculture stocks. Selective breeding to improve resistance to POMS (or OsHV-1) has shown potential as a prevention strategy, with moderate to high heritability for survival achieved during OsHV-1 infection (17–20). Implementation of a breeding program and access to selectively bred stocks can, however, be economically challenging for many end-users, prompting the need for new, accessible, and complementary mitigation strategies to reduce the impact of diseases. For example, vaccination and immune priming have proven to be an effective preventative measure for many major diseases affecting livestock, including fish, and more recently invertebrates, such as crustaceans (21–25). Invertebrates lack a conventional adaptive immune system (i.e., lymphocytes or antibodies) and instead rely on innate immunity to prevent the infection of invading pathogens (26, 27). Numerous studies have reported that invertebrates also possess diverse forms of immune ‘memory’ in which a potentiated immune response (resulting in a reduction of host susceptibility to the infection) has been recorded following a secondary exposure to a pathogen (28– 31). For instance, in the scallop Chlamys farreri, a first short exposure to the pathogen Vibrio anguillarum increased phagocytosis, acid phosphatase activity, and survival following a second encounter to the pathogen (32). Pacific oysters stimulated primarily by heat-killed Vibrio splendidus also displayed stronger immune responses at cellular and molecular levels when they were subjected to a secondary challenge with the live bacteria (33). (34, 35) showed that injection of Polyinosinic: polycytidylic acid or Poly (I:C), a synthetic analog of double stranded RNA with immunostimulant properties, induced a long-lasting antiviral response in Pacific oysters, protecting them against subsequent Frontiers in Immunology 2 Material and methods 2.1 Preparation of OsHV-1 antigens In June 2021, the experiment was conducted to (1) screen multiple preparations of inactivated OsHV-1 (hereafter referred to as antigens) by measuring immune-related functions of hemocytes using flow cytometry (FCM, repeated three times), and (2) confirm the potency of a selected subset of antigen preparations to stimulate immunity using FCM (cytotoxicity and ROS) and molecular analyses (immune- related gene expression, repeated three times, Figure 1). 2.1.1 OsHV-1 stock The OsHV-1 suspension stock was produced in October 2019, as described in Camara et al. (18), from diseased oysters infected with OsHV-1 during a lab challenge (49). Briefly, tissue from high virus load oysters was homogenized, purified by serial filtrations down to 0.22 mm and cryopreserved according to Kirkland et al. (50). On the 3 June 2021, cryopreserved OsHV-1 stock was defrosted by dipping in a 22°C water bath for 10 min. Viral suspension was titrated using qPCR (51) and diluted in 0.22 µmfiltered sterile seawater (SSW) to reach a final concentration of 9.0.105 copies. µl-1. Prepared virus was then inactivated using the methods described below. 2.1.2 BEI inactivation A 0.2M Binary ethylenimine (BEI) solution was prepared by cyclization of 0.2M 2-bromoethylamin-HBr in 0.2M NaOH at 37°C for 1 h (52). The BEI solution was added to OsHV-1 suspension to either a concentration of 0.1% (v/v) and incubated at 22°C for 1 h, 4 h, or 6 h, or to a concentration of 0.04% (v/v), and incubated at 02 frontiersin.org Delisle et al. 10.3389/fimmu.2023.1161145 FIGURE 1 Schematic overview of the in-vitro experimental design, (A) refers to the live infectious OsHV-1, (B) whole virus killed by chemical (BEI, formaldehyde), heat inactivated, or exposed to freeze-thaw cycles, (C) OsHV-1 DNA, (D) OsHV-1 whole protein extract, (E) Poly (I:C, positive control), (F) sterile filtered seawater (negative control). ROS stands for ‘reactive oxygen species’, ORF to ‘open reading frame’. Figure was created using BioRender.com. 22°C for 4 h, 6 h, 9 h, 18 h, or 22 h. Inactivation reactions were stopped by addition of sodium thiosulphate 1M (neutralizing agent) to reach a final concentration of 10% (v/v). Obtained inactivated viral suspensions were stored at 4°C until use. Suspension of neutralized 0.1% (v/v) BEI diluted in filtered (0.22 µm) SSW was used as the vehicle control (VC), (Supplementary Table 1). 2.1.4 Heat inactivation For preparation of heat-inactivated OsHV-1, viral suspensions were incubated for 1 h at 45°C, 50°C, or 52°C, or for 30 min at 54°C, 56°C, and 60°C using a dry bath, and then stored at 4°C until use (Supplementary Table 1). 2.1.5 Freeze-thaw cycles Viral suspensions were placed at -80°C for 12 h, transferred to -20°C for 12 h, and then maintained at 4°C. After complete thawing, viral suspensions were immediately re-frozen at -80°C for 12 h. The freeze-thaw cycles were repeated twice (for Thawing 1) or three times (for Thawing 2), and the antigen suspensions were stored at 4° C until use (Supplementary Table 1). 2.1.3 Formaldehyde inactivation Formaldehyde solution 37% (w/v) was added to the OsHV-1 suspension to a final concentration of 5%, 0.3%, or 0.01%, and incubated at 22°C for 2 h, 4 h, or12 h (5%); for 4 h, 8 h, 12 h, 24 h, 48 h, or 60 h (0.3%); or for 12 h, 24 h, or 60 h (0.01%), respectively. Virus inactivation was stopped by addition of 35% sodium bisulphite to reach a final concentration of 0.035%, and suspensions were then stored at 4°C. Suspension of 5% (v/v) neutralized formalin diluted in filtered (0.22 µm) SSW was used as the VC (Supplementary Table 1). Frontiers in Immunology 2.1.6 OsHV-1 DNA Total viral DNA was extracted from 1 ml of the OsHV-1 stock suspension using blood and tissues kit (QIAGEN) 03 frontiersin.org Delisle et al. 10.3389/fimmu.2023.1161145 immediately added to a 1.5 mL Eppendorf, previously kept on ice. Individual samples were checked under a light microscope (40X magnification) to confirm purity. When ≈16 mL of pure, clean hemolymph had been collected from multiple individuals, hemolymph samples were pooled, diluted (1:4 v/v) with autoclaved 0.2 µm-filtered sterile seawater (FSSW) for flow cytometry or kept undiluted for molecular analyses, and stored on ice until exposure. according to the manufacturer’s protocol. Extracted DNA was resuspended in 10 mM Tris-HCl buffer (pH 7.4) to reach a final concentration of 10 ng DNA µl-1 and stored at -20°C until use. The solution 10 mM Tris-HCl buffer (pH 7.4) was used as a VC (Supplementary Table 1). 2.1.7 Viral proteins Total viral proteins were extracted from 5 ml of the OsHV-1 stock suspension via bead beating for 10 min at 1500 rpm and 4°C using a 1600 MiniG automated tissue homogenizer (SPEX Sample Prep, Metuchen, NJ). Proteins were then solubilized for 45 min by adding 2 ml of extraction buffer (100 mM potassium phosphate, 50 mM NaCl, 0.1 mM EDTA-Na2, 1% polyvinyl pyrrolidone, 2 mM phenylmethylsulfonyl fluoride and 0.1% TritonX-100; pH 7.5 at 4° C). Solubilized proteins were extracted by centrifugation at 13,500 rpm for 15 min at 4°C and ultra-filtered (10 kDa molecular weight cut-off, Amicon Ultra-0.5 10K, Merk-Millipore, Burlington, USA) following manufacturer’s specifications. The semi-purified proteins were reconstituted in 200 µl of sterile Phosphate Buffer Saline (PBS). Total protein content of the lysate was quantified by the Lowry protein assay (53), diluted in sterile PBS to reach a final concentration of 0.2 mg.ml-1, and stored at -80°C until use. Protein extracts were either pure (Protein 1) or diluted at 1:10 (v/ v) in PBS (Protein 2). Sterile PBS was used as a vehicle control (Supplementary Table 1). 2.4 In-vitro exposure Before experimental exposure, each antigen preparation and vehicle control were diluted (1:100 v/v) with FSSW. Polyinosinicpolycytidylic acid [Poly (I:C)], a synthetic analogue of doublestranded RNA (dsRNA), was used as positive control (0.05 mg. ml-1 in SSW; 54) and FSSW was used as a negative control. For flow cytometry, antigens preparations or controls were added (1:80 v/v) to (1:4 v/v) diluted hemolymph (detailed above) and incubated at room temperature (22°C) for 1 h. The expression of five immunerelated genes and the viral gene ORF 87 was evaluated by adding 20 µl of the diluted antigen preparations (1:100 (v/v) or controls to 400 µl of pure hemolymph in 1.5 ml Eppendorf tubes and incubated at 22°C for 1 h. Following experimental exposure, hemocyte reactive oxygen species (ROS) production and viability were determined (detailed below). This process was repeated daily for 3 consecutive days to obtain n = 3 independent replicates. Based on ROS production in hemocytes and an absence of cytotoxicity, a subset of promising antigens was selected for further FCM validation and molecular analyses in a second experiment. Specific antigen preparations which induced significantly less ROS production were also included in the second experiment to maintain a range of contrasted immune responses and improve validation. This second in vitro challenge was repeated daily for 3 consecutive days to obtain n = 3 independent replicates. 2.2 Oysters The experiment was performed using hemolymph of hatcherybred juvenile C. gigas [8 months old, mean live weight 6.7 ± 3.1 g]. These oysters were the offspring of naïve wild stocks and therefore were expected to be highly susceptible to POMS. Prior to sampling, oysters were maintained in flow-through seawater (10 µm filtered) at ambient conditions (10 – 22°C and a salinity of 35 ± 1) and fed ad libitum with hatchery-grown algal food. Experimental oysters were considered naïve to POMS/OsHV-1 due to their rearing with continuous supply of UV-sterilized seawater (80 mJ cm-2) and maintenance under strict biosecurity management to ensure they remained OsHV-1-free. This status was confirmed prior to the experiment by the absence of significant mortality and OsHV-1 DNA detection in tissue (n = 10) using qPCR (51). Oysters were starved for 24 h prior to hemolymph collection to minimize algal contamination of the hemolymph. 2.5 FCM assessments Hemolymph samples that had been exposed to different antigen preparations for 1 h were analyzed using a Guava® EasyCyte™ 5HT flow cytometer equipped with a blue laser (488 nm) and green (525/30 nm), yellow (583/26 nm), and red (695/50) detectors (EMD Millipore, USA). Samples were mixed at medium speed and acquired at a flow rate of 0.24 µL s-1 for 30s. The production of intracellular ROS was measured using 2’,7’Dichlorodihydro-fluorescein diacetate (DCFH-DA, Sigma Aldrich, D6883) according to Donaghy et al. (55). Following 30 min of experimental treatments incubation, DCFH-DA was added at a final concentration of 10 µMand incubated in the dark at room temperature (22°C) for another 30 min until analysis (= total 1 h exposure to treatments). Relative ROS production was expressed as the level of green (FL1) fluorescence. 2.3 Hemolymph collection Hemolymph was sampled from between 22 and 35 oysters collected and pooled daily for experimental exposure. A small notch was made in the shell using wire cutters, and oysters were bled from the adductor muscle sinus using a 25G 1.5-inch needle with 1 mL syringe, previously kept on ice. Between 150 and 1500 µl of hemolymph was withdrawn from each individual and Frontiers in Immunology 04 frontiersin.org Delisle et al. 10.3389/fimmu.2023.1161145 The viability of hemocytes was measured using Fluorescein diacetate (FDA, Invitrogen, F1303) according to Rolton et al. (56). Following 50 min of experimental treatments incubation, FDA was added at a final concentration of 1.25 mg L-1 and incubated in the dark at room temperature for 10 min until analysis (= total 1 h exposure to treatments). Hemocytes were divided into those with high FL1 (corresponding to metabolically active/viable cells) and those with low green fluorescence (non-viable). 3 Results 3.1 79% of the antigenic preparations did not affect the viability of oyster hemocytes Of the 33 antigenic preparations tested, 26 had no cytotoxic effect on oyster hemocytes (as measured by viability). However, hemocytes exposed for 1 h to the following antigen preparations – OsHV-1 previously inactivated using BEI 0.04% for 4 h and 6 h (Figure 2A), formaldehyde 5% for 12 h, formaldehyde 0.3% for 8 h or 24 h, or formaldehyde 0.01% for 12 h (Figure 2C), and heated at 52°C for 1 h (Figure 2D) – showed a significant reduction in viability compared to hemocytes exposed to SSW. The viability of hemocytes that had been heat killed (negative control) was very low (10.2 ± 5.2% mean ± SD, n = 3) compared to hemocytes exposed to SSW (91.3 ± 8.0%, p = 1.7e-13) (Figure 2). 2.6 Molecular analyses RNA was extracted from 400 µl of hemolymph previously exposed to 20 µl of antigen preparation, SSW (negative control), Poly I:C (positive control), or live infectious OsHV-1 using the Quick RNA/ DNA Miniprep plus kit (Zymo Research) according to the manufacturer’s protocol. RNA was eluted in 50 µl DNAse/RNasefree water. As described in Delisle et al. (49), samples were treated with DNAse I (TURBOTM DNase, Invitrogen), the absence of DNA in the samples was confirmed by a 16S PCR assay, purity of the isolated RNA was assessed, and DNAse-treated RNA was transcribed into cDNA. Finally, droplet digital PCR (ddPCR) was conducted in an automated droplet generator (QX200 Droplet Digital PCR SystemTM, Bio-Rad) to determine the expression of five genes (Jak, Stat6, Viperin, IRF2, Myd88) related to oyster innate immunity (44, 57, 58), as well as the ORF 87, an OsHV-1 gene selected from the 39 ORFs described by Segarra et al. (59). Each ddPCR reaction included 1 µl of 3 µM of the primers (Jak, Stat, Viperin, Myd88) or 10 µM (IRF2, ORF87), 10 µl ddPCR Supermix for Evagreen (Bio-Rad), 2 µl cDNA, and 8 µl sterile water for a total reaction volume of 21 µl. As described in Delisle et al. (49), ddPCR was performed using the following cycling protocol: hold at 95°C for 5 s, 45 cycles of 95°C for 30 s, 60°C for 1 min, 4°C for 5 min, and a final enzyme deactivation step at 90°C for 5 min. The plate was then analyzed on the QX200 instrument (Bio-Rad). For each ddPCR plate run, at least one negative control (RNA/DNA-free water; Life Technologies), and one positive control (C. gigas DNA or Gblock for ORF87 diluted 1/10,000) were included. 3.2 Antigenic preparations induced ROS production in hemocytes Hemocytes of C. gigas that had been exposed to 10 antigenic preparations of inactivated OsHV-1 and to the positive control (Poly I:C) showed increased ROS production (Figure 3). Specifically, preparations of virus inactivated using BEI 0.04% at 9, 18, and 22 h (Figure 3A), formaldehyde 5% for 4 h, formaldehyde 0.01% for 24 h (Figure 3B), heat shock at 50°C for 1 h, at 60°C for 0.5 h, or three freeze-thaw cycles (Figure 3C), as well as viral protein extracts pure or diluted 1/10 (v/v, Figure 3D), all significantly increased hemocyte ROS production compared to those exposed to SSW (p < 0.001). Based on an absence of cytotoxicity and high levels of ROS production (using FCM), 10 antigen preparations, as well as eight additional antigen preparations that induced a limited subcellular immune response (Figures 2, 3), were selected for validation (using FCM and molecular analysis). 2.7 Statistical analyses 3.3 All the selected antigen preparations induced upregulation of immune-related genes Statistical analyses were computed using R 4.2.1 (https://www.rproject.org/) and the packages ‘ggpubr’ (60) and ‘rstatix’ (61). One way ANOVA and t tests were performed to evaluate the effects of each antigen preparation on ROS production, hemocytes viability, and gene expression, in comparison to the effects of SSW exposure (negative control); p-values were adjusted with Holm correction. For gene expression, a heatmap was constructed using Multiple Experiment Viewer software (62; http://mev.tm4.org/#/datasets/upload). For all analyses, the threshold significance level was set at 0.05. All the selected antigen preparations induced the upregulation of at least one of the immune- related genes of C. gigas. Exposure of hemocytes to the Poly (I:C) at 0.05 mg. ml-1 for 1 h induced a significant upregulation of MyD88, Viperin, and Stat6 (Figure 4). Exposure of hemocytes to OsHV-1, which had been inactivated using BEI 0.04% for 22 h, resulted in a significant upregulation of all five of the immune- related genes analyzed. Vehicle controls did not induce the expression of the immune- related genes in hemocytes, except the phosphate buffered saline (VC protein, Supplementary Table 1), which induced a significant upregulation of Stat6, Viperin, IRF2, and MyD88. As expected, the expression of ORF87 was only detected in hemocytes exposed to live infectious OsHV-1 (Figure 4). 2.8 Ethics approval The study was conducted according to the guidelines of the Declaration of Helsinki and approval of the Animal Ethics Committee was not applicable for the use of oysters. Frontiers in Immunology 05 frontiersin.org Delisle et al. 10.3389/fimmu.2023.1161145 A B C D FIGURE 2 Viability (in percent) of Crassostrea gigas hemocytes exposed for 1 h to OsHV-1, which had been inactivated with different preparations of (A) Binary ethylenimine BEI, (B) formaldehyde, and (C) temperature treatments. (D) shows viability of hemocytes exposed to two viral protein concentrations and purified viral DNA (see Supplementary Table 1 for details of the different treatments). Box plots indicate the median, upper, and lower quartiles. Whiskers indicate the highest and the lowest percent viable hemocytes and dots indicate outliers. Significance levels are expressed by asterisks: * (p < 0.05), ** (p < 0.01), ***(p < 0.001), **** (p < 0.0001). N = 3/6 replicates. ‘SW’ stands for seawater, ‘VC’ for vehicle control. 33 antigen preparations tested in our study, only seven induced a significant decline in hemocyte viability. Incomplete neutralization of the denaturing agent could explain this observed cytotoxicity. The residual infectivity post-inactivation of the preparations tested was verified by the absence of the open reading frame 87 (ORF87) expression in the hemocytes following 1h of antigen exposure. This ORF codes for an apoptosis inhibitor protein and is expressed during the first few hours post OsHV-1 infection in oysters (42, 59). A 1-hour exposure of hemocytes to 0.5 mg.ml-1 of Poly (I:C) was sufficient to induce cellular ROS production – a proxy of immune response in bivalves (64) – and an upregulation of the genes coding key antiviral effectors: MyD88, Stat 6, and Viperin, validating our screening approach. Increased ROS indicates a stimulation of an immune response, as ROS production is associated with internal chemical destruction of engulfed pathogens or foreign particles within hemocytes (65, 66). The mechanisms of hemocyte activation in oyster immune defense, however, are still largely unknown. In response to foreign particles, oyster hemocytes can: secrete effectors extracellularly; phagocytose particles, where they are destroyed by ROS and 4 Discussion In the present study, we were able to stimulate an antiviral response in juvenile Crassostrea gigas hemocytes using inactivated OsHV-1 and viral extracts, as shown by ROS induction and upregulation of antiviral response-related genes. A previous study, in which C. gigas hemocytes were exposed to live OsHV-1, also resulted in the expression of genes involved in immune-related functions (42); however, this study is the first time inactivated OsHV-1 preparations have been shown to elicit an immune response in-vitro. Virus inactivation transforms antigens from being infectious to non-infectious, and it is important to determine any cytotoxicity of the antigen preparations prior to determining their potency (52). In the absence of bivalve cell line cultures in which to determine cytotoxicity, here, we used a FCM-based assessment of hemocyte viability for rapid in-vitro screening of the OsHV-1 inactivated preparations. Morga et al. (63), similarly used FCM to determine hemocyte viability following an in-vitro exposure of C. gigas hemocytes to the protozoan parasite Bonamia ostreae. Among the Frontiers in Immunology 06 frontiersin.org Delisle et al. 10.3389/fimmu.2023.1161145 A B C D FIGURE 3 Reactive oxygen species (ROS) production (expressed in arbitrary unit, a.u) in Crassostrea gigas hemocytes exposed for 1 h to OsHV-1, which had been inactivated with different preparations of (A) Binary ethylenimine BEI, (B) formaldehyde, and (C) temperature treatments. (D) shows ROS production in hemocytes exposed to two viral protein concentrations and purified viral DNA (see Supplementary Table 1 for details of the different treatments). N = 3/6 replicates, box plots indicate the median, upper and lower quartiles; whiskers indicate the highest and the lowest values of the dataset and dots indicates outliers. Significance levels are expressed by asterisks: * (p < 0.05), ** (p<0.01), ***(p<0.001), **** (p<0.0001). ‘SW’ stands for seawater, ‘VC’ for vehicle control. had been stimulated for 8 h and 3 h, respectively, with 50 µg.ml-1 of Poly (I:C), a much higher concentration than was used in the present study (68, 69). Upregulation of Viperin – an interferoninducible antiviral protein – and Stat 6 – a signal transducer and activator of transcription – have also been observed in oyster hemocytes primed with Poly (I:C), in comparison to those exposed to seawater (46, 70). defense molecules stored in granules; and following stress or recognition of foreign particles by soluble and cellular pattern recognition receptors, induce the expression of specific immune genes (67). An induction of MyD88 gene, an essential signal transducer in the interleukin-1 and Toll-like receptor signaling pathways, was also observed in mussel (Mytilus galloprovincialis) and scallop (Pecten maximus) hemocytes that Frontiers in Immunology 07 frontiersin.org Delisle et al. 10.3389/fimmu.2023.1161145 activity as human Viperin against Dengue virus in-vitro (46, 80). Consequently, its expression in hemocytes could constitute a good indicator of the potency of the antigen preparations. Besides chemical agents, physical methods were used to inactivate OsHV-1 in this study. Thermal inactivation of OsHV-1 at 50°C and 60°C and long thawing cycles caused an increase in ROS production, while the associated molecular responses appeared moderate. These variations in the results could probably be explained by the broad range of stressors able to induce ROS production in bivalves (81), in comparison to the specificity of the antiviral response. Nonetheless, we obtained a good correlation overall between immune-related markers measured via flow cytometry (ROS production) and qPCR analyses (immune-gene expression), with the ranking of the 10 best antigen preparations maintained when upregulation of immunity genes was considered. Of note, protein extracts obtained from OsHV-1 and resuspended in PBS also induced a marked positive expression of MyD88, IRF2, and Viperin; however, the confounding effect of the vehicle control PBS on the hemocytes cannot be excluded. Similar induction of immune response following PBS exposure has been reported (82, 83). To conclude, these findings are significant as they evidence for the first time the potential for stimulating oyster’s innate immunity using OsHV-1 antigens, which may enable cost-effective therapeutic treatment to mitigate the economic impacts of OsHV1/POMS. For instance, chemical inactivation using the Binary ethylenimine at 0.04% for 22 h was identified as the best candidate preparation requiring additional research. However, it is essential to test these antigen preparations further using in-vivo infection models to validate promising candidate pseudo-vaccines. Nonetheless, we demonstrated that the use of flow cytometry-based cellular assays was an effective and rapid screening tool to select treatments for ‘pseudo-vaccine’ development. FIGURE 4 Heatmap focusing on the expression of 5 genes related to the innate immune response in Crassostrea gigas and ORF87, an OsHV1 ORF expressed early after infection. The intensities of the colors indicate the magnitude of the differential expression (log2 foldchange), N = 3 replicates. The fold-changes were calculated by comparing the expression of each gene in hemocytes exposed to Poly (I:C), live infectious OsHV-1, or OsHV-1 inactivated, with its expression in negative control (hemocytes exposed to SSW). Significance levels are expressed by asterisks: * (p < 0.05), ** (p < 0.01), ***(p < 0.001), **** (p < 0.0001). The interaction between OsHV-1 and the oyster host cells has not been fully elucidated, and the functions of proteins coded by OsHV-1 genome are largely unknown. However, antibody blocking and pull-down assays suggest the potential implication of three putative OsHV-1 membrane proteins (ORF 25, ORF 41, and ORF 72) in the virus/host interaction by binding of host cytoskeleton (71, 72). Interestingly, antigen preparations resulting from BEI inactivation induced the strongest immune and antiviral response. Binary ethylenimine is an aziridine preparation commonly used in veterinary vaccine production as an inactivating agent (73). At 1 mM, BEI induces an alkylation of the nucleic acids without damaging proteins (74). In the present work, high ROS production coupled with upregulation of the five tested genes were recorded when using OsHV-1 inactivated with BEI at [0.04%] for 22h, supporting the role of viral proteins in the stimulation of antiviral response in oysters. Conversely, we observed a reduced potency of antigen preparations that were inactivated by formaldehyde, one of the most widely used inactivating agents, for an extended exposure time (12 and 48h), suggesting an irreversible denaturation of proteins induced by formaldehyde (75). Three antigen preparations inactivated with BEI induced a strong upregulation of the transcript coding for Viperin. Viperin is a highly conserved evolutionary host protein (76), which restricts the replication of a range of RNA and DNA viruses [e.g., human cytomegalovirus (77), immunodeficiency virus (78), and Hepatitis C virus (79)], by interacting with viral protein and altering the site of virus budding. In C. gigas, Viperin has been reported to be one of the earliest and most regulated genes in response to OsHV-1 exposure (54, 80), and it also exhibits the same level of antiviral Frontiers in Immunology Data availability statement The original contributions presented in the study are included in the article/Supplementary Materials. Further inquiries can be directed to the corresponding authors. Author contributions LD, AR, JV, conception and design. LD and AR lab trial and data analyses. All authors, writing, revision/editing. LD, JV funding. All authors contributed to the article and approved the submitted version. Funding This work was supported by the Cawthron Shellfish Aquaculture Research Platform (contract no. CAWX1801) funded by the New Zealand Ministry of Business, Innovation and Employment. 08 frontiersin.org Delisle et al. 10.3389/fimmu.2023.1161145 Acknowledgments Publisher’s note We would like to thank Ruth Garcia Gomez, Hayley Chapman, Carol Peychers, and Karthiga Kumanan for their technical assistance and Kirsty Smith for her advice. Thank you also to Nick King and Seumas Walker for reviewing this manuscript. We are also grateful to William McKay and Jim Dollimore at Biomarine Ltd for the provision of wild oysters, and the hatchery team at the Cawthron Aquaculture Park for maintaining and growing the experimental animals. All claims expressed in this article are solely those of the authors and do not necessarily represent those of their affiliated organizations, or those of the publisher, the editors and the reviewers. 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Assessment of the association of the rs12329760 polymorphism of the &lt;i&gt;TMPRSS2&lt;/i&gt; gene with acute coronary syndrome in patients with new coronavirus infection
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Оценка ассоциации полиморфизма rs12329760 гена TMPRSS2 с острым коронарным синдромом у пациентов, перенесших новую коронавирусную инфекцию В.А. Козик1, Л.А. Шпагина1, И.С. Шпагин1, С.В. Максимова1, 2, Н.Г. Ложкина3, В.Н. Максимов1, 2 1 Новосибирский государственный медицинский университет Министерства здравоохранения Российской Федерации Россия, 630091, г. Новосибирск, Красный пр., 52 Козик В.А., Шпагина Л.А., Шпагин И.С., Максимова С.В., Ложкина Н.Г., Максимов В.Н., 2024 А Т Е Р О С К Л Е Р О З / A T E R O S C L E R O Z А Т Е Р О С К Л Е Р О З / A T E R O S C L E R O Z 2024 Т. 20, № 1 Научно-практический журнал DOI 10. 52727/2078-256Х-2024-20-1-35-41 Аннотация Обоснование. Сердечно-сосудистые заболевания (ССЗ) остаются на первом месте по уровню заболеваемости и смертности как в Российской Федерации, так и во всeм мире. С конца 2019 – начала 2020 г. планету охватила пандемия новой коронавирусной инфекции (НКИ), которая внесла свой вклад в развитие и течение ССЗ. Поэтому стали рассматривать молекулярно-генетические маркеры как факторы, потенциально влияющие на ССЗ, их развитие и тяжесть течения. Цель исследования – оценить ассоциацию варианта нуклеотидной последо- вательности rs12329760 гена ТMPRSS2 с острым коронарным синдромом (ОКС) у пациентов, перенесших НКИ. Материал и методы. В исследование включены 100 пациентов (женщин – 50, мужчин – 50) с ОКС и перенесенной НКИ, которые отбирались на основе положитель- ного ПЦР-теста на наличие SARS-CoV-2 в анамнезе, госпитализированных в региональный сосудистый центр №  7 Городской клинической больницы №  2 г. Новосибирска. Возраст женщин составил 59,5  ±  7,2  года, мужчин — 53,5  ±  9,3 года. Диагноз ОКС устанавливался по совокупности критериев, предложенных и установленных Российским кардиологическим обществом и в соответствии с обновленными клиническими рекомендациями Министерства здравоохранения Российской Федерации от 2020 г. Пациентам проводились клинико- инструментальное обследование, коронароангиография со стентированием, предусмотрен- ные стандартами оказания медицинской помощи и клиническими рекомендациями. У па- циентов определяли вариант нуклеотидной последовательности rs12329760 гена TMPRSS2 с помощью ПЦР с последующим анализом полиморфизма длин рестрикционных фрагментов. Группу сравнения составили 200 пациентов с ОКС без перенесенной НКИ (нет положи- тельного ПЦР-теста, нет антител к SARS-CoV-2). Результаты. Носительство генотипов СС, CT, TT rs12329760 гена TMPSS2 не ассоциировано с повышенным риском развития ОКС у пациентов, перенесших НКИ. При сравнении частот генотипов rs12329760 гена TMPSS2 в группах с ОКС и НКИ и ОКС без НКИ не получено статистически значимых различий. В группе с ОКС и НКИ чаще встречается носительство гомозиготного генотипа СС. Заключение. Вариант нуклеотидной последовательности rs12329760 гена TMPRSS2 не ассоциирован с ОКС у пациентов, перенесших НКИ. Ключевые слова: острый коронарный синдром, новая коронавирусная инфекция, вариант нуклеотидной последовательности, ген TMPRSS2, генетические маркеры, rs12329760. Ключевые слова: острый коронарный синдром, новая коронавирусная инфекция, вариант нуклеотидной последовательности, ген TMPRSS2, генетические маркеры, rs12329760. Конфликт интересов. Авторы заявляют об отсутствии конфликта интересов. Финансирование. Молекулярно-генетический фрагмент исследования выполнен в рамках бюджетной темы, рег. № 122031700094-5. Автор для переписки: Козик В.А., e-mail valiyta90@mail.ru Для цитирования: Козик В.А., Шпагина Л.А., Шпагин И.С., Максимова С.В., Ложки- на Н.Г., Максимов В.Н. Оценка ассоциации полиморфизма rs12329760 гена TMPRSS2 с острым коронарным синдромом у пациентов, перенесших новую коронавирусную инфекцию. Атеросклероз, 2024; 20 (1): 35–41. doi: 10. Assessment of the association of the rs12329760 polymorphism of the TMPRSS2 gene with acute coronary syndrome in patients with new coronavirus infection V.A. Kozik1, L.A. Shpagina1, I.S. Shpagin1, S.V. Maksimova1, 2, N.G Lozhkina3, V.N. Maksimov1, 2 1 Novosibirsk State Medical University of Mivzdrav of Russia 52, Krasny av., Novosibirsk, 630091, Russia 2 Research Institute of Internal and Preventive Medicine – Branch of the Institute of Cytology and Genetics, Siberian Branch of Russian Academy of Sciences 175/1, Boris Bogatkov str., Novosibirsk, 630089, Russia 3 Federal Research Center for Fundamental and Translational Medicine 2, Timakov str., Novosibirsk, 630117, Russia Abstract The authors declare no conflict of interest. Funding. The molecular genetic fragment of the study was carried out within the framework of the budget topic reg. No. 122031700094-5. Conflict of interest. The authors declare no conflict of interest. Funding. The molecular genetic fragment of the study was carried out within the framework of the budget topic reg. No. 122031700094-5. Conflict of interest. The authors declare no conflict of interest. Funding. The molecular genetic fragment of the study was carried out within the framework of the budget topic reg. No. 122031700094-5. Funding. The molecular genetic fragment of the study was carried out within the framework of the budget topic reg. No. 122031700094-5. g p g Correspondence: Kozik V.A., e-mail valiyta90@mail.ru Correspondence: Kozik V.A., e-mail valiyta90@mail.ru Citation: Kozik V.A., Shpagina L.A., Shpagin I.S., Maksimova S.V., Lozhkina N.G, Maksimov V.N. Assessment of the association of the rs12329760 polymorphism of the TMPRSS2 gene with acute cor- onary syndrome in patients with new coronavirus infection. Ateroscleroz, 2024; 20 (1): 35–41. doi: 10.52727/2078-256X-2024-20-1-35-41 Abstract Introduction. Cardiovascular diseases (СVD) rank first in terms of morbidity and mortality not only in the Russian Federation but throughout the world. Since the end of 2019 a pandemic of Se- vere Acute Respiratory Syndrome (SARS) or new coronavirus infection (NCI) has spread throughout the world, which contributed to the development and course of CVD. Therefore, genetic markers began to be considered as factors potentially influencing CVD, its development and severity. Objec- tive: To evaluate the association of the nucleotide sequence variant rs12329760 of the TMPRSS2 gene with acute coronary syndrome in patients who have had a new coronavirus infection. Material and methods. The study included 100 patients (women – 50, men – 50) with ACS and previous NCI, who were selected on the basis of a positive PCR test for the presence of SARS-CoV-2 in the anamnesis, hospitalized at the regional vascular center No. 7 of the City Clinical Hospital No. 2 of the city of Novosibirsk. Women age was 59.5 ± 7.2 years, men age was 53.5 ± 9.3 years. The diagnosis of ACS was established according to a set of criteria proposed and established by the Rus- sian Society of Cardiology and in accordance with the updated clinical recommendations of the Ministry of Health of the Russian Federation of 2020. Patients underwent clinical and instrumental examination, coronary angiography with possible stenting, as provided for in the standards of medical care and clinical guidelines. The nucleotide sequence variant rs12329760 of the TMPSS2 gene was determined in patients using PCR with further analysis of restriction fragment length polymorphism. The comparison group consisted of 200 patients with ACS without previous NCI (no positive PCR test, no positive antibodies). Results. Carriage of CC, СТ, TT, genotype variants of the nucleotide sequence rs12329760 of the TMPSS2 gene are not associated with an increased risk of developing ACS in patients with previous NCI. When comparing the frequencies of the rs12329760 genotypes of the TMPSS2 gene in groups with ACS with NCI and ACS without NCI, no statistically significant differences were obtained. In the group with ACS with NCI, carriage of the homozygous genotype was more common (p = 0.011). Conclusions. Variants of the nucleotide sequence rs12329760 of the TMPSS2 gene are not associated with ACS with previous NCI. Keywords: acute coronary syndrome, new coronavirus infection (NCI), nucleotide sequence vari- ant (NSV), TMPRSS2, genetic markers, rs12329760. Conflict of interest. Аннотация 52727/2078-256Х-2024-20-1-35-41 © Козик В.А., Шпагина Л.А., Шпагин И.С., Максимова С.В., Ложкина Н.Г., Максимов В.Н., 2024 35 Атеросклероз. Т. 20. № 1. 2024 / Ateroscleroz. Vol. 20. N 1. 2024 Введение но, несмотря на это, смертность от ССЗ оста- ется крайне высокой [2]. Свой вклад в развитие данной патологии внесла новая коронавирусная инфекция (НКИ), что обусловливает актуаль- ность данной проблемы [3]. Уже известно, что НКИ поражает сердечную мышцу, активирует гиперкоагуляцию и способствует микротромбо- зам, вызывая тем самым как в госпитальном, так и в постгоспитальном периоде ОКС, мио- Как в Российской Федерации, так и во всем мире сердечно-сосудистые заболевания (ССЗ) остаются на первом месте по уровню заболева- емости и смертности [1]. Новые лекарственные препараты, новые технологии лечения внесли значительный вклад в течение острого коронар- ного синдрома (ОКС) и прогноз для пациентов, 36 В.А. Козик, Л.А. Шпагина, И.С. Шпагин и др. Из исследования исключались пациенты с любой онкологической патологией, тяжелой печеночной и почечной недостаточностью, инсулинзависимым сахарным диабетом 1 и 2 типа, воспалительными заболеваниями печени, почек, эндокринных желез, половых органов, психическими расстройствами, алкоголизмом, токсикоманией, ВИЧ. От каждого пациента получено информированное согласие на участие в исследовании в соответствии с этическими требованиями ВОЗ. Исследование одобрено локальным этическим комитетом ФГБОУ ВО «Новосибирский государственный медицин- ский университет» Минздрава России (протокол № 152 от 25.05.2023). кардит, инфаркт миокарда (ИМ) с подъемом и без подъема сегмента ST [4]. Особое внимание ученые всего мира уделяют молекулярно-гене- тическим маркерам как факторам, потенциаль- но влияющим на ССЗ, их развитие, тяжесть те- чения. Так, например, определенные пациенты лучше реагируют на лечение и кардиореабили- тацию, а у кого-то чаще случаются осложнения. тацию, а у кого-то чаще случаются осложнения. Ген трансмембранной сериновой протеа- зы-2 (transmembrane serine protease 2, TMPRSS2) кодирует одноименный белок из семейства се- риновых протеаз [5], широко распространен- ных в природе и составляющих около трети всех известных протеолитических ферментов, которые участвуют в различных процессах, та- ких как пищеварение, оплодотворение, акти- вация комплемента, апоптоз, иммунный ответ [6]. Ген TMPRSS2 ассоциирован с раком про- статы и повышенным риском инфицирования коронавирусами SARS-CoV и SARS-CoV-2. В результатах большого количества исследований определяются ассоциации варианта нуклеотид- ной последовательности (ВНП) rs12329760 гена TMPRSS2 с различными ССЗ [7]. Целью насто- ящего исследования стала оценка ассоциации ВНП rs12329760 гена TMPSS2 с ОКС у пациен- тов, перенесших НКИ. Первую запись ЭКГ пациенту проводила бригада скорой медицинской помощи, а за- тем – дежурная служба регионального сосудис- того центра. При выполнении настоящего исследования по ЭКГ, которую регистриро- вали в 12 стандартных отведениях с помощью шестиканального аппарата Megacart (Simens- Elema АВ, Германия), определяли динамику, локализацию и стадию ОКС. Введение Селективную ко- ронарографию выполняли на 1–3-и сутки от развития симптомов острого ИМ на ангиогра- фической установке Optima IGS 330 (General Electric, США) с фиксацией изображения на компьютере. Всем пациентам ставились коро- нарные стенты с лекарственным покрытием си- ролимус BioMime (Meril Life Sciences, Индия), система доставки быстрой смены rapid exchange (RX) с раздуваемым полуподдатливым балло- ном, минимально выходящим за пределы стен- та. Всем пациентам с ОКС и НКИ в анамнезе при поступлении были проведены холтеровское мониторирование ЭКГ и все остальные лабора- торные исследования согласно стандарту обсле- дования ОКС. Материал и методы Дизайн исследования: сравнительное опи- сательное исследование двух параллельных групп с ретроспективным компонентом. Ос- новную группу составили 100 больных с ОКС и перенесенной НКИ (наличие положительного ПЦР-теста на наличие SARS-CoV-2 в анамне- зе, повышенное содержание IgG в крови), 50 женщин (возраст 59,5  ±  7,2  года) и 50 мужчин (возраст 53,5  ±  9,3 года). В группу сравнения вошли 200 пациентов с ОКС без перенесенной НКИ в анамнезе, сопоставимых по полу и воз- расту, она была набрана  на базе ГБУЗ НСО «Городская клиническая больница №  1» г. Но- восибирска и включала больных с острым ИМ с подъемом сегмента ST, последовательно посту- павших в отделение реанимации и интенсивной терапии регионального сосудистого центра № 1. Больные основной группы (с НКИ в анамне- зе) поступали в региональный сосудистый центр №  7 ГБУЗ НСО «Городская клиниче- ская больница №  2» г. Новосибирска. Диагноз ОКС устанавливался по совокупности критери- ев, предложенных и установленных Российским кардиологическим обществом и в соответствии с обновленными клиническими рекомендация- ми Министерства здравоохранения Российской Федерации от 2020 г. ДНК из образцов венозной крови выделялась с помощью фенол-хлороформного метода. Всем исследуемым пациентам определяли ВНП rs12329760 гена TMPRSS2 с помощью полимеразной цепной реакции с последующим анализом полиморфизма длин рестрикционных фрагментов. Для амплификации использовали праймеры: 5'-ctacggaccaaacttcatccttccg-3'(F) и 5'-gtctgttactgtcactcggcgggt-3'(R). Смесь для ПЦР объемом 25 мкл включала: Трис-HCI (pH 9,0) 75  мM, (NH4)2SO4 20 мM, Тween-20 0,01  %, 3,0  мМ MgCl2, по 2,0  мкл каждого праймера, 0,5  мМ смеси dNTP, 2  мкг ДНК, 1 единицу Тaq-полимеразы, вода до объема 25  мкл. Ам- плификацию проводили в следующем тем- пературном режиме: 33 цикла, включающих денатурацию 95 °С 30  с, отжиг праймеров 60 °С 30 с и элонгацию 72 °С 30 с. Рестрикцию 37 Атеросклероз. Т. 20. № 1. 2024 / Ateroscleroz. Vol. 20. N 1. 2024 циентов), генотипа CC – 60 % (30 пациентов). В группе сравнения у мужчин частота генотипа ТТ составила 5,2 % (6 пациентов), частота ге- нотипа СТ – 26,1 % (30 пациентов), частота ге- нотипа СС – 68,7 % (79 пациентов). У женщин в группе с ОКС и НКИ частота генотипа ТТ составила 14 % (7 пациентов), частота геноти- па СТ – 40 % (20 пациентов), частота генотипа СС – 46 % (23 пациента). В группе сравнения у женщин частота генотипа ТТ составила 8,2 % (7 пациентов), частота генотипа СТ – 35,3 % (30 пациентов), частота генотипа СС – 56,5 % (48 пациентов). Материал и методы Таким образом, носительство гомозигот как в исследуемой группе, так и в группе сравнения было существенно выше гетерозигот в процентном соотношении. Статистически значимых различий выявлено не было. проводили с 10  ед. активности рестриктазы Msp I («СибЭнзим», Новосибирск) при 37 °С в течение 16 ч. Размер продукта амплификации 160  п.н. Детекцию продуктов амплификации и рестрикции осуществляли методом электро- фореза в 4  % полиакриламидном геле с после- дующим окрашиванием бромистым этидием. После проведения рестрикции при генотипе ТТ детектировался продукт 160 п.н., при генотипе СС – продукты 137 п.н. и 23 п.н., при гетеро- зиготном генотипе все перечисленные продук- ты: 160 п.н., 137 п.н., 23 п.н. проводили с 10  ед. активности рестриктазы Msp I («СибЭнзим», Новосибирск) при 37 °С в течение 16 ч. Размер продукта амплификации 160  п.н. Детекцию продуктов амплификации и рестрикции осуществляли методом электро- фореза в 4  % полиакриламидном геле с после- дующим окрашиванием бромистым этидием. После проведения рестрикции при генотипе ТТ детектировался продукт 160 п.н., при генотипе СС – продукты 137 п.н. и 23 п.н., при гетеро- зиготном генотипе все перечисленные продук- ты: 160 п.н., 137 п.н., 23 п.н. При выполнении статистического анализа рассчитывали частоту качественных признаков, средние значения количественных показателей (М) и их стандартное отклонение (SD). Срав- нение групп по качественным признакам проводили с использованием критерия χ2, ко- личественных – с помощью критерия Стью- дента. Оценивали отношение шансов (ОШ) с 95%-м доверительным интервалом (95 % ДИ) для каждой переменной. Клинико-анамнестическая характеристика пациентов основной группы и группы сравнения представлена в табл. 2, из которой видно, что у больных ОКС с НКИ чаще, чем в отсутствие последней, наблюдались однососудистое пора- жение коронарных артерий, артериальная гипер- тензия, чрескожные коронарные вмешательства со стентированием, смерть родственников от ССЗ и дислипидемия в анамнезе (табл. 2). Результаты Всем пациентам определяли ВНП rs12329760 гена трансмембранной сериновой протеазы-2 с помощью полимеразной цепной реакции, по результатам которого в группе c ОКС и НКИ генотип TT выявлен у 9 (9 %) пациентов, генотип СT – у 38 (38 %), генотип СС – у 53 (53 %). В группе сравнения: генотип TT – у 13 (6,5 %) пациентов, генотип CT – у 60 (30 %), генотип СС – у 127 (63,5 %) пациентов соот- ветственно. Частоты генотипов представлены в табл. 1. Обсуждение В настоящем исследовании чаще всего наблюдалось носительство генотипа CC гена TMPRSS2 rs12329760. Не выявлено связи ВНП с развитием ОКС у пациентов, перенесших НКИ, в отличие от данных зарубежных работ, в которых ВНП изучались на больших выборках пациентов. Так, например, ученые Бразилии, включившие в исследование 402 пожилых пациента с подтвержденным COVID-19 [6], по- С разделением по полу в исследуемой группе у мужчин частота генотипа TT составила 4 % (2 пациента), генотипа СT – 36 % (18 па- Т аблица 1 Частоты генотипов rs12329760 гена TMPRSS2 с острым коронарным синдромом у пациентов, перенесших новую коронавирусную инфекцию и группы сравнения T able 1 Frequencies of rs12329760 genotypes of the TMPRSS2 gene with acute coronary syndrome in patients who had a new coronavirus infection and comparison groups TMPRSS2, rs12329760 ОКС и НКИ / ACS with NCI Контрольная группа с ОКС / Control group with ACS p n % n % Генотип TT / Genotype TT 9 9 13 6,5 р = 0,432 Генотип СТ / Genotype CT 38 38 60 30 р = 0,207 Генотип СС / Genotype CC 53 53 127 63,5 р = 0,104 Всего / Total 100 100,0 200 100,0 ц Частоты генотипов rs12329760 гена TMPRSS2 с острым коронарным синдромом у пациентов, перенесших новую коронавирусную инфекцию и группы сравнения T able 1 Частоты генотипов rs12329760 гена TMPRSS2 с острым коронарным синдромом у пациентов, пере коронавирусную инфекцию и группы сравнения s12329760 гена TMPRSS2 с острым коронарным синдромом у пациентов, перенесших новую коронавирусную инфекцию и группы сравнения 38 В.А. Козик, Л.А. Шпагина, И.С. Шпагин и др. Обсуждение Т аблица 2 Клинико-анамнестическая характеристика ОКС у пациентов, перенесших НКИ, и группы сравнения T able 2 Clinical and anamnestic characteristics of acute coronary syndrome in patients who suffered a new coronavirus infection and comparison group Показатель / Characteristic ОКС с НКИ / ACS with NCI (n = 100) ОКС без НКИ / ACS with- out NCI (n = 200) p ОШ [95% ДИ] / OR [95% CI] Возраст, лет/ Age, years 55,5 ± 9,5 56,5 ± 7,2 0,933 – Сроки поступления: до 2 ч /1 сут, n (%) / Timing of patient admission: up to 2 hours/1 day, n (%) 79/21 (79/21) 118/82 (59/41) <0,001 2,614 [1,497; 4,566] Тромболизис, n (%) / Thrombolysis, n (%) 8 (8) 14 (7) 0,755 1,155 [0,468; 2,853] Предшествующая стенокардия напряжения, функциональный класс 2–3, n (%) / Previous angina pectoris, functional class 2–3, n (%) 23 (23) 56 (28) 0,4354 0,768 [0,439; 1,343] Острый ИМ в анамнезе, n (%) / History of acute MI, n (%) 7 (7) 12 (6) 0,738 1,179 [0,449; 3,094] Чрескожные коронарные вмешательства в анамнезе, n (%) / History of percutaneous coronary interventions, n (%) 9 (9) 6 (3) 0,025 3,198 [1,105; 9,254] Аортокоронарное шунтирование в анамнезе, n (%) / History of coronary artery bypass grafting, n (%) 1 (1) 4 (2) 0,524 0,495 [0,055; 4,488] Однососудистое поражение по коронароангиографии, n (%) / Single-vessel lesion ac- cording to coronary angiography, n (%) 72 (72) 68 (34) <0,001 4,992 [2,951; 8,442] Многососудистое поражение по коронароангиографии, n (%) / Multivessel lesion according to coronary angiography, n (%) 28 (28) 51 (25,5) 0,644 1,136 [0,662; 1,950] Артериальная гипертензия, n (%) / Arterial hypertension, n (%) 72 (72) 104 (52) 0,001 2,374 [1,415; 3,981] Подтвержденный сахарный диабет 2 типа, n (%) / Confirmed type 2 diabetes mellitus, n (%) 21 (21) 44 (22) 0,843 0,942 [0,524; 1,694] Курение, n (%) / Smoking, n (%) 34 (34) 77 (38,5) 0,447 0,823 [0,498; 1,360] Смерть родственников от ССЗ, n (%) / Relatives death’s from CVD, n (%) 20 (20) 72 (36) 0,005 0,444 [0,252; 0,785] Дислипидемия в анамнезе, n (%) / History of dyslipidemia, n (%) 17 (17) 57 (28,5) 0,030 0,514 [0,280; 0,941] Жизнеугрожающие нарушения ритма, n (%) / Life-threatening rhythm disturbances, n (%) 11 (11) 32 (16) 0,244 0,649 [0,312; 1,349] Кillip > II, n (%) 12 (12) 38 (19) 0,126 0,581 [0,289; 1,170] Т аблица 2 я характеристика ОКС у пациентов, перенесших НКИ, и группы сравнения Т аблица 2 Клинико-анамнестическая характеристика ОКС у пациентов, перенесших НКИ, и группы сравнения T able 2 Т Клинико-анамнестическая характеристика ОКС у пациентов, перенесших НКИ, и группы сравн предыдущих исследований этого варианта в популяциях европейского происхождения, ко- торые предполагали, что носительство данного аллеля, наоборот, является защитным факто- ром от тяжелого течения НКИ. Список литературы/ References 1. Barbarash O.L., Duplyakov D.V., Zateish- chikov D.A., Shakhnovich R.M., Yavelov I.S., Ya- kovlev A.N., Abugov S.A., Alekyan B.G., Arkhi- pov M.V., Vasilieva E.Yu., Galyavich A.S., Ganyukov V.I., Gilyarevskiy S.R., Golubev E.P., Golukhova E.Z., Gratsianskiy N.A., Karpov Yu.A., Kosmacheva E.D., Lopatin Yu.M., Markov V.A., Nikulina N.N., Pevzner D.V., Pogosova N.V., Pro- topopov A.V., Skrypnik D.V., Tereshchenko S.N., Ustyugov S.A., Khripun A.V., Shalaev S.V., Shpe- ktor A.V., Yakushin S.S. Acute coronary syndrome without ST segment elevation electrocardiogram. Clinical guidelines 2020. Russ. J. Cardiol., 2021; 26 (4): 149–202. (In Russ). doi: 10.15829/1560-4071- 2021-4449 [Барбараш О.Л., Дупляков Д.В., Затей- щиков Д.А., Шахнович Р.М., Явелов И.С., Яков- лев А.Н., Абугов С.А., Алекян Б.Г., Архипов М.В., Васильева Е.Ю., Галявич А.С., Ганюков В.И., Гиляревский С.Р., Голубев Е.П., Голухова Е.З., Грацианский Н.А., Карпов Ю.А., Космачева Е.Д,, Лопатин Ю.М., Марков В.А., Никулина Н.Н., Певзнер Д.В., Погосова Н.В., Протопопов А.В,, Скрыпник Д.В., Терещенко С.Н., Устюгов С.А., Хрипун А.В., Шалаев С.В., Шпектор А.В., Яку- 1. Barbarash O.L., Duplyakov D.V., Zateish- chikov D.A., Shakhnovich R.M., Yavelov I.S., Ya- kovlev A.N., Abugov S.A., Alekyan B.G., Arkhi- pov M.V., Vasilieva E.Yu., Galyavich A.S., Ganyukov V.I., Gilyarevskiy S.R., Golubev E.P., Golukhova E.Z., Gratsianskiy N.A., Karpov Yu.A., Kosmacheva E.D., Lopatin Yu.M., Markov V.A., Nikulina N.N., Pevzner D.V., Pogosova N.V., Pro- topopov A.V., Skrypnik D.V., Tereshchenko S.N., Ustyugov S.A., Khripun A.V., Shalaev S.V., Shpe- ktor A.V., Yakushin S.S. Acute coronary syndrome without ST segment elevation electrocardiogram. Clinical guidelines 2020. Russ. J. Cardiol., 2021; 26 (4): 149–202. (In Russ). doi: 10.15829/1560-4071- 2021-4449 [Барбараш О.Л., Дупляков Д.В., Затей- щиков Д.А., Шахнович Р.М., Явелов И.С., Яков- лев А.Н., Абугов С.А., Алекян Б.Г., Архипов М.В., Васильева Е.Ю., Галявич А.С., Ганюков В.И., Гиляревский С.Р., Голубев Е.П., Голухова Е.З., Грацианский Н.А., Карпов Ю.А., Космачева Е.Д,, Лопатин Ю.М., Марков В.А., Никулина Н.Н., Певзнер Д.В., Погосова Н.В., Протопопов А.В,, Скрыпник Д.В., Терещенко С.Н., Устюгов С.А., Хрипун А.В., Шалаев С.В., Шпектор А.В., Яку- /j p 5. Kozik V.A., Shpagin I.S., Zolotukhina E.V., Panacheva L.A., Shpagina L.A. Molecular genetic marcers of cardiovascular pathology in patients with a new coronavirus infection COVID-19. Modern Probl. Sci. Educаt., 2023; 3: 98. (In Russ). doi: 10.17513/ spno.32563 [Козик В.А., Шпагин И.С., Золотухи- на Е.В., Паначева Л.А., Шпагина Л.А. Молеку- лярно-генетические маркеры кардиоваскулярной патологии у пациентов, перенесших новую коро- навирусную инфекцию COVID-19. Соврем. пробл. науки и образования, 2023; 3: 98. doi: 10.17513/ spno.32563]. 6. Заключение ВНП rs12329760 гена TMPRSS2 не ассоци- ирован с ОКС у пациентов, перенесших НКИ. 4. 4. Yaghoobi A., Lord J.S., Rezaiezadeh J.S., Yekanine- jad M.S., Amini M., Izadi P. TMPRSS2 polymor- phism (rs12329760) and the severity of the COVID-19 in Iranian population. PLoS One, 2023; 18 (2): e0281750. doi: 10.1371/journal.pone.0281750 Обсуждение ontgomery H., Openshaw P.J.M., Begg C., Walsh T., nesa A., Flores C., Riancho J.A., Rojas-Martinez A., Lapunzina P.; GenOMICC Investigators; SCOURGE Consortium; ISARICC Investigators; 23andMe Vitart V., Abedalthagafi M., Luchessi A.D., Parra E.J., Cruz R., Carracedo A., Fawkes A., Murphy L., Rowan K., Pereira A.C., Law A., Fairfax B., Hendry S.C., Baillie J.K. GWAS and meta-analysis identifies 49 genetic variants underlying critical COVID-19. Nature. 2023; 617 (7962): 764–768. doi: 10.1038/s41586-023-06034-3 Обсуждение В украинской популяции изучался ген-кандидат rs12329760 гена TMPRSS2 и его ассоциация с повышен- ным риском развития НКИ, а также смертно- стью [8]. Всего исследовали три группы боль- ных: 62 человека без кислородной терапии, 92 с неинвазивной и 35 с инвазивной кисло- казали, что выживаемость носителей генотипа GG была меньше по сравнению с больными с другими генотипами (p = 0,009). При много- факторном анализе генотип GG был фактором, независимо связанным с риском смерти у по- жилых людей (относительный риск 4,03, 95 % ДИ 1,49–10,84). В исследовании иранских уче- ных, включавшем 251 пациента с НКИ, пока- зано, что аллель Т rs12329760 в гене TMPRSS2 представляет собой аллель риска тяжелой фор- мы заболевания [4], в отличие от большинства 39 Атеросклероз. Т. 20. № 1. 2024 / Ateroscleroz. Vol. 20. N 1. 2024 родной терапией. Обращает на себя внимание преобладание носительства гомозигот (СС и ТТ) над гетерозиготой (СТ), что сопоставимо с результатами нашего исследования. В работе [9] доказана связь rs12329760 гена-кандидата TMPRSS2 с повышением риска развития более тяжелых форм НКИ: при исследовании 693 пациентов с положительным исходом болезни (выздоровление) и 592 человек с негативным исходом (смерть) выявлено, что генотип CC rs12329760 TMPRSS2 связан с повышенным уровнем смертности от COVID-19, т. е. пред- ставляет собой полиморфизм, связанный со значительно более высокой заболеваемостью и тяжелым течением НКИ. родной терапией. Обращает на себя внимание преобладание носительства гомозигот (СС и ТТ) над гетерозиготой (СТ), что сопоставимо с результатами нашего исследования. В работе [9] доказана связь rs12329760 гена-кандидата TMPRSS2 с повышением риска развития более тяжелых форм НКИ: при исследовании 693 пациентов с положительным исходом болезни (выздоровление) и 592 человек с негативным исходом (смерть) выявлено, что генотип CC rs12329760 TMPRSS2 связан с повышенным уровнем смертности от COVID-19, т. е. пред- ставляет собой полиморфизм, связанный со значительно более высокой заболеваемостью и тяжелым течением НКИ. шин С.С. Острый коронарный синдром без подъема сегмента ST электрокардиограммы. Клинические рекомендации 2020. Рос. кардиол. журн., 2021; 26, (4): 149–202. doi: 10.15829/1560- 4071-2021-4449]. шин С.С. Острый коронарный синдром без подъема сегмента ST электрокардиограммы. Клинические рекомендации 2020. Рос. кардиол. журн., 2021; 26, (4): 149–202. doi: 10.15829/1560- 4071-2021-4449]. родной терапией. Обращает на себя внимание преобладание носительства гомозигот (СС и ТТ) над гетерозиготой (СТ), что сопоставимо с результатами нашего исследования. Обсуждение В работе [9] доказана связь rs12329760 гена-кандидата TMPRSS2 с повышением риска развития более тяжелых форм НКИ: при исследовании 693 пациентов с положительным исходом болезни (выздоровление) и 592 человек с негативным исходом (смерть) выявлено, что генотип CC rs12329760 TMPRSS2 связан с повышенным уровнем смертности от COVID-19, т. е. пред- ставляет собой полиморфизм, связанный со значительно более высокой заболеваемостью и тяжелым течением НКИ. 2. 2. Kozik V.A., Shpagin I.S., Shpagina L.A., Loktin E.M. Damage of the cardiovascular system in patients with a new coronavirus infection: current state of the problem. Modern Probl. Sci. Educt., 2023; 4: 135. (In Russ). doi: 10.17513/spno.32651 [Козик В.А., Шпа- гин И.С., Шпагина Л.А., Локтин Е.М. Пораже- ние сердечно-сосудистой системы у пациентов, перенесших новую коронавирусную инфекцию: современное состояние проблемы. Соврем. пробл. науки и образования, 2023; 4: 135. doi: 10.17513/ spno.32651]. 3. 3. Pairo-Castineira E., Rawlik K., Bretherick A.D., Qi T., Wu Y., Nassiri I., McConkey G.A., Zechner M., Klaric L., Griffiths F., Oosthuyzen W., Kousathanas A., Richmond A., Millar J., Russell C.D., Malinauskas T., Thwaites R., Morrice K., Keating S., Maslove D., Nichol A., Semple M.G., Knight J., Shankar-Hari M., Summers C., Hinds C., Horby P., Ling L., McAuley D., Montgomery H., Openshaw P.J.M., Begg C., Walsh T., Tenesa A., Flores C., Riancho J.A., Rojas-Martinez A., Lapunzina P.; GenOMICC Investigators; SCOURGE Consortium; ISARICC Investigators; 23andMe COVID-19 Team; Yang J., Ponting C.P., Wilson J.F., Vitart V., Abedalthagafi M., Luchessi A.D., Parra E.J., Cruz R., Carracedo A., Fawkes A., Murphy L., Rowan K., Pereira A.C., Law A., Fairfax B., Hendry S.C., Baillie J.K. GWAS and meta-analysis identifies 49 genetic variants underlying critical COVID-19. Nature. 2023; 617 (7962): 764–768. doi: 10.1038/s41586-023-06034-3 Неоднородные результаты научных иссле- дований еще раз доказывают, что влияние на тяжесть развития заболеваний оказывает не один полиморфизм гена, а множество. Существенно ограничивает интерпретацию результатов тот факт, что в подавляющем большинстве работ используется подход подбора отдельных генов-кандидатов. Возможно, в будущих исследованиях, где будут изучаться полиморфизмы всего генома (GWAS) у боль- шей выборки людей, разных мест проживания, рас, возрастов, результаты будут более показа- тельными. Неоднородные результаты научных иссле- дований еще раз доказывают, что влияние на тяжесть развития заболеваний оказывает не один полиморфизм гена, а множество. Существенно ограничивает интерпретацию результатов тот факт, что в подавляющем большинстве работ используется подход подбора отдельных генов-кандидатов. Возможно, в будущих исследованиях, где будут изучаться полиморфизмы всего генома (GWAS) у боль- шей выборки людей, разных мест проживания, рас, возрастов, результаты будут более показа- тельными. Сведения об авторах: Валентина Александровна Козик, канд. мед. наук, ассистент кафедры госпитальной терапии и медицинской реабилитации, Новосибирск, Россия, ORCID: 0000-0001-7128-7887, e-mail: valiyta90@mail.ru р , р , , , y Любовь Анатольевна Шпагина, д-р мед. наук, проф., зав. кафедрой госпитальной терапии и медицинской реабилитации, Новосибирск, Россия, ORCID: 0000-0003-3446-8018, e-mail: mkb-2@yandex.ru Любовь Анатольевна Шпагина, д-р мед. наук, проф., зав. кафедрой госпитальной терапии реабилитации, Новосибирск, Россия, ORCID: 0000-0003-3446-8018, e-mail: mkb-2@yandex.ru Любовь Анатольевна Шпагина, д-р мед. наук, проф., зав. кафедрой госпитальной терапии и медицинской реабилитации, Новосибирск, Россия, ORCID: 0000-0003-3446-8018, e-mail: mkb-2@yandex.ru Илья Семенович Шпагин, д-р мед. наук, проф. кафедры госпитальной терапии и медицинской реабилитации, Новосибирск, Россия, ORCID: 0000-0002-3109-9811, e-mail: dr.ilya.shpagin@gmail.com Илья Семенович Шпагин, д-р мед. наук, проф. кафедры госпитальной терапии и медицинской реабилитации, Новосибирск, Россия, ORCID: 0000-0002-3109-9811, e-mail: dr.ilya.shpagin@gmail.com , , y p g g димировна Максимова, ординатор кафедры медицинской генетики и биологии, младший научны восибирск, Россия, ORCID: 0000-0002-2472-181X, e-mail: 99naruto@mail.ru р , , , y p g g Софья Владимировна Максимова, ординатор кафедры медицинской генетики и биологии, м сотрудник, Новосибирск, Россия, ORCID: 0000-0002-2472-181X, e-mail: 99naruto@mail.ru ру р Наталья Геннадьевна Ложкина, д-р мед. наук, проф., рук. группы «Клиническая и экспериментальная кардиология», Новосибирск, Россия, ORCID: 0000-0002-4832-3197, e-mail: lozhkina.n@mail.ru Наталья Геннадьевна Ложкина, д-р мед. наук, проф., рук. группы «Клиническая и экспериментальная кардиология», Новосибирск, Россия, ORCID: 0000-0002-4832-3197, e-mail: lozhkina.n@mail.ru Владимир Николаевич Максимов, д-р мед. наук, проф., зав. лабораторией молекулярно-генетических исследований терапевтических заболеваний, проф. кафедры медицинской генетики и биологии, Новосибирск, Россия, ORCID: 0000-0002-7165-4496, e-mail: medik11@mail.ru Список литературы/ References Andrade C.C., Silva A.T.P., Vasconcelos L.R.S., Oliveira P.R.S., de Souza C.D.F., da Costa Armstrong A., do Carmo R.F. A Polymorphism in the TMPRSS2 gene increases the risk of death in older patients hospitalized with COVID-19. Viruses, 2022; 14 (11): 2557. doi: 10.3390/v14112557 ( ) / 7. Sekiya T., Ogura Y., Kai H., Kawaguchi A., Oka- wa S., Hirohama M., Kuroki T., Morii W., Hara A., Hiramatsu Y., Hitomi S., Kawakami Y., Arakawa Y., Maruo K., Chiba S., Suzuki H., Kojima H., Tachika- wa H., Yamagata K. TMPRSS2 gene polymorphism common in East Asians confers decreased COVID-19 40 В.А. Козик, Л.А. Шпагина, И.С. Шпагин и др. Ukrainian population. Acta Biomed., 2023; 94 (1): e2023030. doi: 10.23750/abm.v94i1.13543 Ukrainian population. Acta Biomed., 2023; 94 (1): e2023030. doi: 10.23750/abm.v94i1.13543 / 8. Аidashev I., Izmailova O., Shlykova O., Kabaliei A., Vatsenko A., Ivashchenko D., Dudchenko M., Vo- lianskyi A., Zelinskyy G., Koval T., Dittmer U. Poly- morphism of tmprss2 (rs12329760) but not ace2 (rs4240157), tmprss11a (rs353163) and cd147 (rs8259) is associated with the severity of COVID-19 in the / 9. Beheshti Shirazi S.S., Sakhaee F., Sotoodeh- nejadnematalahi F., Zamani M.S., Ahmadi I., An- vari E., Fateh A. rs12329760 Polymorphism in transmembrane serine protease 2 gene and risk of coronavirus disease 2019 mortality. Biomed. Res. Int., 2022; 2022: 7841969. doi: 10.1155/2022/7841969 Ukrainian population. Acta Biomed., 2023; 94 (1): e2023030. doi: 10.23750/abm.v94i1.13543 9. Beheshti Shirazi S.S., Sakhaee F., Sotoodeh- nejadnematalahi F., Zamani M.S., Ahmadi I., An- vari E., Fateh A. rs12329760 Polymorphism in transmembrane serine protease 2 gene and risk of coronavirus disease 2019 mortality. Biomed. Res. Int., 2022; 2022: 7841969. doi: 10.1155/2022/7841969 Статья поступила 03.12.2023 После доработки 03.01.2024 Принята к печати 14.01.2024 susceptibility. Front. Microbiol., 2022; 13: 943877. doi: 10.3389/fmicb.2022.943877 8. Аidashev I., Izmailova O., Shlykova O., Kabaliei A., Vatsenko A., Ivashchenko D., Dudchenko M., Vo- lianskyi A., Zelinskyy G., Koval T., Dittmer U. Poly- morphism of tmprss2 (rs12329760) but not ace2 (rs4240157), tmprss11a (rs353163) and cd147 (rs8259) is associated with the severity of COVID-19 in the susceptibility. Front. Microbiol., 2022; 13: 943877. doi: 10.3389/fmicb.2022.943877 8. Аidashev I., Izmailova O., Shlykova O., Kabaliei A., Vatsenko A., Ivashchenko D., Dudchenko M., Vo- lianskyi A., Zelinskyy G., Koval T., Dittmer U. Poly- morphism of tmprss2 (rs12329760) but not ace2 (rs4240157), tmprss11a (rs353163) and cd147 (rs8259) is associated with the severity of COVID-19 in the Ukrainian population. Acta Biomed., 20 e2023030. doi: 10.23750/abm.v94i1.13543 9. Beheshti Shirazi S.S., Sakhaee F., nejadnematalahi F., Zamani M.S., Ahm vari E., Fateh A. rs12329760 Polym transmembrane serine protease 2 gene coronavirus disease 2019 mortality. Biom 2022; 2022: 7841969. doi: 10.1155/2022/7 Ukrainian population. Acta Biomed., 2023; 94 (1): e2023030. doi: 10.23750/abm.v94i1.13543 susceptibility. Front. Microbiol., 2022; 13: 943877. doi: 10.3389/fmicb.2022.943877 Information about the authors: Valentina A. Kozik, candidate of medical sciences, assistant at the department of hospital therapy and medical rehabilitation, Novosibirsk, Russia, ORCID: 0000-0001-7128-7887, e-mail: valiyta90@mail.ru Lyubov A. Shpagina, doctor of medical sciences, professor, head of the department of hospital therapy and medical rehabilitation, Novosibirsk, Russia, ORCID: 0000-0003-3446-8018, e-mail: mkb-2@yandex.ru pagin, doctor of medical sciences, professor of the department of hospital therapy and medic Novosibirsk, Russia, ORCID: 0000-0002-3109-9811, e-mail: dr.ilya.shpagin@gmail.com Ilya S. Shpagin, doctor of medical sciences, professor of the department of hospital the rehabilitation, Novosibirsk, Russia, ORCID: 0000-0002-3109-9811, e-mail: dr.ilya.shpagin@gmail.com Sofya V. Maksimova, resident of the department of medical genetics and biology, junior researcher, Novosibirsk, Russia, ORCID: 0000-0002-2472-181X, e-mail: 99naruto@mail.ru Sofya V. Maksimova, resident of the department of medical genetics and biology, junior researcher, Novosibirsk, Russia, ORCID: 0000-0002-2472-181X, e-mail: 99naruto@mail.ru Natalya G. Lozhkina, doctor of medical sciences, professor, head of the clinical and experimental cardiology group, Novosibirsk, Russia, ORCID: 0000-0002-4832-3197, e-mail: lozhkina.n@mail.ru Vladimir N. Maksimov, doctor of medical sciences, professor, head laboratory of molecular genetic research of therapeutic diseases, professor of the department of medical genetics and biology, Novosibirsk, Russia, ORCID: 0000- 0002-3157-7019, e-mail: medik11@mail.ru Received 03.12.2023 Revision received 03.01.2024 Accepted 14.01.2024 Статья поступила 03.12.2023 После доработки 03.01.2024 Принята к печати 14.01.2024 Received 03.12.2023 Revision received 03.01.2024 Accepted 14.01.2024 Статья поступила 03.12.2023 После доработки 03.01.2024 Принята к печати 14.01.2024 41
https://openalex.org/W2894688931
https://revistas.ucm.es/index.php/CIYC/article/download/60692/4564456547542
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"Teoría e historia de la propaganda" (2017), Adrián Huici Módenes, Madrid, Editorial Síntesis, 2017, pp. 278.
Cuadernos de información y comunicación
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RESEÑAS CIC. Cuadernos de Información y Comunicación ISSN: 1135-7791 http://dx.doi.org/10.5209/CIYC.60692 Teoría e historia de la propaganda (2017), Adrián Huici Módenes, Madrid, Editorial Síntesis, 2017, pp. 278. Adrián Huici Módenes es profesor titular en la Facultad de Comunicación de la Universidad de Sevilla. Durante su carrera académica se ha especializado en el campo de la comunicación pública y la propaganda, investigando en torno a la relación de ésta última con el mito y la cultura popular. En su último libro nos presenta amplio repaso por la teoría y el desarrollo histórico de los fenómenos propagandísticos, desde sus primeros antecedentes en las culturas antiguas, hasta sus más modernas expresiones en torno a las nuevas herramientas que nos ofrece internet. A lo largo de los dos primeros capítulos, en los cuales se presenta la propaganda en términos teóricos, el autor conecta sus posiciones con una serie de obras fundamentales tanto dentro de la academia española como en las bases de la doctrina en términos científicos a principios del siglo XX. Algunas de las mencionadas en este sentido son Alejandro Pizarroso, Mario Herreros, Maria V. Reyzábal, Juan Rey y Antonio Pineda. Respecto de las principales contribuciones desde el ámbito internacional, el autor menciona a numerosos autores entre los que se encuentran pioneros como Edward Bernays o Harold Lasswell y otros autores algo posteriores como Jacques Ellul, Jean-Marie Domenach, Leonard Doob, Paul Watzlawick, Elliot Aronson y Anthony Pratkanis. Intercalando su saber con las aportaciones de éstos, el autor expone en el segundo capítulo, el más extenso, el contenido propagandístico sustancial, enfatizando su naturaleza práctica. De ese modo se introduce la relación entre ésta y otros modelos de enunciados persuasivos, subrayando la importancia de la imagen como elemento fundacional de la misma y su relación con el símbolo y el mito. De igual modo se presentan una serie de reglas básicas para la articulación de discursos propagandísticos, tanto en un contexto de gestión de la opinión pública en tiempo de paz como durante la guerra. Se pone fin al desarrollo de la teoría de la propaganda teniendo en cuenta de que modos se articulan cada uno de los lenguajes de los cuales esta se sirve y sus diferentes códigos y canales. Resulta muy reseñable el esfuerzo llevado a cabo por el autor en su intento de abarcar, aunque de una manera esquemática, la casi totalidad de la historia de Occidente. De ese modo, el autor nos invita en el capítulo tres a ver nuestra historia, desde los sumerios hasta la protopropaganda de los imperios napoleónicos, a través de los ojos del experto en propaganda. Éste último término, “protopropaganda”, es utilizado como forma de conceptualizar todos aquellos elementos propagandísticos propios de la política a lo largo de toda la historia previa al establecimiento de los modernos medios de comunicación de masas: “…esto no significa que antes del siglo XVIII no existieran formas persuasivas vinculadas con el poder y la ideología, solo que carecían de los medios y de la sofisticación que aportan las nuevas tecnologías de la comunicación.” (p. 81) El extensísimo recorrido histórico al que se nos invita puede inducirnos a ser conscientes de dos elementos problemáticos, que sin CIC. Cuad. inf. com. 23, 2018: 235-238 235 236 Reseñas. CIC. Cuad. inf. comu. 23, 2018: 235-238 embargo no restan valor a la propuesta del autor. En primer lugar, debemos ser conscientes de que no podemos abordar el capítulo tres ni como una historia propiamente dicha, ya que cada uno de los asuntos esquemáticamente tratados merecería un abordaje mucho más extenso y detallado, ni como una historia de las ideas políticas. En segundo lugar, la obra parece en ocasiones conducirnos hacía una visión de la política como mera canalización de mensajes propagandísticos. Ejemplos de ello es la posibilidad de entender cualquier ámbito político como un terreno para la expresión de la manipulación, desde la arquitectura monumental egipcia (p. 82) o la propagación del modelo democrático hecho por los atenienses (p. 85) hasta el uso de la guillotina por parte de los revolucionarios franceses (p. 113). Otro ejemplo se encuentra en el tratamiento del nacionalismo, enfocado desde la perspectiva de las Comunidades imaginadas de Anderson, y por tanto enfatizando su carácter contingente: “debemos situar a la propaganda en la génesis misma del nacionalismo” (p. 126) Uno de los puntos fuertes de la obra es la posibilidad de dar cuenta de algunos de los elementos de los que se está nutriendo la propaganda en nuestros días. El libro trata por ello de tener en cuenta, aunque de una manera provisoria, algunos fenómenos experimentados en los últimos cinco o diez años entre los que podemos citar la irrupción de las redes sociales como un nuevo agente dentro de la comunicación política o la maquinaria propagandística de la que se sirve el ISIS. Resulta evidente que respecto de los fenómenos novedosos la obra no puede profundizar, de tal modo que sólo el futuro nos dirá si las intuiciones plasmadas por el autor se cumplen o no. En cualquier caso, algunas de éstas podrían ser matizadas: “…YouTube como un nuevo ágora político, que en cierto sentido, superaba a la idealizada democracia ateniense.” (p. 270) También resulta adecuado señalar cómo la importancia de la imagen dentro de las técnicas propagandísticas, lo cual es enfatizado en diversas ocasiones, encuentra su paralelismo con la importancia que dentro del recorrido argumentativo del libro tienen las diversas ilustraciones que acompañan cada una de los puntos elaborados. De este modo podemos encontrar imágenes que da cuenta desde el poder simbólico de los cuadros de Napoleón a la relevancia del debate televisado entre Nixon y Kennedy en el año 1960. Por otro lado, la obra contiene un evidente tono crítico hacia la propaganda como técnica de condicionamiento social. Ciertamente, como es de esperar en alguien que se ha especializado en este campo, escapa de las visiones maniqueas que se refieren a la propaganda como un elemento propio de los regímenes totalitarios en tanto que se define por el uso de la desinformación, manipulación y mentira. Frente a esto, el autor nos advierte desde un inicio en que los regímenes democráticos hacen un uso constante de la propaganda ya que ésta es indisociable de la competición política, de tal modo que, si bien por medio de eufemismos como “marketing político, publicidad política, marketing electoral o relaciones públicas…la propaganda está tan vigente hoy como en os oscuros tiempos de Goebbels o Stalin…” (12). Este fragmento nos permite de hecho dar cuenta de que, si bien el autor es crítico con la relación entre democracia y propaganda, el verdadero blanco de sus críticas parecen ser los nefastos usos de la propaganda por parte de modelos políticos monstruosos surgidos en el periodo de entreguerras. La propaganda se convierte así en un elemento técnico al servicio de regímenes políticos buenos y malos. Si los malos son los totalitarios, en especial el nazismo y el comunismo, los cuáles parecen juzgarse en los mismos términos, con una continua apelación a las Reseñas. CIC. Cuad. inf. comu. 23, 2018: 235-238 237 similitudes entre Hitler y Stalin, los buenos, aunque con matices, son las democracias liberales del mundo occidental. De este modo el libro se posiciona claramente a favor de una opción, simplificando ciertos elementos, lo cual le da precisamente un leve tiente propagandístico a su defensa de los valores hegemónicos (“En este caso, el proceso de demonización parece un tanto gratuito, puesto que el carácter intrínsecamente demoniaco de Hitler no necesitaba de mayores explicaciones: el hombre se retrataba solo”, p. 51. “…procuraban identificar la figura poco agraciada de Franco con el mismísimo Campeador, algo que hoy resulta irrisorio, tanto como los increíbles calificativos que la literatura del régimen prodigaba a Stalin: los extremos siempre acaban tocándose.”, p. 59. “Afortunadamente, existen ejemplos en positivo y, sin duda, el más espectacular es el caso del rugby en Sudáfrica. Recordemos que Nelson Mandela utilizó…” p. 70. “Naturalmente, cuando Lenin dice “educación” o “ideología”, en lo que está pensando es en la propaganda.” p. 164.) Por tanto, el desarrollo argumentativo de la obra nos induce a planear lo siguiente: si aceptamos, tal y como se sostiene en la obra, que habitualmente se pretende convencer de que propaganda es “lo de los otros”, mientras que “nosotros propagamos la verdad”, ¿por qué el autor, que debiera enfatizar su visión crítica del fenómeno, acaba limitándose a considerar que existen propagandas “buenas” y propagandas “malas”? En este sentido parece que se refiere implícita y frecuentemente a propagandas que se justificarían por sus beneficiosos resultados y otras que, tal y como podemos observar desde el prisma histórico, no solo no podemos justificar sino que debemos contrarrestar en tanto que partícipes de un proyecto aberrante. A pesar de esto, incluso en algunos fragmentos el autor parece inclinarse finalmente al argumento central de aquellos que “luchan contra la propaganda en favor de la democracia”, sosteniendo, a colación de la habitual frivolidad con la que se hace uso del marketing político y la campañas negativas, que “todo ello, sin duda, va en desmedro de la calidad de los mensajes, que no pueden profundizar sobre ningún tema, y de la democracia misma, cuyo combustible fundamental es la información amplia y de calidad” (p. 265) Frente a los ejemplos anteriores, en los cuales el autor parece seguir a pies juntillas la ortodoxia cultural occidental, resulta interesante el modo en que reivindica una lectura crítica en torno al modo en que el cristianismo se ha comportado históricamente, tratando a su vez de señalar cómo el islam ha sido y sigue siendo atacado desde unos planteamientos que no se aplican en el caso de los seguidores de Jesucristo. En este sentido resulta muy reseñable el modo en que argumenta en torno al modo en que el aparato mediático estadounidense y europeo se ha esforzado sistemáticamente, tras la caída del muro de Berlín, por construir un nuevo monstruo, en este caso el fanático islamista. En esta empresa el imperialismo cultural estadounidense, encabezado por la industria cinematográfica, ha sido capaz de construir una imagen del musulmán a través de películas citadas como Aladín, Mentiras arriesgadas o Delta Force, de las cuales se desprende el estereotipo del “hombre barbudo, con un turbante, un cinturón de explosivos y un Corán y un fusil de asalto en cada mano.”(p. 273) A modo de valoración general podemos considerar que la obra nos ofrece una interesante recopilación de un material amplísimo tanto en términos teóricos como históricos. Por tanto, podríamos considerarlo como un buen punto de partida para llevar a cabo una investigación sistemática, teniendo en cuenta que introduce gran 238 Reseñas. CIC. Cuad. inf. comu. 23, 2018: 235-238 cantidad de temas y da cuenta de muchos de los más significativos estudiosos en torno a la propaganda. Miguel Fernández de la Peña Universitat de Barcelona miguelmixel@hotmail.com
https://openalex.org/W4311505148
https://www.frontiersin.org/articles/10.3389/fragi.2022.1063760/pdf
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Living alone reduces the decline of calf circumference among Chinese older adults: A 4-year longitudinal study
Frontiers in aging
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cc-by
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TYPE Original Research PUBLISHED 15 December 2022 DOI 10.3389/fragi.2022.1063760 TYPE Original Research PUBLISHED 15 December 2022 DOI 10.3389/fragi.2022.1063760 TYPE Original Research PUBLISHED 15 December 2022 DOI 10.3389/fragi.2022.1063760 OPEN ACCESS OPEN ACCESS EDITED BY Alan Bruno Silva Vasconcelos, University Center—AGES, Brazil REVIEWED BY Marcos Raphael Pereira Monteiro, Federal University of Sergipe, Brazil Leandro Henrique Albuquerque Brandão, Federal University of Minas Gerais, Brazil Xiaolei Liu, Sichuan University, China *CORRESPONDENCE Jun Zhang, who626zj@126.com SPECIALTY SECTION This article was submitted to Musculoskeletal Aging, a section of the journal Frontiers in Aging RECEIVED 07 October 2022 ACCEPTED 06 December 2022 PUBLISHED 15 December 2022 CITATION Wang D and Zhang J (2022), Living alone reduces the decline of calf circumference among Chinese older adults: A 4-year longitudinal study. Front. Aging 3:1063760. doi: 10.3389/fragi.2022.1063760 OPEN ACCESS EDITED BY Alan Bruno Silva Vasconcelos, University Center—AGES, Brazil REVIEWED BY Marcos Raphael Pereira Monteiro, Federal University of Sergipe, Brazil Leandro Henrique Albuquerque Brandão, Federal University of Minas Gerais, Brazil Xiaolei Liu, Sichuan University, China *CORRESPONDENCE Jun Zhang, who626zj@126.com SPECIALTY SECTION This article was submitted to Musculoskeletal Aging, a section of the journal Frontiers in Aging RECEIVED 07 October 2022 ACCEPTED 06 December 2022 PUBLISHED 15 December 2022 CITATION Wang D and Zhang J (2022), Living alone reduces the decline of calf circumference among Chinese older adults: A 4-year longitudinal study. Front. Aging 3:1063760. doi: 10.3389/fragi.2022.1063760 Dong Wang and Jun Zhang* Department of Neurology, Peking University People’s Hospital, Beijing, China Dong Wang and Jun Zhang* Department of Neurology, Peking University People’s Hospital, Beijing, China Background: Calf circumference (CC) is regarded as a surrogate marker of skeletal muscle mass with high sensitivity and specificity for predicting sarcopenia. A cross-sectional study reported older adults living alone were at high risk of developing sarcopenia. Whether living alone affects the change of calf circumference is unknown and there is no evidence from longitudinal study. The purpose of this study was to investigate the relationship between living arrangements and the change of calf circumferences among older adults in China. CITATION Wang D and Zhang J (2022), Living alone reduces the decline of calf circumference among Chinese older adults: A 4-year longitudinal study. Front. Aging 3:1063760. doi: 10.3389/fragi.2022.1063760 Methods: The data were from the Chinese Longitudinal Healthy Longevity Survey. A total of 2,203 older adults (age ≥65 years, mean age: 80.61 ± 8.30 years, 50.0% female) who were interviewed in 2014 and then 2018 follow-up survey were finally included for analysis. Living arrangements and other information were collected in 2014. living alone, calf circumference, older adults, sarcopenia, longitudinal study 1 Introduction FIGURE 1 Participant flow in this study. Sarcopenia is defined as age-related loss of skeletal muscle mass, accompanied by decline in muscle strength and/or reduced physical function (Chen et al., 2020). Sarcopenia increases the risks of falls, fractures, disability, and mortality among older adults. Globally, the prevalence of sarcopenia in adults over the age of 60 is 10% (Shafiee et al., 2017), and the prevalence of sarcopenia among older adults over the age of 80 is about 50% (Meng et al., 2014). With an increasingly ageing population, sarcopenia has become a serious public health issue in modern society. Skeletal muscle mass is one of the core dimensions for assessing sarcopenia. Calf circumference (CC) can be used as a surrogate marker of skeletal muscle mass, which has high sensitivity and specificity for predicting sarcopenia (Kawakami et al., 2015). Measures of CC may be used as a diagnostic proxy in settings where no other muscle mass diagnostic methods are available, and CC < 34 cm for men and <33 cm for women for screening sarcopenia is recommended in the Asian Working Group for Sarcopenia (AWGS) 2019 consensus (Cruz-Jentoft et al., 2019; Chen et al., 2020). In addition, CC is also an important variable in predictive models of other disorders, such as cardiovascular disease (Wu et al., 2018). The CC decline is often regarded as a risk factor affecting health, especially among older adults. Therefore, it is of great significance to explore the factors influencing the change of the CCs among older adults. cross-sectional studies suggested that living alone was a risk factor for frailty in older adults, especially in men (Yamanashi et al., 2015; Kojima et al., 2020); however, meta-analysis of cohort studies did not yield statistically significant results (OR = 0.88, 95 %CI = 0.76–1.03) (Kojima et al., 2020). As for sarcopenia, previous cross-sectional study suggested that older adults living alone are at higher risk for sarcopenia (Cheng et al., 2021), while evidence from cohort studies is still lacking. CC decline among older adults means reduction of skeletal muscle mass and increased risk of developing sarcopenia. There is no report about the associations of some social factors with CC decline among older adults. OPEN ACCESS Calf circumferences were measured and recorded in the questionnaires of two waves and the differences were calculated. Logistic regression analyses were conducted to evaluate the association of living arrangements (living alone or not living alone) with the change of calf circumferences (decline or no decline). COPYRIGHT © 2022 Wang and Zhang. 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. Results: There were 446 (20.2%) participants living alone and 1,757 (79.8%) participants not living alone. After about 4 years, calf circumferences of 866 (39.3%) older adults declined. Compared to not living alone, living alone was negatively associated with calf circumference decline, even after adjustment for covariates (OR: 0.702, 95% CI: 0.547–0.901, p = 0.005). This significant association was found in 65–79 years old (OR: 0.619, 95% CI: 0.407–0.942, p = 0.025), female (OR: 0.567, 95% CI: 0.408–0.790, p = 0.001) and city/town (OR: 0.461, 95% CI: 0.310–0.685, p < 0.001) subgroups but not in advanced age (≥80 years old), male and rural subgroups. Conclusion: This study revealed that living alone was associated with a decreased risk of calf circumference decline among older adults, and might be a protective factor for sarcopenia. KEYWORDS living alone, calf circumference, older adults, sarcopenia, longitudinal study Frontiers in Aging 01 frontiersin.org Wang and Zhang 10.3389/fragi.2022.1063760 1 Introduction Therefore, we used the cohort data from the Chinese Longitudinal Healthy Longevity Survey (CLHLS) to explore the relationship between living arrangements and the change of CC among older adults, and analyze the effect of living alone on the changes in skeletal muscle mass and the development of sarcopenia. Living arrangement is an important observational variable in many older adults-related studies about mental health, cognitive impairment and other diseases (Beghi et al., 2021; Rosenwohl- Mack et al., 2021). Living arrangements are influenced by regional culture and the economic development of different times. Under the influence of Confucianism, the traditional Chinese family is of large size and multigenerational. However, in the new era, the influence of the traditional family concept has declined. Due to the needs of work and the development of urbanization, many young people live far away from their parents, and the traditional family model is gradually deconstructed (Phillips and Feng, 2015). Among older adults, the phenomenon of living alone due to the death of one’s spouses is becoming more and more common. With an increasingly aging population, the older adults living alone has received more and more attention from the society. 2.3 Living arrangements Living arrangements were assessed by the item “co-residence of interviewee” in the questionnaire. The options were “with household member(s),” “alone,” and “in an institution,” with 1,735 (78.8%), 446 (20.2%), 22 (1.0%) respondents respectively. The living arrangements were dichotomized as living alone (LA) and not living alone [NLA, including “with household member(s)” and “in an institution”] for analysis. 2.2 Assessment of CC In this survey, CCs were recorded and rounded up to the nearest integer in centimeters. The CC difference was defined as CC at 2018 wave minus CC at 2014 wave. Given the possible measurement errors and the round-off recording principles, the decline of CC in this survey was defined as CC difference <−1 cm, and CC difference ≥−1 cm means no decline. Finally, there were 866 (39.3%) subjects who had declined CCs. 2.1 Study population A modified Mini Mental State Examination (mMMSE) score was calculated by 23 items in original questionnaire to assess the cognitive function. A detailed introduction to this mMMSE has been published elsewhere (Guo et al., 2022). Every interviewee was asked about diagnoses (hypertension, diabetes, heart disease, stroke, respiratory diseases, tuberculosis, cancer, Parkinson’s disease, arthritis) by hospital. to answer questions, the interview was finished by a proxy interviewer, often a spouse or other close relative. At first wave, 7,192 respondents were interviewed in 2014, out of which 1,525 respondents were lost to follow-up at second wave in the 2018 survey and 2,226 died before 2018. Subjects under the age of 65 were excluded. We checked and cleaned the missing data and registration errors of important variables. Finally, 2,203 cases were included for analysis (Figure 1). 2.5 Statistical analysis IBM SPSS statistics 26.0 (IBM Corporation, Armonk, NY, United States) was used for statistical analysis. The mean, standard deviation, median, and ratio were calculated for statistical descriptions according to different types of variables. When data was normally distributed, the differences of continuous variables between LA and NLA group at 2014 wave were determined by the independent-sample t test, and the homogeneity of variance was tested by Levene’s test. The Mann-Whitney U test was used when data was not normally distributed. The statistical analyses of categorical variables were performed by Pearson χ2 test. Both univariate and multivariate logistic regression analyses were used to examine the associations of living arrangements with CC difference. Odds ratios (ORs) and 95% confidence intervals (CIs) were calculated. The statistical significance was defined as p < .05, and all were two-tailed tests. 2.4 Covariates The sociodemographic variables including age, sex, weight (kg), height (cm), education (uneducated or educated), marital status, residence (rural or city/town) and financial support (sufficient or insufficient) were selected from the dataset. The options of current marital status were “currently married and living with spouse,” “married but not living with spouse,” “divorced,” “widowed,” and “never married.” They were dichotomized as “separated/divorced/widowed/single” (SDWS) or not. Body mass index (BMI) was calculated as weight in kilograms divided by the square of height in meters. The sleep quality was categorized as “very good,” “good,” “so,” “bad,” and “very bad” in primitive questionnaire and was dichotomized as bad and not bad for analysis. Sleep duration were recorded in this survey as short (<5 h), medium (5–9 h) and long (>9 h) according to the recommendation (Hirshkowitz et al., 2015). For smoking and drinking, respondents were categorized as current, ex-smoker/drinker, or never. By asking questions: “Exercise or not at present?” and “Have you done physical labor regularly?,” data about exercise/physical labor or not 2.1 Study population The physical and mental health of older adults living alone may be affected when the level of support from family or society changes (Lou and Ng, 2012). It is generally believed that lack of family companionship is harmful to the physical and mental health, but the effects of living alone on the health of older adults are currently inconsistent in different literatures (Holt-Lunstad et al., 2015; Zhou et al., 2018; Gu et al., 2019). Both sarcopenia and frailty are age-related syndromes with some overlap in clinical manifestations (Roberts et al., 2021). Meta-analysis of The data were from CLHLS 2014–2018 longitudinal dataset, which is a nationally representative study of Chinese older adults and covers 23 out of 31 provinces in Mainland China. The CLHLS study was approved by the Research Ethics Committee of Peking University (IRB00001052-13074). The interview was conducted at participants’ homes by well-trained investigators using a structured questionnaire. When a participant was unable Frontiers in Aging frontiersin.org 02 Wang and Zhang 10.3389/fragi.2022.1063760 10.3389/fragi.2022.1063760 were recorded. Depression was measured using two questions: “Have you felt sad, blue, or depressed for 2 weeks or more in last 12 months?” and “Have you lost interest in most things like hobbies, work, or similar activities?.” Answer of “yes” to any question is considered a representation of depression (Su et al., 2021). Activities of daily living (ADL) disability was defined as needs for support in one or more of the five activities (bathing, dressing, toileting, indoor transferring, and feeding) or being incontinent. A modified Mini Mental State Examination (mMMSE) score was calculated by 23 items in original questionnaire to assess the cognitive function. A detailed introduction to this mMMSE has been published elsewhere (Guo et al., 2022). Every interviewee was asked about diagnoses (hypertension, diabetes, heart disease, stroke, respiratory diseases, tuberculosis, cancer, Parkinson’s disease, arthritis) by hospital. were recorded. Depression was measured using two questions: “Have you felt sad, blue, or depressed for 2 weeks or more in last 12 months?” and “Have you lost interest in most things like hobbies, work, or similar activities?.” Answer of “yes” to any question is considered a representation of depression (Su et al., 2021). Activities of daily living (ADL) disability was defined as needs for support in one or more of the five activities (bathing, dressing, toileting, indoor transferring, and feeding) or being incontinent. 3 Results At 2014 wave, there were 446 (20.2%) participants living alone and 1,757 (79.8%) participants not living alone. Table 1 presents the baseline characteristics by two types of living arrangements. No significant differences were found among BMI, sleep quality, exercise, physical labor, depression, ADL disability, and comorbidities. Compared to subjects not living alone, older, women, uneducated, SDWS, rural, insufficient financial support, short-sleeper, non-smoker, non-drinker, and lower mMMSE scorer were more common in subjects living alone. The outcomes of logistic regression models are illustrated in Table 2. Compared to NLA group, the OR of the LA group for crude model was 0.728 (95% CI: 0.585–0.905, p = .004). After adjustments for covariates, the results were also statistically Frontiers in Aging frontiersin.org 03 10.3389/fragi.2022.1063760 Wang and Zhang TABLE 1 Baseline characteristics by living arrangements at 2014 wave. TABLE 1 Baseline characteristics by living arrangements at 2014 wave. DWS, separated/divorced/widowed/single; ADL, activities of daily living; mMMSE, modified Mini Mental state examination. *median and 25th–75th percentile. tistical difference. 3 Results Living alone (n = 446) Not living alone (n = 1,757) p Age (year) 82.19 ± 7.77 80.21 ± 8.38 <.001 Advanced age (≥80 years), n (%) 267 (59.9%) 809 (46.0%) <.001 Female, n (%) 267 (59.9%) 834 (47.5%) <.001 BMI (kg/m2) 22.37 ± 3.81 22.54 ± 3.98 .410 Uneducated, n (%) 277 (62.1%) 786 (44.7%) <.001 Marital status (SDWS), n (%) 422 (94.6%) 656 (37.3%) <.001 Residence (Rural), n (%) 264 (59.2%) 939 (53.4%) .029 Financial support (insufficient), n (%) 96 (21.5%) 269 (15.3%) .002 Sleep quality (Bad), n (%) 52 (11.7%) 201 (11.4%) .897 Sleep duration .024 Short, n (%) 46 (10.3%) 115 (6.5%) Long, n (%) 72 (16.1%) 290 (16.5%) Medium, n (%) 328 (73.5%) 1,352 (76.9%) Smoking .020 Current, n (%) 78 (17.5%) 349 (19.9%) Ex-smoker, n (%) 42 (9.4%) 237 (13.5%) Never, n (%) 326 (73.1%) 1,171 (66.6%) Drinking .034 Current, n (%) 78 (17.5%) 346 (17.9%) Ex-drinker, n (%) 33 (7.4%) 191 (10.9%) Never, n (%) 335 (75.1%) 1,220 (69.4%) Exercise, n (%) 145 (32.5%) 619 (35.2%) .281 Physical labor, n (%) 382 (85.7%) 1,481 (84.3%) .478 Depression, n (%) 50 (11.2%) 195 (11.1%) .946 ADL disability, n (%) 27 (6.1%) 133 (7.6%) .271 mMMSE 22.00 (19.00–22.25) p 22 (20.00–23.00) p .006 Disease Hypertension, n (%) 184 (41.3%) 730 (41.5%) .911 Diabetes, n (%) 75 (16.8%) 282 (16.1%) .695 Heart disease, n (%) 98 (22.0%) 348 (19.8%) .309 Stroke, n (%) 67 (15.0%) 257 (14.6%) .833 Respiratory diseases, n (%) 66 (14.8%) 281 (16.0%) .536 Tuberculosis, n (%) 47 (10.5%) 135 (7.7%) .051 Cancer, n (%) 23 (5.2%) 114 (6.5%) .298 Parkinson’s disease, n (%) 22 (4.9%) 92 (5.2%) .796 Arthritis, n (%) 60 (13.5%) 235 (13.4%) .966 BMI, body mass index; SDWS, separated/divorced/widowed/single; ADL, activities of daily living; mMMSE, modified Mini Mental state examination. *median and 25th–75th percentile. Frontiers in Aging 04 frontiersin.org 10.3389/fragi.2022.1063760 Wang and Zhang significant. Living alone was independently and negatively associated with CC decline in adjusted model 2: the adjusted OR of LA group was 0.702 (95% CI: 0.547–0.901, p = .005). In adjusted model 2, advanced age (≥80 years) and stroke were two covariates that were significantly associated with CC decline (Table 2). TABLE 2 Associations between CC decline and living arrangements by logistic regression models. 3 Results Crude model Adjusted model 1 Adjusted model 2 OR (95% CI) p OR (95% CI) p OR (95% CI) p Living alone 0.728 (0.585–0.905) 0.004 0.711 (0.570–0.887) 0.002 0.702 (0.547–0.901) 0.005 Age (≥80 years) 1.246 (1.048–1.483) 0.013 1.230 (1.013–1.494) 0.037 Sex (female) 0.938 (0.789–1.116) 0.471 0.869 (0.693–1.091) 0.226 Uneducated 0.893 (0.727–1.098) 0.283 Marital status (SDWS) 1.096 (0.883–1.362) 0.405 Residence (Rural) 0.992 (0.826–1.191) 0.928 Financial support (insufficient) 1.026 (0.806–1.306) 0.835 Sleep quality (bad) 1.097 (0.798–1.506) 0.570 Sleep duration Short 0.719 (0.485–1.068) 0.102 Long 1.046 (0.824–1.328) 0.710 Medium Ref. Smoking Current, n (%) 0.832 (0.641–1.079) 0.165 Ex-smoker, n (%) 0.873 (0.643–1.184) 0.383 Never, n (%) Ref. Drinking Current 0.983 (0.767–1.261) 0.894 Ex-drinker 0.941 (0.682–1.298) 0.710 Never Ref. Exercise 0.969 (0.799–1.177) 0.753 Physical labor 0.860 (0.673–1.099) 0.228 Depression 0.829 (0.621–1.106) 0.202 ADL disability 1.003 (0.713–1.412) 0.985 mMMSE 0.992 (0.970–1.015) 0.490 Hypertension 1.122 (0.918–1.372) 0.261 Diabetes 0.847 (0.608–1.179) 0.324 Heart disease 1.126 (0.852–1.487) 0.405 Stroke 1.486 (1.085–2.035) 0.014 Respiratory diseases 1.122 (0.840–1.498) 0.435 tuberculosis 0.607 (0.342–1.079) 0.089 Cancer 1.050 (0.527–2.093) 0.889 Parkinson’s disease 1.517 (0.710–3.241) 0.281 Arthritis 0.812 (0.588–1.121) 0.206 OR, odds ratio; CI, confidence interval; SDWS, separated/divorced/widowed/single; ADL, activities of daily living; mMMSE, modified Mini Mental state examination. Bold values represent statistical difference. TABLE 2 Associations between CC decline and living arrangements by logistic regression models. adjusted model 2, advanced age (≥80 years) and stroke were two covariates that were significantly associated with CC decline (Table 2). adjusted model 2, advanced age (≥80 years) and stroke were two covariates that were significantly associated with CC decline (Table 2). significant. Living alone was independently and negatively associated with CC decline in adjusted model 2: the adjusted OR of LA group was 0.702 (95% CI: 0.547–0.901, p = .005). In significant. Living alone was independently and negatively associated with CC decline in adjusted model 2: the adjusted OR of LA group was 0.702 (95% CI: 0.547–0.901, p = .005). In 05 Frontiers in Aging 05 frontiersin.org 10.3389/fragi.2022.1063760 Wang and Zhang TABLE 3 Associations between CC decline and LA by subgroup analysis. Table 3 Table 3 shows the results of subgroup analysis. LA was significantly associated with CC decline in 65–79 years old, female and city/town subgroups: the adjusted ORs of LA group were 0.619 (95% CI: 0.407–0.942, p = .025), 0.567 (95% CI: 0.408–0.790, p = .001) and 0.461 (95% CI: 0.310–0.685, p < .001) respectively. LA was not significantly associated with CC decline in advanced age (≥80 years old), male and rural subgroups. Gu et al. presented a theoretical framework of reciprocal causality between LA and health. That is, the solitary living arrangement needs to be conceptualized as the cause of subsequent health outcomes as well as the outcome of the prior health status (Gu et al., 2019). Health status of older adults affected their preference for living arrangements and both physical and mental health conditions play a role in the transitions of living arrangements (Brown et al., 2002). A disabled older adult who loses the capability to live independently would be more inclined to live with others. In addition, older adults living alone need to cope with more jobs of daily life by themselves, helping them get rid of their bad sedentary habits; and a better cognitive or physical function is needed to support them in handling the daily jobs. Physical activity, especially resistance exercise, which can improve skeletal muscle mass and strength, is the primary method for sarcopenia prevention and treatment (Cruz-Jentoft and Sayer, 2019). However, the BMI, exercise and physical labor, ADL disability, depression status, and comorbidities were all comparable at baseline and didn’t change the result in the adjusted model. And the mMMSE score was lower in the LA group probably due to lower education level. It suggests that the reciprocal causality alone cannot explain the findings. 3 Results y g p y Crude model Adjusted model OR (95% CI) p OR (95% CI) p 65–79 years old 0.628 (0.443–0.891) 0.009 0.619 (0.407–0.942) 0.025 ≥80 years old 0.764 (0.575–1.016) 0.065 0.760 (0.553–1.043) 0.090 Female 0.556 (0.412–0.750) <0.001 0.567 (0.408–0.790) 0.001 Male 1.034 (0.746–1.432) 0.841 0.960 (0.646–1.428) 0.841 Rural 0.875 (0.659–1.317) 0.354 0.943 (0.676–1.317) 0.731 City/town 0.560 (0.396–0.794) 0.001 0.461 (0.310–0.685) <0.001 Adjusted model: adjustment for age, sex, education, marital status, residence, financial, sleep quality, sleep duration, smoking, drinking, exercise, physical labor, depression, ADL disability, mMMSE and comorbidities, the grouping variable were excluded in each subgroup analysis. Bold values represent statistical difference. Adjusted model: adjustment for age, sex, education, marital status, residence, financial, sleep quality, sleep duration, smoking, drinking, exercise, physical labor, depression, ADL disability, mMMSE and comorbidities, the grouping variable were excluded in each subgroup analysis. Bold values represent statistical difference. 2020). In addition, LA was often accompanied by decreased family and social support and social isolation for older adults (Lou and Ng, 2012; Teerawichitchainan et al., 2015), and thus modified the associations of loneliness with adverse health outcomes in previous study (Wei et al., 2022). Maybe less tobacco use could be protective (Locquet et al., 2021). However, the results were sustained in multivariate logistic analyses adjusted for these covariates. There must be some other mediators that make LA protective for CC decline. 4 Discussion To the best of our knowledge, this is the first longitudinal study to evaluate the association of living arrangements with CC decline among older adults. Both univariate and multivariate logistic regression results of our study showed that LA was a protective factor for CC decline, which might help preserve skeletal muscle mass and reduce the risk of sarcopenia. The results of cross-sectional and longitudinal study about living arrangements and frailty were different (Woods et al., 2005; Yamanashi et al., 2015), and the result of our longitudinal study was also inconsistent with the result of cross-sectional study (Cheng et al., 2021). The association of LA with CC decline seems to be specific for older adults in 65–79 years old, female and city/ town subgroups when conducting the subgroup analysis. The reason why LA was protective was not clear yet. In our results, the age structure, gender ratio, education level, marital status, urban-rural distribution, economic status, sleep duration, smoking and drinking status, and mMMSE score were different between LA and NLA groups; older, women, uneducated, SDWS, rural, insufficient financial support, short-sleeper, non-smoker, non-drinker, and lower mMMSE scorer were more common in subjects living alone. These variables with statistical difference above, such as lower economic status and educational level in LA group, didn’t seem to be beneficial for skeletal muscle mass maintenance and sarcopenia prevention (Brennan-Olsen et al., LA does not always have adverse effects on physical and mental health, depending on the different context (Yeung and Cheung, 2015). Previous studies suggested that LA in older adults was not always a risk factor for health, and adverse health Frontiers in Aging frontiersin.org 06 Wang and Zhang 10.3389/fragi.2022.1063760 outcomes among older adults living alone might be confounded by poor social network (Sakurai et al., 2019). Women live longer than men and are more likely to be widowed. Compared to men, women tend to have larger social networks (Cornwell, 2011), which may explain the protective effect specific in women in our result. Another advantage of LA is that older adults who live alone can be relieved of family obligations and have more free time (Eshbaugh, 2008; Gu et al., 2019), which may offset the adverse effects of LA for physical and mental health. Actually, other study also found that women living alone have better psychologic function or lower frailty risk than women living with a spouse (Michael et al., 2001; Trevisan et al., 2016). 4 Discussion Compared to very old adults over the age of 80 or rural residents, people aged 65 to 79 or urban residents in China have more energy or better conditions to do the things they love or to be socially active (Liu et al., 2019). This may explain the protective effect specific for older adults in 65–79 years old and city/town subgroups in our result. It reminds us that the public health policy should be specific for different area groups, different age groups, and different gender groups. The implementation of the newly proposed rural revitalization strategy of China will bridge the urban-rural gap and might reduce the risk of sarcopenia among rural older adults. And future research needs better quantification of social factors to differentiate between different context. specific for different area groups, different age groups, and different gender groups. The results of this study will also provide a theoretical basis for the rural revitalization strategy of China to improve the health of rural older adults. Further studies are required to confirm these findings and identify the specific mechanisms that can be applied to improve the public health of older adults. 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. Acknowledgments The authors wish to thank the participants of the CLHLS. Data availability statement The datasets presented in this study can be found in online repositories. The names of the repository/repositories and accession number(s) can be found below: https://opendata. pku.edu.cn/dataverse/CHADS. Author contributions The strength of this study was that it was the first longitudinal study to investigate the relationship between living arrangements and CC decline, revealing the causality better. However, there were also limitations. First, the measurement of the CCs was not accurate enough, which were rounded up to the nearest integer in centimeters. And CC is recommended as one of the methods for sarcopenia screening in AWGS 2019 but not a gold standard for diagnosis. It could reflect but not substitute for skeletal muscle mass when it comes to sarcopenia. Second, we did not consider the change of living arrangements between two waves. The advantage, however, is that compared to other variables, such as sleep quality or sleep duration at different time, living arrangement is a more stable exposure in a 4-year time span (Nakakubo et al., 2021). 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Identifying and managing malnutrition, frailty and sarcopenia in the community: A narrative review. Nutrients 13 (7), 2316. doi:10.3390/nu13072316 Cheng, L., Sit, J. W. H., Chan, H. Y. L., Choi, K. C., Cheung, R. K. Y., Wong, M. M. H., et al. (2021). Author Correction: Sarcopenia risk and associated factors among Chinese community-dwelling older adults living alone. Sci. 5 Conclusion In summary, this study revealed that living alone was associated with a reduced risk of calf circumference decline among older adults, especially for older adults of 65–79 years old, women and city/town dwellers. It suggests that living alone may be a protective factor for skeletal muscle mass maintenance and sarcopenia prevention. Public health policy should be All claims expressed in this article are solely those of the authors and do not necessarily represent those of their affiliated organizations, or those of the publisher, the editors and the reviewers. Any product that may be evaluated in this article, or claim that may be made by its manufacturer, is not guaranteed or endorsed by the publisher. Frontiers in Aging frontiersin.org 07 Wang and Zhang Wang and Zhang 10.3389/fragi.2022.1063760 References National sleep foundation’s sleep time duration recommendations: Methodology and results summary. Sleep. Health 1 (1), 40–43. doi:10.1016/j.sleh. 2014.12.010 Wei, K., Liu, Y., Yang, J., Gu, N., Cao, X., Zhao, X., et al. (2022). Living arrangement modifies the associations of loneliness with adverse health outcomes in older adults: evidence from the CLHLS. BMC Geriatr. 22 (1), 59. doi:10.1186/s12877-021-02742-5 Holt-Lunstad, J., Smith, T. B., Baker, M., Harris, T., and Stephenson, D. (2015). Loneliness and social isolation as risk factors for mortality: a meta-analytic review. Perspect. Psychol. Sci. 10 (2), 227–237. doi:10.1177/1745691614568352 Woods, N. F., LaCroix, A. Z., Gray, S. L., Aragaki, A., Cochrane, B. B., Brunner, R. L., et al. (2005). Frailty: emergence and consequences in women aged 65 and older in the women’s health initiative observational study. J. Am. Geriatr. Soc. 53 (8), 1321–1330. doi:10.1111/j.1532-5415.2005.53405.x Kawakami, R., Murakami, H., Sanada, K., Tanaka, N., Sawada, S. S., Tabata, I., et al. (2015). Calf circumference as a surrogate marker of muscle mass for diagnosing sarcopenia in Japanese men and women. Geriatr. Gerontol. Int. 15 (8), 969–976. doi:10.1111/ggi.12377 Wu, C. J., Kao, T. W., Chang, Y. W., Peng, T. C., Wu, L. W., Yang, H. F., et al. (2018). Does the additional component of calf circumference refine metabolic syndrome in correlating with cardiovascular risk? J. Clin. Endocrinol. Metab. 103 (3), 1151–1160. doi:10.1210/jc.2017-02320 Kojima, G., Taniguchi, Y., Kitamura, A., and Fujiwara, Y. (2020). Is living alone a risk factor of frailty? A systematic review and meta-analysis. Ageing Res. Rev. 59, 101048. doi:10.1016/j.arr.2020.101048 Yamanashi, H., Shimizu, Y., Nelson, M., Koyamatsu, J., Nagayoshi, M., Kadota, K., et al. (2015). The association between living alone and frailty in a rural Japanese population: the nagasaki islands study. J. Prim. Health Care 7 (4), 269–273. doi:10. 1071/hc15269 Liu, J., Rozelle, S., Xu, Q., Yu, N., and Zhou, T. (2019). Social engagement and elderly health in China: Evidence from the China health and retirement longitudinal survey (CHARLS). Int. J. Environ. Res. Public Health 16 (2), 278. doi:10.3390/ijerph16020278 Yeung, W. J. J., and Cheung, A. K. L. (2015). Living alone: One-person households in asia. Demogr. Res. 32, 1099–1112. doi:10.4054/demres.2015.32.40 Locquet, M., Bruyere, O., Lengele, L., Reginster, J. Y., and Beaudart, C. (2021). Relationship between smoking and the incidence of sarcopenia: The SarcoPhAge cohort. Public Health 193, 101–108. doi:10.1016/j.puhe.2021.01.017 Zhou, Z., Mao, F., Ma, J., Hao, S., Qian, Z. M., Elder, K., et al. (2018). References A longitudinal analysis of the association between living arrangements and health among older adults in China. Res. Aging 40 (1), 72–97. doi:10.1177/ 0164027516680854 Lou, V. W., and Ng, J. W. (2012). Chinese older adults’ resilience to the loneliness of living alone: a qualitative study. Aging Ment. Health 16 (8), 1039–1046. doi:10. 1080/13607863.2012.692764 Frontiers in Aging 08 frontiersin.org
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Presence of Spodoptera frugiperda Multiple Nucleopolyhedrovirus (SfMNPV) Occlusion Bodies in Maize Field Soils of Mesoamerica
Insects
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Presence of Spodoptera frugiperda Multiple Nucleopolyhedrovirus (SfMNPV) Occlusion Bodies in Maize Field Soils of Mesoamerica Trevor Williams 1,* , Guadalupe del Carmen Melo-Molina 2, Jaime A. Jiménez-Fernández 2, Holger Weissenberger 3, Juan S. Gómez-Díaz 1, Laura Navarro-de-la-Fuente 1 and Andrew R. R 1 Instituto de Ecología AC (INECOL), Xalapa 91073, Veracruz, Mexico 2 El Colegio de la Frontera Sur (ECOSUR), Tapachula 30700, Chiapas, Mexico 3 El Colegio de la Frontera Sur (ECOSUR), Chetumal 77014, Quintana Roo, Mexico 4 CSIRO Division of Entomology, Canberra, ACT 2601, Australia * Correspondence: trevor.williams@inecol.mx Simple Summary: Nucleopolyhedroviruses of caterpillars (Lepidoptera) produce occlusion bodies (OBs) that protect the virions within a protein matrix. This allows the virus to persist in the envi- ronment until ingested by a susceptible insect. The soil is an important reservoir of OBs that can be transported to plants for transmission to the host caterpillar. The Spodoptera frugiperda multiple nucleopolyhedrovirus is a lethal pathogen of the fall armyworm, a major pest of maize, rice, and sorghum. Analysis of 186 soil samples collected from maize fields in southern Mexico, Belize, and Guatemala revealed that almost 19% of samples had OBs present at low concentrations. Genetic analysis revealed the presence of genetic diversity in the soil OB populations. The presence of OBs was higher in maize fields with living crops and in specific types of soils. These findings suggest that the soil could be a valuable source of genetic diversity for the design of virus-based insecticides to control this important pest. insects insects insects insects insects Citation: Williams, T.; Melo-Molina, G.d.C.; Jiménez-Fernández, J.A.; Weissenberger, H.; Gómez-Díaz, J.S.; Navarro-de-la-Fuente, L.; Richards, A.R. Presence of Spodoptera frugiperda Multiple Nucleopolyhedrovirus (SfMNPV) Occlusion Bodies in Maize Field Soils of Mesoamerica. Insects 2023, 14, 80. https://doi.org/10.3390/ insects14010080 Abstract: The occlusion bodies (OBs) of lepidopteran nucleopolyhedroviruses can persist in soil for extended periods before being transported back on to the foliage for transmission to the host insect. A sensitive insect bioassay technique was used to detect OBs of Spodoptera frugiperda multiple nucleopolyhedrovirus (SfMNPV) in 186 soil samples collected from maize fields in the southern Mexican states of Chiapas, Tabasco, Campeche, Yucatán, and Quintana Roo, as well Belize and Guatemala. Overall, 35 (18.8%) samples proved positive for SfMNPV OBs. The frequency of OB-positive samples varied significantly among Mexican states and countries (p < 0.05). Between 1.7 and 4.4% of S. frugiperda larvae that consumed OB-positive samples died from polyhedrosis disease. Restriction endonuclease analysis using PstI and HindIII confirmed that the soil-derived isolates were strains of SfMNPV and that genetic diversity was evident among the isolates. The prevalence of OB-positive soil samples did not differ with altitude or extension (area) of the maize field, but it was significantly higher in fields with the presence of living maize plants compared to those containing dead plants or crop residues (p < 0.05). Georeferenced soil samples were used to identify soil types on digitized soil maps. Lithosol and Luvisol soils had a higher than average prevalence of OB-positive samples (42–45% positive) (p = 0.006), as did Andosol, Gleysol, and Vertisol soils (33–60% OB-positive), although the sample sizes were small (<5 samples) for the latter three soils. In contrast, Cambisol soils had a lower than average prevalence of OB-positive samples (5% positive). Bioassays on Acrisol, Fluvisol, Phaeozem, and Rendzina soils resulted in intermediate levels of OB-positive samples. We conclude that certain soil types may favor OB persistence and virus-mediated biological pest control. The soil is also likely to provide a valuable source of genetic diversity for the design of virus-based insecticides against this pest. Academic Editors: Wataru Mitsuhashi and Kotaro Konno Received: 30 December 2022 Accepted: 11 January 2023 Published: 13 January 2023 Received: 30 December 2022 Accepted: 11 January 2023 Published: 13 January 2023 1. Introduction Nucleopolyhedrovirus occlusion bodies (OBs) that are released from virus-killed insects are washed off the plant by the action of rainfall or by the senescence and shedding of OB-contaminated leaves and subsequently enter the soil [1,2]. As a result, the soil represents an important environmental reservoir of nucleopolyhedrovirus OBs [3], although the persistence of OB populations in soil is poorly understood [4]. The OB structure allows the occlusion-derived virions (ODVs) to remain viable for months or years in soil, given favorable conditions of pH and temperature [5]. OBs in soil are also protected from the adverse effects of solar ultraviolet (UV) radiation that can rapidly inactivate these viruses [6]. OBs in the soil are transported back on to host plants during plant growth through the soil, through the action of wind-blown dust and rain-splash [7–9], or they are carried on the bodies of foraging invertebrates [10]. As OBs adhere strongly to soil particles, it is difficult to separate them by conventional techniques [11]. Techniques have been developed to detect the presence of OBs in soil by polymerase chain reaction (PCR), although amplification can be hindered by a diversity of soil components [12–14]. A sensitive bioassay technique has been developed involving feeding early instar larvae with mixtures of soil and semi-synthetic diet [15]. This technique has been used to obtain novel occluded virus isolates [16] and to study the abundance and genetic composition of soil OB populations [17,18] and their interaction with soil-dwelling invertebrates [19,20]. The Spodoptera frugiperda multiple nucleopolyhedrovirus (SfMNPV) (family Bac- uloviridae; genus Alphabaculovirus) is a common pathogen in natural populations of the fall armyworm, Spodoptera frugiperda (Lepidoptera: Noctuidae) in the Americas, and it has now been reported in China [21], India [22], and Indonesia [23], where the pest has recently invaded. The virus usually causes mortality of between 1% and 5% in S. frugiperda larvae [24–26], although some populations can experience seasonal epizootics [27,28]. This virus has attracted attention as the active ingredient in the development of biological insecticides against this pest, and several commercial products have been developed [29]. As part of a long-term study on the biology, ecology, and bioinsecticide potential of the S. frugiperda—SfMNPV pathosystem, we examined the prevalence of SfMNPV OBs in maize field soils in southern Mexico and part of Central America, over an area of >2000 km2. 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/). Keywords: Baculoviridae; Alphabaculovirus; virus persistence; insect bioassay; genetic diversity; soil type; fall armyworm https://www.mdpi.com/journal/insects Insects 2023, 14, 80. https://doi.org/10.3390/insects14010080 2 of 15 Insects 2023, 14, 80 1. Introduction The sampling program generated multiple isolates, some of which were gifted to other researchers who examined their insecticidal characteristics [30]. As digitalized maps of soil types in this region recently became available, we used our data, collected two decades ago, to analyze the relationship between soil type and the presence of SfMNPV OBs in maize fields in the region. 2. Materials and Methods 2.1. Insect Colonies and Reference Virus Isolate 2.2. Soil Sampling Between 30 June and 20 October 2000, a total of 186 soil samples were collected from maize fields in the southern Mexican states of Chiapas (120 samples), Tabasco (1 sample), Campeche (8 samples), Yucatán (16 samples), and Quintana Roo (10 samples), as well as parts of Belize (4 samples) and Guatemala (27 samples). Only a single sample was taken in Tabasco as the road to Campeche passed through this state for only a very short distance (~5 km). Sampling was non-systematic and consisted of driving along roads and selecting accessible fields with evidence of maize cultivation (planted crop or crop residues). In areas of intense maize production, fields were sampled at intervals of hundreds of meters (shortest distance 100 m) or a few kilometers, whereas in other areas, such as Belize where the dominant crop is sugarcane, maize fields were sampled at intervals of tens of kilometers (longest distance ~80 km). The aim was to obtain a range of samples in each area that we passed through. A soil sample was taken by scraping away the upper 5 cm of soil and collecting ~100 g of soil at a depth of 5–10 cm at five random points in each field to produce a sample of ~500 g that was placed in a black plastic bag, mixed by shaking, sealed, and labeled with a site code. Following the procedures of Richards & Christian [15], the uppermost 5 cm of soil was not sampled as OBs in this layer would be exposed to extremes of solar UV radiation and high temperatures that may have affected their viability. The geographical coordinates and altitude of each field were recorded using a GPS locator (Garmin III Plus, Garmin International Inc., Olathe, KS, USA); the area of the field was estimated (based on the approximate dimensions and shape of the field); the state of the crop was classified in terms of growth status (living or dead), as maize is commonly folded over and allowed to dry prior to harvesting in this region, or the presence of plant residues from a previously harvested crop (Supplemental File S1). Soil samples were taken to the laboratory in ECOSUR, Tapachula, Chiapas, spread on sheets of paper, and allowed to air dry for 2–5 days in a dark room at 26–28 ◦C and then prepared for insect bioassay. 2.1. Insect Colonies and Reference Virus Isolate A laboratory colony was started in 1996 using larvae of S. frugiperda collected from maize fields close to the city of Tapachula, Chiapas, Mexico. Larvae were reared individ- ually in 30 mL plastic cups with a piece of semi-synthetic diet based on soya, yeast, and maize flour without formaldehyde [31]. For oviposition, groups of adults were placed in paper bags with a plastic dish containing a cotton pad soaked in 10% honey solution and maintained at 25–28 ◦C. This colony was used for the isolation of SfMNPV OBs from soil samples. In the year 2000, a sample from this colony was sent to the Universidad Pública de Navarra, Spain, reared, and analyzed by reverse transcription PCR using primers tar- geting the ie-0 and polh genes and was found to be free of sublethal nucleopolyhedrovirus infection [32]. A second colony of S. frugiperda was started in 2021 using larvae collected from maize plants in a field close to Cempoala, Veracruz. This colony was treated identically to that of the first colony except that it was maintained at a constant 26 ± 1 ◦C in a temperature- controlled room. This colony was used to amplify isolates prior to restriction endonuclease analysis. PCR analysis using primers targeted at the sf58 gene [33] or the polh gene [34] did Insects 2023, 14, 80 3 of 15 3 of 15 not result in amplification of DNA samples taken from larvae, pupae, or adults, suggesting that this colony did not harbor sublethal nucleopolyhedrovirus disease. y p y The Nicaraguan isolate of SfMNPV (SfMNPV-NIC) was used as a reference isolate that has been characterized in detail in previous studies [35–37]. This isolate naturally comprises nine genotypic variants that are present in varying proportions. 2.2. Soil Sampling Following the bioassay procedure, all soils were autoclaved prior to disposal to eliminate soil-borne pathogens. 2.3. Bioassay of Soil Samples The soil bioassay procedure involves larvae that are fed mixtures of soil and semi- synthetic diet [15]. Air-dried soil samples were passed through a 1 mm sieve to eliminate stones and large pieces of organic material. Sieves were washed and decontaminated by treatment with 0.5% sodium hypochlorite solution to avoid cross-contamination. A 10 g sample of sieved soil was placed in a 250 mL plastic cup and mixed thoroughly with 90 g of semi-synthetic diet using a disposable wooden spatula to produce a smooth uniform paste. The soil–diet mixture was then distributed evenly among 30 plastic cups (30 mL capacity), and two S. frugiperda first instars from the first laboratory colony were placed in each cup. An additional group of 10 larvae per sample was reared individually with a mixture of sterilized soil and diet as controls. Larvae were incubated at 25 ◦C and were checked at 48 h intervals until death or pupation. A small portion of each dead larvae was taken with a toothpick, smeared on a microscope slide, stained using Giemsa stain, and examined for the presence of OBs under a phase-contrast microscope at ×1000 with oil immersion [38]. The efficacy of the soil sterilization procedure was checked by autoclaving samples of 10 g soil that had been spiked with 1.9 × 106 OB/g soil (equivalent to the estimated LC50 concentration mentioned in Section 3.1). None of the larvae that were Insects 2023, 14, 80 4 of 15 treated with soil+diet mixtures of autoclaved soil succumbed to polyhedrosis disease (3 replicates of 24 larvae each). OB-positive larva were macerated in 50 µL of distilled water, mixed with 10% sucrose and 0.05% Fluorella blue, and used to inoculate fourth instar larvae that had been starved overnight. Larvae that consumed the inoculum within 15 min were individualized in the wells of a 24-well cell culture plate with a piece of diet. Larvae were incubated at 25 ± 1 ◦C and were checked daily for signs of polyhedrosis disease until death. Virus-killed larvae were placed in 1.5 mL centrifuge tubes and stored at −80 ◦C for 21 years. Some of the samples were gifted to other researchers to perform comparative studies on the insecticidal characteristics of the isolates. A selection of 32 isolates was subjected to PCR amplification using primers Sf58.1 (5′-GTCCTCGGTGCTGAATCAGG-3′) and Sf58.2 (5′-TTACGTAGGTGCTGGAGGAG-3′) targeted at the sf58 gene of SfMNPV [33]. 2.3. Bioassay of Soil Samples For this, viral DNA was extracted from OBs as follows. Each infected larva was homogenized, filtered through an 80 micron pore steel mesh, and centrifuged at 2500 rpm for 5 min to pellet debris. The OB suspension was placed on a cushion of 50% glycerol and centrifuged at 12,000 rpm for 10 min. The resulting OB pellet was washed with distilled water and resuspended in 200 µL of distilled water. A 100 µL volume of 3xDAS buffer (0.3 M Na2CO3, 0.5 M NaCl, 0.03 M EDTA; pH 10.5) was added to the OB suspension and incubated at 45 ◦C for 45 min to release occlusion- derived virions (ODVs). The suspension was centrifuged at 3000 rpm for 5 min to remove undissolved OBs, and the supernatant was centrifuged at 12000 rpm for 10 min. The resulting pellet of ODVs was resuspended in 100 µL MilliQ water, 10 µL of 10% SDS, and 3 µL of proteinase K (20 mg/mL), followed by incubation at 45 ◦C for 45 min. Viral genomic DNA was then extracted by treatment with phenol-chloroform and precipitated in absolute ethanol with 1/10 vol 3 M sodium acetate at −20 ◦C overnight. The DNA pellet was washed with 70% ethanol and resuspended in 50 µL MilliQ water, and the concentration was measured in a UV-spectrometer at 260 nm (BioSpec-Nano, Shimadzu, Japan). p p p A 1 µL sample of the resulting DNA was amplified at 95 ◦C for 3 min, followed by 35 cycles of 95 ◦C for 30 s, 59 ◦C for 30 s, 72 ◦C for 1 min, and final extension at 72 ◦C for 7 min. Electrophoresis was performed in 1.2% agarose in TBE buffer (100 mM Tris, 90 mM boric acid, 1 mM EDTA; pH 8.3) containing 35 µL/liter of ethidium bromide (10 mg/mL) and photographed on a transilluminator (UV ChemiDoc XRS+ System with Image Lab Software; Bio-Rad, Hercules, CA, USA). A sample of SfMNPV-NIC (106 OBs) was included as a positive control and water as a negative extraction control. The frequency of OB-positive soil samples was compared across the states of southern Mexico and Belize and Guatemala by Fisher’s exact test, whereas the prevalence of virus- induced disease in bioassay larvae exposed to soil samples was compared across states and countries by fitting a generalized linear model with a binomial error structure in GLIM 4 [39]. 2.3. Bioassay of Soil Samples To quantify the sensitivity of the soil bioassay, the procedure was performed using a characterized clay soil (pH 5.2) from the grounds of the Instituto de Ecología AC, Xalapa, Mexico, which was studied previously [20]. For this, 1 mL suspensions of SfMNPV-NIC OBs were mixed with 10 g soil samples to produce concentrations of 2 × 104, 2 × 105, 2 × 106, 2 × 107 OBs/g soil. Control samples were treated with 1 mL distilled water. Each OB-contaminated soil was mixed with 90 g semi-synthetic diet and distributed among the wells of 24 well cell culture plates, and a single S. frugiperda first instar larva from the Cempoala colony was placed in each well and incubated at 27 ± 1 ◦C in darkness. Larvae that died from polyhedrosis disease (confirmed by examination of Giemsa-stained smears) were counted and recorded. The procedure was replicated four times using different batches of insects. The results were subjected to logit regression in GLIM 4 [39]. 3.1. Calibration of the Soil Bioassay Mortality in the soil bioassay ranged from 1% in the 2 × 104 OB/g soil concentration to 99% in the 2 × 107 OB/g soil concentration (χ2 = 310.2, df = 1, p < 0.001). No virus-induced deaths were observed in the control. The slope (± SE) of the logit regression was 1.383 ± 0.153, and the estimated LC50 was 1.9 × 106 OBs/g soil (range of 95% C.I.: 1.48 × 106–2.48 × 106). There was no evidence of overdispersion in the data (residual deviance/residual d.f. = 1.02). 2.5. Relationship between Soil OBs and Environmental Factors 2.5. Relationship between Soil OBs and Environmental Factors Data collected on the altitude (GPS data) and the estimated size of maize fields were compared for OB-positive and OB-negative soil samples by Kruskal–Wallis test and were reported as medians ± interquartile range (IQR). The stage of the crop cycle was classified as (i) green living stages from seedling to maize cob production or (ii) dry, dead plants and crop residues, as farmers twist and bend the maize stem to allow the cobs to dry in the field before they are harvested. The frequency of positive and negative samples was compared for each crop stage by contingency table analysis. The GPS-referenced location of each sample was plotted on digitized maps produced using vector data on southern Mexico [40] or Belize and Guatemala produced by the Food and Agriculture Organization of the United Nations [41]. Maps were drawn, and sample points were plotted using ArcGIS 10.4 software [42]. Intensive sampling was performed in the southernmost Soconusco region of Chiapas State, so this region was plotted on a separate map for clarity. Mapped information was used to identify soil type and subtype for each of the 186 samples analyzed. The prevalence of OB-positive samples was compared among soil types by contingency table analysis. Soils with fewer than five samples were excluded from this analysis. 2.4. Restriction Endonuclease Analysis of Soil Isolates To examine the presence of genetic diversity in soil isolates, eight isolates were selected at random. These isolates were amplified in S. frugiperda fourth instars from the Cempoala Insects 2023, 14, 80 5 of 15 5 of 15 colony. Larvae were individualized, starved overnight, and then allowed to drink a suspension of 108 OBs/mL in 10% sucrose solution with 0.05% Fluorella blue. Larvae that consumed the inoculum within 15 min were individualized in the wells of a 24-well cell culture plate with a piece of diet. Larvae were incubated at 27 ± 1 ◦C and were checked daily for signs of polyhedrosis disease. Virus-killed larvae were placed in 1.5 mL centrifuge tubes and stored at −20 ◦C. To obtain viral DNA, larvae were individually homogenized in 1 mL distilled water. OBs were then purified, and DNA was extracted, purified by phenol-chloroform treatment, and resuspended in 100 µL of MilliQ water as described in Section 2.3. Samples of 1 µg DNA of each isolate were digested with restriction endonucleases PstI or HindIII (New England Biolabs, Ipswich, MA, USA) at 37 ◦C following the manufacturer’s recommendations. After 6 h, the reaction was stopped by addition of loading buffer, and samples were loaded in a 0.6% (PstI) or 0.8% (HindIII) agarose gel in TBE buffer containing 35 µL/liter of ethidium bromide (10 mg/mL). Electrophoresis was performed overnight at 30 V. Each gel was then photographed on a transilluminator (UV ChemiDoc XRS+ System with Image Lab Software; Bio-Rad, Hercules, CA, USA). 3.2. Bioassay of Soil Samples from Maize Fields Of the 186 soil samples that were collected from maize fields, 35 (18.8%) proved posi- tive for SfMNPV OBs by insect bioassay (Figure 1, Table 1). The prevalence of OB-positive samples varied significantly by state and country (Fisher’s exact p = 0.003). A single neg- ative sample from Tabasco state was not included in the analysis. The highest number of OB-positive samples (N = 14) was obtained from Chiapas state, reflecting the inten- sive sampling in this state. The remaining states in Mexico had intermediate numbers of OB-positive samples (N = 3–6). Two positive samples were obtained from Belize, and six positive samples were collected in Guatemala (Table 1). 6 of 15 the am- emental Insects 2023, 14, 80 Figure 1. Distribution of soils samples that proved positive (orange points) or negative (black points) for SfMNPV OBs in the soil-diet bioassay. Samples were distributed across fives states of southern Mexico, Belize, and Guatemala. Intensive sampling was performed in the Soconusco region in the south of Chiapas State, Mexico (shown enclosed in a black rectangle). Figure 1. Distribution of soils samples that proved positive (orange points) or negative (black points) for SfMNPV OBs in the soil-diet bioassay. Samples were distributed across fives states of southern Mexico, Belize, and Guatemala. Intensive sampling was performed in the Soconusco region in the south of Chiapas State, Mexico (shown enclosed in a black rectangle). Figure 1. Distribution of soils samples that proved positive (orange points) or negative (black points) for SfMNPV OBs in the soil-diet bioassay. Samples were distributed across fives states of southern Mexico, Belize, and Guatemala. Intensive sampling was performed in the Soconusco region in the south of Chiapas State, Mexico (shown enclosed in a black rectangle). Figure 1. Distribution of soils samples that proved positive (orange points) or negative (black points) for SfMNPV OBs in the soil-diet bioassay. Samples were distributed across fives states of southern Mexico, Belize, and Guatemala. Intensive sampling was performed in the Soconusco region in the south of Chiapas State, Mexico (shown enclosed in a black rectangle). Table 1. Results of insect bioassays on soil samples according to Mexican State and country. Table 1. Results of insect bioassays on soil samples according to Mexican State and country. State/Country 1 No. of Samples Tested No. of Positive Samples (%) No. of Larvae Tested Against OB-Positive Samples No. 3.3. Restriction Endonuclease Analysis of Soil Isolates 3.3. Restriction Endonuclease Analysis of Soil Isolates 3.3. Restriction Endonuclease Analysis of Soil Isolates 3.3. Restriction Endonuclease Analysis of Soil Isolates Restriction endonuclease analysis of eight randomly selected isolates indicated con- siderable levels of genetic diversity across these isolates. Compared to the SfMNPV-NIC reference isolate, all the soil-derived isolates differed in the number and size of restriction fragments and also in the presence of sub-molar fragments that suggest that individual isolates likely comprise mixtures of genotypes (clear differences are marked with asterisks in Figure 2A,B). Digestion with PstI and HindIII revealed polymorphism that was apparent across the range of fragment lengths. The results of these analyses should be viewed with caution as the largest fragments (>48 Kb) may be difficult to distinguish from undigested genomic DNA, or partially digested DNA in the case of some sub-molar bands. The three short fragments of between 0.5 and 1 Kb produced by HindIII treatment were similar across all isolates, but they are not easily visualized in Figure 2B. Restriction endonuclease analysis of eight randomly selected isolates indicated con- siderable levels of genetic diversity across these isolates. Compared to the SfMNPV-NIC reference isolate, all the soil-derived isolates differed in the number and size of restriction fragments and also in the presence of sub-molar fragments that suggest that individual isolates likely comprise mixtures of genotypes (clear differences are marked with asterisks in Figure 2A,B). Digestion with PstI and HindIII revealed polymorphism that was appar- ent across the range of fragment lengths. The results of these analyses should be viewed with caution as the largest fragments (>48 Kb) may be difficult to distinguish from undi- gested genomic DNA, or partially digested DNA in the case of some sub-molar bands. The three short fragments of between 0.5 and 1 Kb produced by HindIII treatment were similar across all isolates, but they are not easily visualized in Figure 2B. Figure 2. Restriction endonuclease analysis of a selection of soil-derived isolates of SfMNPV (lanes A-H) compared to the SfMNPV-NIC reference isolate (Nic). Genomic DNA was digested with (A) PstI or (B) HindIII and subjected to electrophoresis in 0.6% and 0.8% agarose, respectively. The Figure 2. Restriction endonuclease analysis of a selection of soil-derived isolates of SfMNPV (lanes A-H) compared to the SfMNPV-NIC reference isolate (Nic). Genomic DNA was digested with (A) PstI or (B) HindIII and subjected to electrophoresis in 0.6% and 0.8% agarose, respectively. 3.3. Restriction Endonuclease Analysis of Soil Isolates 3.3. Restriction Endonuclease Analysis of Soil Isolates The isolates used for this analysis were SfMNPV-NIC (labeled Nic) sample #121 (labeled A), #36 (B), #37 (C), #19 (D), #152 (E), #92 (F), #177 (G), and #50 (H), following the sample numbers given in Supplemental File S1. Restriction fragments and sub-molar bands that differed from those of SfMNPV-NIC are labeled with an asterisk. Molecular markers were NEB 1 Kb ladder (M1) and NEB λ mono-cut DNA (M2). Figure 2. Restriction endonuclease analysis of a selection of soil-derived isolates of SfMNPV (lanes A-H) compared to the SfMNPV-NIC reference isolate (Nic). Genomic DNA was digested with (A) PstI or (B) HindIII and subjected to electrophoresis in 0.6% and 0.8% agarose, respectively. The Figure 2. Restriction endonuclease analysis of a selection of soil-derived isolates of SfMNPV (lanes A-H) compared to the SfMNPV-NIC reference isolate (Nic). Genomic DNA was digested with (A) PstI or (B) HindIII and subjected to electrophoresis in 0.6% and 0.8% agarose, respectively. The isolates used for this analysis were SfMNPV-NIC (labeled Nic) sample #121 (labeled A), #36 (B), #37 (C), #19 (D), #152 (E), #92 (F), #177 (G), and #50 (H), following the sample numbers given in Supplemental File S1. Restriction fragments and sub-molar bands that differed from those of SfMNPV-NIC are labeled with an asterisk. Molecular markers were NEB 1 Kb ladder (M1) and NEB λ mono-cut DNA (M2). 3.2. Bioassay of Soil Samples from Maize Fields of Virus Killed Larvae (%) Chiapas 120 14 (11.7%) 765 22 (2.9%) Tabasco 1 0 (0%) - - Campeche 8 4 (50.0%) 240 8 (3.3%) Yucatán 16 6 (37.5%) 360 15 (4.2%) Quintana Roo 10 3 (30.0%) 180 8 (4.4%) Belize 4 2 (50.0%) 120 2 (1.7%) Guatemala 27 6 (22.2%) 360 6 (1.7%) Totals: 186 35 (18.8%) 2025 61 (3.0%) 1 A single soil sample from Tabasco state was negative in the insect bioassay and was not included in the statistical analyses. On average (±SE), 1.74 ± 0.18 larvae died from polyhedrosis disease in the OB-positive samples (range 1–4 larvae/sample). Lethal polyhedrosis disease was confirmed by exam- ination of Geimsa smears. The prevalence of infection (range 1.7–4.4%; Table 1) did not differ significantly among samples from different states and countries (GLM χ2 = 6.245, df = 5, p = 0.283). An average (±SE) of 2.14 ± 0.38 larvae (range 0–8) died from other causes in OB-positive samples, mainly bacterial deaths, and these larvae were not considered further. None of the control larvae that consumed sterilized soil samples succumbed to polyhedrosis disease. p y All 32 isolates tested proved positive for PCR amplification of the sf58 gene, resulting in the expected 306 bp product, although there appeared to be slight variation in the amplicon size, suggesting the presence of genotypic variation among isolates (Supplemental Figure S3). 7 of 15 ncluded 7 of 15 ncluded Insects 2023, 14, 80 Totals: 3.4. Relationship between Soil OBs and Environmental Factors Percentage values shown in gray rectangles are based on the indicated sample sizes (N). Figure 3. Prevalence of OB-positive and -negative samples taken from maize fields with (A) living green crops and (B) dry crops or post-harvest crop residues. Percentage values shown in gray rec- tangles are based on the indicated sample sizes (N). Figure 3. Prevalence of OB-positive and -negative samples taken from maize fields with (A) living green crops and (B) dry crops or post-harvest crop residues. Percentage values shown in gray rectangles are based on the indicated sample sizes (N). Georeferenced information was used to locate samples taken from twelve types of soil, and 16 soil subtypes that are present in this region. The soil type of all but five sam- ples could be identified by plotting sample points on a combined map of the region incor- porating data from southern Mexico, Belize, and Guatemala (Figure 4A). The five samples from unknown soil types were classified as “uncharacterized”. Intensive sampling per- formed in the Soconusco region of Chiapas State, Mexico was plotted on a separate map for clarity (Figure 4B). Georeferenced information was used to locate samples taken from twelve types of soil, and 16 soil subtypes that are present in this region. The soil type of all but five samples could be identified by plotting sample points on a combined map of the region incorporating data from southern Mexico, Belize, and Guatemala (Figure 4A). The five samples from unknown soil types were classified as “uncharacterized”. Intensive sampling performed in the Soconusco region of Chiapas State, Mexico was plotted on a separate map for clarity (Figure 4B). y g The prevalence of OB-positive samples varied significantly among soil types (χ2 = 22.998, df = 9, p = 0.006; soil types with a sample size of <5 were not included in the analysis, namely Andosol, Nitisol, and Regosol). Compared to the average of 18.8% of OB-positive samples (shown in Table 1), Lithosol and Luvisol soils had a clearly higher than average prevalence of OB-positive samples (Figure 5). This was also the case for Andosol, Gleysol, and Vertisol soils (33–60% OB-positive), but the sample sizes were small for those soils (4–6 samples each). In contrast, Cambisol soils were well represented (21 sam- ples) but had a lower than average prevalence of OB-positive samples. 3.4. Relationship between Soil OBs and Environmental Factors Maize fields were sampled at altitudes between 8 and 2630 m above mean sea level, but altitude did not differ significantly between OB-positive soil samples (median [IQR]: 57 m [105]) and OB-negative samples (69 m [197]) (Kruskal–Wallis H = 1.039, df = 1, p = 0.308). Maize fields varied in estimated area from 0.04 to 15 ha, but the size the field did not differ significantly between OB-positive soil samples (median [IQR]: 1 ha [2.5]) and OB-negative samples (1 ha [1.25]) (Kruskal–Wallis H = 0.00, df = 1, p = 0.997). The presence of the crop in the green living stages (from seedling to cob production) was associated with an increased probability of OB-positive soil samples (Figure 3A,B). Overall, 29 out of 120 (24%) maize fields with living crops had OB-positive soils, whereas Insects 2023, 14, 80 8 of 15 uction) 3A B) 8 of 15 uction) 3A B) only 6 out of 66 (9%) maize fields with dead, dry crops and crop residues proved OB-positive in the insect bioassay (χ2 = 6.335, df = 1, p = 0.012). Looking at these results in another way, 83% (29 out of 35) of OB-positive soils originated from fields with green living crops, whereas only 17% (6 out of 35) of the OB-positive soils originated from fields with dry crops and crop residues. In this region, farmers twist and fold the maize stem to allow the cobs to dry before harvesting. only 6 out of 66 (9%) maize fields with dead, dry crops and crop residues proved OB- positive in the insect bioassay (χ2 = 6.335, df = 1, p = 0.012). Looking at these results in another way, 83% (29 out of 35) of OB-positive soils originated from fields with green living crops, whereas only 17% (6 out of 35) of the OB-positive soils originated from fields with dry crops and crop residues. In this region, farmers twist and fold the maize stem to allow the cobs to dry before harvesting. Figure 3. Prevalence of OB-positive and -negative samples taken from maize fields with (A) living green crops and (B) dry crops or post-harvest crop residues. Percentage values shown in gray rec- tangles are based on the indicated sample sizes (N). Figure 3. Prevalence of OB-positive and -negative samples taken from maize fields with (A) living green crops and (B) dry crops or post-harvest crop residues. 3.4. Relationship between Soil OBs and Environmental Factors The other soil types (Acrisol, Fluvisol, Phaeozem, and Rendzina soils) had intermediate levels of OB-positive samples that were similar to the overall average prevalence (Figure 5). The numbers of OB-positive samples among the soil subtypes are listed in Supplemental Material Table S1, and a brief description of soil characteristics and properties is provided in Table S2. Insects 2023, 14, 80 9 of 15 (A) (B) Figure 4. Soil type composite maps for (A) southern Mexico, Belize, and Guatem Soconusco region of Chiapas State, Mexico. Georeferenced soil samples were plotted samples (open black circles) and OB-negative samples (black circles with a cross). H versions of these maps are available as Supplemental Material (Figures S1 and S2). Figure 4. Soil type composite maps for (A) southern Mexico, Belize, and Guatemala and (B) the Soconusco region of Chiapas State, Mexico. Georeferenced soil samples were plotted as OB-positive samples (open black circles) and OB-negative samples (black circles with a cross). High resolution versions of these maps are available as Supplemental Material (Figures S1 and S2) (A) (A) (B) Figure 4. Soil type composite maps for (A) southern Mexico, Belize, and Guatemala Soconusco region of Chiapas State, Mexico. Georeferenced soil samples were plotted as samples (open black circles) and OB-negative samples (black circles with a cross). Hig versions of these maps are available as Supplemental Material (Figures S1 and S2). Figure 4. Soil type composite maps for (A) southern Mexico, Belize, and Guatemala and (B) the Soconusco region of Chiapas State, Mexico. Georeferenced soil samples were plotted as OB-positive samples (open black circles) and OB-negative samples (black circles with a cross). High resolution versions of these maps are available as Supplemental Material (Figures S1 and S2). (A) (B) (B) Figure 4. Soil type composite maps for (A) southern Mexico, Belize, and Guatemala Soconusco region of Chiapas State, Mexico. Georeferenced soil samples were plotted as samples (open black circles) and OB-negative samples (black circles with a cross). Hig versions of these maps are available as Supplemental Material (Figures S1 and S2). Figure 4. Soil type composite maps for (A) southern Mexico, Belize, and Guatemala and (B) the Soconusco region of Chiapas State, Mexico. Georeferenced soil samples were plotted as OB-positive samples (open black circles) and OB-negative samples (black circles with a cross). High resolution versions of these maps are available as Supplemental Material (Figures S1 and S2). Discussion 4. Discussion Discussion A 2600 km-long soil sampling trip around southern Mexico, Belize, and Guatemala was followed by intensive sampling in the Soconusco coastal region of the state of Chia- as. Fall armyworm is a major pest of maize in this region, especially during the rainy eason (May–October) when this study was conducted. In general, the ecology of OBs in he soil reservoir is poorly understood, which prompted the present study on the preva- f OB i i fi ld i M i A 2600 km-long soil sampling trip around southern Mexico, Belize, and Guatemala was followed by intensive sampling in the Soconusco coastal region of the state of Chiapas. Fall armyworm is a major pest of maize in this region, especially during the rainy season (May–October) when this study was conducted. In general, the ecology of OBs in the soil reservoir is poorly understood, which prompted the present study on the prevalence of OBs in maize fields in Mesoamerica. ence of OBs in maize fields in Mesoamerica. Overall, almost one in five soil samples (35 out of 186 samples; 18.8%) proved positive or SfMNPV OBs by insect bioassay, indicating that this could be a productive strategy for btaining novel virus isolates. A selection of 32 isolates all proved positive for PCR am- lification of the sf58 gene, which encodes a per os infection factor (PIF9) present in al- habaculoviruses and betabaculoviruses [33,43] (Supplemental Figure S3). This provides dditional evidence that the majority of isolates were SfMNPV, although we were unable Overall, almost one in five soil samples (35 out of 186 samples; 18.8%) proved positive for SfMNPV OBs by insect bioassay, indicating that this could be a productive strategy for obtaining novel virus isolates. A selection of 32 isolates all proved positive for PCR amplification of the sf58 gene, which encodes a per os infection factor (PIF9) present in alphabaculoviruses and betabaculoviruses [33,43] (Supplemental Figure S3). This provides additional evidence that the majority of isolates were SfMNPV, although we were unable to test the isolates that had been gifted to other researchers. o test the isolates that had been gifted to other researchers. The soil bioassay contrasts with the conventional method of collecting large numbers f S. frugiperda larvae from infested fields and rearing them in the laboratory to detect atural SfMNPV infections [24–26,28]. 3.4. Relationship between Soil OBs and Environmental Factors 10 of 15 brief Insects 2023, 14, 80 Figure 5. Prevalence of samples from different types of soil that were positive for SfMNPV OBs. Values above columns indicate the number of samples tested (N). Figure 5. Prevalence of samples from different types of soil that were positive for SfMNPV OBs. Values above columns indicate the number of samples tested (N). gure 5. Prevalence of samples from different types of soil that were positive for SfMNPV OBs. alues above columns indicate the number of samples tested (N). Figure 5. Prevalence of samples from different types of soil that were positive for SfMNPV OBs. Values above columns indicate the number of samples tested (N). Discussion 4. Discussion The reason why the LC50 values differed by approximately 100-fold between droplet feeding and soil-diet bioassay techniques is likely due to two reasons: (i) the OB-contaminated soil was diluted ten-fold by the addition of the diet prior to the soil-diet bioassay, and (ii) OBs adhere strongly to components of the soil, particularly clay minerals [49], and so may be less readily solubilized in the insect midgut than purified OBs in water. g p The soil + diet bioassay technique was successfully applied in a study of natural OB populations in greenhouse crops attacked by the beet armyworm, Spodoptera exigua (Lepidoptera: Noctuidae) in southern Spain. Of a total of 267 substrate samples from green- houses, 34% proved positive for the homologous nucleopolyhedrovirus (SeMNPV) [18]. Following this, a slightly modified soil + diet bioassay technique was used on early instars of S. frugiperda to detect OB-positive samples (8% positive) in soils taken from maize fields in northern Mexico [16]. Subsequent analysis indicated that some of these isolates were mixtures of SfMNPV and a granulovirus, suggesting that the insect colony may have harbored a sublethal granulovirus infection [50]. An alternative explanation involving simultaneous acquisition of both types of virus from a single soil sample is unlikely given the low concentration of OBs in soil. Nevertheless, granuloviruses can also persist in soil for extended periods [51,52]. p A selection of the SfMNPV isolates from the present study was gifted to A.M. Martínez- Castillo (Universidad Michoacana de San Nicolás de Hidalgo, Mexico), and two of them, from Chiapas and Yucatán, were subjected to analysis of insecticidal characteristics in different Mexican populations of S. frugiperda. The Yucatán isolate was also used for field tests with promising results [30], underlining the value of soil OB populations as a source of new and highly insecticidal isolates. Larvae that became infected after consuming soil samples probably only consumed a single OB, as consumption of one OB was previously demonstrated to result in 2.7–5.4% mortality in S. frugiperda second instars [53]. Nonetheless, restriction endonuclease analysis indicated that genetic diversity was present in each of the isolates tested, as evidenced by the presence of fragment size polymorphisms and sub-molar fragments (Figure 4A,B). This was likely due to the presence of genotypic diversity present within individual OBs present in the soil. Discussion 4. Discussion The soil bioassay approach has the additional ben- fit that it could be used for isolate prospection during periods when the pest is not The soil bioassay contrasts with the conventional method of collecting large numbers of S. frugiperda larvae from infested fields and rearing them in the laboratory to detect natural SfMNPV infections [24–26,28]. The soil bioassay approach has the additional benefit that it could be used for isolate prospection during periods when the pest is not present in the crop, or to quantify soil OB persistence over extended periods of time, even when non-host crop plants have been planted as part of a crop rotation cycle. The prevalence of OB-positive samples varied across the sampled region, but the prevalence of infection in larvae that consumed soil samples was invariably low (average 3%). Calibration of the soil bioassay indicated that the concentrations of OBs in virus- positive maize field samples were likely between 104 and 105 OBs/g soil. This is not an insignificant amount as the 50% lethal concentration of SfMNPV OBs by droplet feeding bioassay is approximately 2 × 104 OBs/mL in second instar larvae of S. frugiperda [44]. y pp y f g p The estimated concentration of SfMNPV in soil samples in our study (104–105 OBs/g) was very similar to the concentration of OBs reported in other pest–virus pathosystems in Spain [18], Botswana [45], Canada [46], and Connecticut, USA [47], although much higher concentrations (7 × 107 OBs/g) were reported from soil under trees infested by Hyphantria cunea in Japan [48]. It is important to note that these studies all used different methodologies to quantify OBs in soil samples. In the case of the soil-diet bioassay in first instars, the estimated LC50 was 1.9 × 106 OBs/g soil, which compared favorably with values derived from soil-diet bioassays on second Insects 2023, 14, 80 11 of 15 11 of 15 instars of 1.4 × 106 OBs/g soil [20] and 2.7 × 106 OBs/g soil, albeit with shorter periods (2–4 days) of feeding on the soil–diet mixture [19]. Discussion 4. Discussion Restriction endonucleases provide a rapid visual assessment of genetic similarities between isolates but are of limited value for determining the presence of genetic diversity as large fragments, and sub-molar bands may be the result of incomplete digestion of genomic DNA. Nonetheless, we performed several digestions of viral DNA with different enzymes such as BamHI and EcoRI (data not shown), as well as several digestions using HindIII and PstI (shown in Figure 2A,B), and in all cases sub-molar bands were present, which lends support to the idea that the isolates comprised mixtures of genotypic variants. pp p g yp The SfMNPV-NIC isolate comprises a mixture of nine genotypic variants in various proportions that are enveloped together within ODVs and co-occluded in OBs [53,54]. Genotypic diversity has a marked influence on the transmission and persistence of nu- cleopolyhedroviruses [54–57]. A study on OB populations in greenhouse soil substrate revealed that the prevalence of different genotypic variants of SeMNPV varied seasonally and across different parts of the study area, suggesting that certain variants may be better adapted than others to persist outside the host [18]. However, the genotypic diversity of OBs on plant surfaces, in infected insects, and in the soil has not yet been subjected to systematic comparison. y p The viable OB population in the soil reflects the result of processes that introduce OBs and those that eliminate OBs from the soil habitat. OBs released from virus-killed insects that fall off the plant and from OB-contaminated foliage are washed onto the soil by rainfall [1] and through the shedding of OB-contaminated leaves [2,48]. As a result, there is a clear correlation between the presence of the pest on the crop and the abundance of OBs present in the soil [18,46,51,58]. This was confirmed in the present study as we detected a positive association between OBs in soil samples and the living stage of the crop cycle, Insects 2023, 14, 80 12 of 15 12 of 15 prior to harvest, that is the stage attacked by S. frugiperda larvae (Figure 3A,B). Contrarily, processes that eliminate OBs include UV-irradiation and high temperatures at the soil surface, loss through OB transportation onto plant surfaces by wind and rain-splash, and chemical and microbial degradation of OBs in the soil [59–61]. Discussion 4. Discussion In addition, processes such as water percolation, ploughing, the movement of livestock or the activity of the soil fauna can be responsible for altering the vertical distribution of OBs in soil [20,58,61]. p g An important finding was that the prevalence of OB-positive samples varied signifi- cantly among soil types with higher than average prevalence in Lithosol, Luvisol, Andosol, Gleysol, and Vertisol soils, although sample sizes were reduced for the later three soil types. Soils are extraordinarily variable in their physico-chemical and biological properties. How- ever, it is worth noting that Luvisols, Andosols, Gleysols, and Vertisols are characterized by high clay content, which is known to strongly bind to OBs [49]. Surface charge and hydrophobic surface attributes appear to be involved in the interaction of OBs with soil minerals [62,63]. Lithosols are also known as Leptosols and are characterized by very shal- low soils over rock, highly calcareous or stony material without clearly expressed horizons. They comprise a wide variety of soils with a diversity of chemical and physical properties. Interestingly, Cambisol soils had a lower than average prevalence of OB-positive samples. Cambisols are known as “brown soils”, that are in the beginning of soil formation and have weak horizon differentiation. Cambisols are characterized by the absence of a layer of accumulated clay, humus, soluble salts, and iron or aluminum oxides [64–67]. The cause of the low prevalence of OBs in Cambisols may be related to the low clay content or some other factor(s) (see Supplemental Table S2). Finally, although we did not measure soil pH, it is known that pH affects the long-term persistence of OBs in soil both at low pH (acidic) and high pH (alkaline) [51]. Soil pH may also affect surface charge and the tendency of OBs to bind to soil particles [63], which is another field of research that merits systematic examination using modern analytical chemistry techniques. These findings have intriguing implications in that they suggest that soil type could have a direct effect on the persistence of lepidopteran nucleopolyhedroviruses in the envi- ronment. However, on a note of caution, additional factors such as the local maize planting practices, the phenological development of the crop and the density of the S. frugiperda infestation could also affect the likelihood of detecting OBs in soil samples, although we did not measure these variables in the present study. Discussion 4. Discussion It would be interesting to examine sys- tematically the influence of soil type on the prevalence of virus-induced disease in similar pest–crop combinations grown in different types of soil, to explicitly test the hypothesis that soils that favor OB persistence can augment the biological control of lepidopteran pests. 5. Conclusions We conclude that the soil represents an enormous and largely untapped source of novel nucleopolyhedrovirus isolates. Such isolates harbor genotypic diversity that could be useful in the development of biological insecticides. The probability of isolating novel virus strains increased during periods of vegetative growth when the pest was likely present in the maize crop. Certain soil types appeared to be more amenable than others to the persistence of soil OB populations. That soil type could manifestly influence biological pest control by these viruses is a notion that requires systematic evaluation. Finally, the soil-diet bioassay technique has clear potential for the study of the ecology of soil OB populations and the factors that mediate OB persistence in soil, such as soil composition, agronomic practices, and the role of soil invertebrates in OB dispersal. Supplementary Materials: The following supporting information can be downloaded at: https:// www.mdpi.com/article/10.3390/insects14010080/s1, (FileS1.xlxs); Figure S1: High-resolution version of Figure 4A; Figure S2: High-resolution version of Figure 4B; Figure S3: PCR amplification of soil isolates using primers targeted at the sf58 gene of SfMNPV; Table S1: Number of OB-positive samples according to soil type and subtype; Table S2: Properties and characteristics of soil types that differed in the prevalence of OB-positive samples. 13 of 15 13 of 15 Insects 2023, 14, 80 Author Contributions: Conceptualization, A.R.R. and T.W.; methodology, A.R.R., T.W., J.S.G.-D. and H.W.; formal analysis, T.W.; investigation, G.d.C.M.-M., J.A.J.-F., A.R.R., T.W., J.S.G.-D. and L.N.-d.-l.-F.; resources, A.R.R., T.W. and J.S.G.-D.; data curation, T.W.; writing—original draft preparation, G.d.C.M.-M., J.A.J.-F. and T.W.; writing—review and editing, T.W., visualization, H.W. and L.N.-d.-l.-F.; supervision, T.W., project administration, T.W.; funding acquisition, T.W. and A.R.R. All authors have read and agreed to the published version of the manuscript. Funding: The soil collection and virus detection study was funded by CONACYT-SIBEJ 0501047 and the Australian Academy of Science via a travel grant to A.R.R. In addition, L.N.-d.-l.-F. received an Ayudante de Investigador III award from CONACYT, Mexico. Data Availability Statement: All the data presented in this study are available in the file Supplementary data file FileS1.xlxs. Acknowledgments: We thank Juan Cisneros and Dora I. Penagos (ECOSUR) for assistance with soil bioassays and Cindy S. Molina (INECOL) for sharing PCR amplification results on the insect colony. Conflicts of Interest: The authors declare no conflict of interest. Conflicts of Interest: The authors declare no conflict of interest. References 1. D’Amico, V.; Elkinton, J.S. Rainfall effects on transmission of gypsy moth (Lepidoptera: Lymantriidae) n Environ. Entomol. 1995, 24, 1144–1149. [CrossRef] 1. D’Amico, V.; Elkinton, J.S. 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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/W3174449198
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English
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Unsupervised feature selection with least-squares quadratic mutual information
Indonesian journal of electrical engineering and computer science
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Corresponding Author: Janya Sainui Division of Computational Science, Faculty of Science Prince of Songkla University Songkhla, Thailand Email: janya.s@psu.ac.th Indonesian Journal of Electrical Engineering and Computer Science Vol. 22, No. 3, June 2021, pp. 1619∼1628 ISSN: 2502-4752, DOI: 10.11591/ijeecs.v22i3.pp1619-1628 Indonesian Journal of Electrical Engineering and Computer Science Vol. 22, No. 3, June 2021, pp. 1619∼1628 ISSN: 2502-4752, DOI: 10.11591/ijeecs.v22i3.pp1619-1628 1619 Ì ABSTRACT We propose the feature selection method based on the dependency between features in an unsupervised manner. The underlying assumption is that the most important feature should provide high dependency between itself and the rest of the features. Therefore, the top m features with maximum dependency scores should be selected, but the redundant features should be ignored. To deal with this problem, the objective function that is applied to evaluate the dependency between features plays a crucial role. However, previous methods mainly used the mutual information (MI), where the MI estimator based on the k-nearest neighbor graph, resulting in its estimation de- pendent on the selection of parameter, k, without a systematic way to select it. This implies that the MI estimator tends to be less reliable. Here, we introduce the least- squares quadratic mutual information (LSQMI) that is more sensible because its tuning parameters can be selected by cross-validation. We show through the experiments that the use of LSQMI performed better than that of MI. In addition, we compared the pro- posed method to the three counterpart methods using six UCI benchmark datasets. The results demonstrated that the proposed method is useful for selecting the informative features as well as discarding the redundant ones. Unsupervised feature selection with least-squares quadratic mutual information Janya Sainui, Chouvanee Srivisal Division of Computational Science, Faculty of Science, Prince of Songkla University, Songkhla, Thailand Article Info Article history: Received Dec 27, 2020 Revised May 10, 2021 Accepted May 24, 2021 Keywords: Least-squares quadratic mutual information Mutual information Unsupervised feature selection This is an open access article under the CC BY-SA license. This is an open access article under the CC BY-SA license. 1. INTRODUCTION Feature selection aims to select the most informative subset of features to capture the structure of the original data. It usually appears as a pre-processing step for various tasks such as classification [1], [2], clustering [3]-[5], data mining [6], resulting in better results. In a supervised scenario, the idea is to select the features that are the most relevant to the class labels [7], [8]. However, in this paper, we focus on an unsupervised feature selection that is more difficult to achieve than the supervised feature selection due to the absence of the class labels [9]-[14]. In unsupervised manner, feature selection is important as it helps improve the performance as well as reduce the computational time of clustering [15]-[19]. Moreover, unsupervised feature selections may be used for various purposes such as visualization and pre-processing step for supervised learning [7], [15], [20]. Unsupervised feature selection can be divided into two categories, namely wrapper method and filter method [10], [15]. Many existing methods of unsupervised feature selection are based on the wrapper method such that the selected features are dependent on the specific clustering algorithm. The main drawbacks of the wrapper method are its computational complexity and limited use with a particular clustering Journal homepage: http://ijeecs.iaescore.com 1620 Ì ISSN: 2502-4752 algorithm. In this paper, we focus on the filter method which evaluates the important features without using any clustering algorithm [21]-[25]. Thus, the filter method is more general and useful than the wrapper method. We review some existing unsupervised feature selection methods relating to our work as follows. algorithm. In this paper, we focus on the filter method which evaluates the important features without using any clustering algorithm [21]-[25]. Thus, the filter method is more general and useful than the wrapper method. We review some existing unsupervised feature selection methods relating to our work as follows. Laplacian score (LS) [10] is based on Laplacian Eigenmap and locality preserving projection. For each feature, the Laplacian score is estimated using the nearest neighbor graph. If it is a good feature, its LS tends to be small. The LS is based on the observation that, two data points are probably related to the same cluster if they are close to each other. The drawbacks of this method are that there is no systematic way to tune the number of nearest neighbor k or Gaussian width, and the redundant features are not observed. 1. INTRODUCTION multi-cluster feature selection (MCFS) [11] selects the features while preserving the cluster structure of the data. The authors proposed to use multiple eigenvectors of graph Laplacian, which are defined on the affinity matrix of data points to capture the multi-cluster structure of data. The algorithm performs especially well when the number of selected features is small (e.g., <= 50). However, it performs best when the number of used eigenvectors is equal to the number of clusters, but in an unsupervised manner, the number of clusters remains unknown. Moreover, we observed through the experiments that this method tends to be sensitive to noise features. Unsupervised feature selection based on mutual information (UFSMI) [9] is derived from the observation that good features share information in common, and noisy features are less correlated with the other features. Mutual information (MI) is then used as the objective function to capture the shared information between a feature and the rest of features, and they choose the first m features that achieve the higher mutual information. The higher level of noise leads to a smaller average value of the score function because the addition of noise reduces the mutual information between features. The desired property of the feature selection algorithm aims at removing noisy features. However, the estimation of MI is less reliable as it is computed using the k-nearest neighbor graph. Thus, its performance is based on the tuning parameter, k, resulting in that a subset of the selected features may not be the best. All methods mentioned above including several existing methods such as [12]-[14] are based on the k-nearest neighbor graph or other tuning parameters, and there is no systematic way to choose such parameters (i.e., k). In addition, they did not observe the redundant features. Therefore, in this paper, we would like to deal with these problems. Our idea is inspired by [9] as they proved that a good feature is expected to be dependent on the rest of features. However, the method did not take the redundancy of features into account, and thus may not produce an optimal feature subset. Moreover, in [9], they used the mutual information (MI) as their dependent measure, and the MI is estimated based on the k-nearest neighbors which lacks of the systematic way to choose the appropriate k. So that the estimation of MI tends to be less reliable. 2. THE LEAST-SQUARE QUADRATIC MUTUAL INFORMATION In this section, we first review the least-square quadratic mutual information (LSQMI) [27] that can be used to measure a statistical dependence among features. As LSQMI is ranged from 0 to ∞, we need to normalize LSQMI to [0,1] range. We then review the normalization of LSQMI [28] that is finally used as the objective function for our proposed unsupervised feature selection method. 1. INTRODUCTION In order to solve these problems, we propose to apply the L2-distance variant of MI called the quadratic mutual information (QMI) [26], where the least-square method is used to approximate QMI [27]. Therefore, the used parameters can be obtained automatically by cross-validation. The contribution of this paper is a novel method of unsupervised feature selection based on the least-squares quadratic mutual information (LSQMI), which is powerful for selecting the most important features as well as rejecting the redundant features. In the experimental results, we show that the LSQMI is more reliable than MI. In addition, we demonstrate that the proposed method is promising through the experimental results on six UCI machine learning repositories. The rest of paper is organized as follows. In section 2, we review the least-square quadratic mutual information and its normalization. We describe the proposed method in section 3. The experimental setup and results are shown in section 4. In section 5, we provide the conclusion. Indonesian J Elec Eng & Comp Sci, Vol. 22, No. 3, June 2021 : 1619 – 1628 2.1. LSQMI estimation Suppose that we are given a set of {(xi, yi)}n i=1 independently drawn from a joint probability dis- tribution with density p(x, y). Here, x ∈Rn is a featrue vector and y ∈Rn×(d−1) is the rest of feature vectors, where n denotes the number of samples and d denotes the number of features. The quadratic mutual information (QMI) [26] between x and y is defined as donesian J Elec Eng & Comp Sci, Vol. 22, No. 3, June 2021 : 1619 – 1628 Indonesian J Elec Eng & Comp Sci ISSN: 2502-4752 Ì 1621 Indonesian J Elec Eng & Comp Sci ISSN: 2502-4752 Ì QMI := ZZ  f(x, y) 2 dxdy, QMI := ZZ  f(x, y) 2 dxdy, QMI := ZZ  f(x, y) 2 dxdy, where where where f(x, y) := p(x, y) −p(x)p(y), f(x, y) := p(x, y) −p(x)p(y), f(x, y) := p(x, y) −p(x)p(y), and p(x) and p(y) denote the marginal densities of x and y, respectively. In LSQMI [27], the density difference f(x, y) is modeled as and p(x) and p(y) denote the marginal densities of x and y, respectively. In LSQMI [27], the density difference f(x, y) is modeled as g(x, y) = n X ℓ=1 θℓK(x, xℓ)L(y, yℓ), g(x, y) = n X ℓ=1 θℓK(x, xℓ)L(y, yℓ), g(x, y) = n X ℓ=1 θℓK(x, xℓ)L(y, yℓ), where K(x, xℓ) and L(y, yℓ) are kernel functions for x and y, respectively. Then, θ = (θ1, . . . , θn)⊤is learned by least-squares as where K(x, xℓ) and L(y, yℓ) are kernel functions for x and y, respectively. Then, θ = (θ1, . . . , θn)⊤is learned by least-squares as min θ ZZ  g(x, y) −f(x, y) 2 dxdy. min θ ZZ  g(x, y) −f(x, y) 2 dxdy. An empirical and regularized version of the above optimization problem is given as bθ := argmin θ h θ⊤Hθ −2θ⊤bh + λθ⊤θ i , where λ ≥0 is the regularization parameter, and H and bh are defined as where λ ≥0 is the regularization parameter, and H and bh are defined as Hℓ,ℓ′ := Z K(x, xℓ)K(x, xℓ′)dx Z L(y, yℓ)L(y, yℓ′)dy, (1) bhℓ:= 1 n n X i=1 K(xi, xℓ)L(yi, yℓ) −1 n2 n X i,j=1 K(xi, xℓ)L(yj, yℓ). 2.2. The normalisation of LSQMI 2.2. The normalisation of LSQMI As LSQMI is ranged from 0 to ∞, we here normalize it as follow: As LSQMI is ranged from 0 to ∞, we here normalize it as follow: As LSQMI is ranged from 0 to ∞, we here normalize it as follow: \ NQMI := [ QMI(x, y) max([ QMI(x, x), [ QMI(y, y)) . (2) (2) The higher \ NQMI indicates the more information shared between the feature x and the rest of features y, while it is closed to zero if the feature x is a noise feature. We assume that the values of [ QMI(y, y) and [ QMI(x, x) are larger than zero and that of [ QMI(x, y), because they are the dependency of themselves. Now, the range of \ NQMI is [0, 1]. However, in practice, \ NQMI may be higher than 1. The higher \ NQMI indicates the more information shared between the feature x and the rest of features y, while it is closed to zero if the feature x is a noise feature. We assume that the values of [ QMI(y, y) and [ QMI(x, x) are larger than zero and that of [ QMI(x, y), because they are the dependency of themselves. Now, the range of \ NQMI is [0, 1]. However, in practice, \ NQMI may be higher than 1. 2.1. LSQMI estimation (1) In this paper, we use the Gaussian kernel for both K(x, xℓ) and L(y, yℓ) as both x and y are continuous: In this paper, we use the Gaussian kernel for both K(x, xℓ) and L(y, yℓ) as both x and y are continuous: K(x, xℓ) := exp  −∥x −xℓ∥2 2σ2  , L(y, yℓ) := exp  −∥y −yℓ∥2 2σ2  , so that the integral in H (1) can be computed analytically as so that the integral in H (1) can be computed analytically as Hℓ,ℓ′ = (πσ2)dx/2 exp  −∥xℓ−xℓ∥2 4σ2  × (πσ2)dy/2 exp  −∥yℓ−yℓ′∥2 4σ2  . Thus, the solution bθ can be obtained as Thus, the solution bθ can be obtained as bθ = (H + λI)−1bh, bθ = (H + λI)−1bh, where I denotes the identity matrix. Finally, the least-squares quadratic mutual information (LSQMI) is given by [ QMI := max(0, 2bθ ⊤bh −bθ ⊤Hbθ). [ QMI := max(0, 2bθ ⊤bh −bθ ⊤Hbθ). [ QMI := max(0, 2bθ ⊤bh −bθ ⊤Hbθ). Unsupervised feature selection with least-squares quadratic mutual information (Janya Sainui) ISSN: 2502-4752 1622 Ì 2.2. The normalisation of LSQMI The inputs of the algorithm are a set of features, {f1, ..., fd}, the number of needed features, m, and a threshold, τ. While the output is a subset of selected features S = {f ∗ 1 , . . . , f ∗ m}. The algorithm starts by 3. THE PROPOSED UNSUPERVISED FEATURE SELECTION METHOD In an unsupervised scenario, the objective function that is used to evaluate the important feature is very important. However, the objective function of the existing methods tends to be less reliable because its approximation depends on the parameters included in the objective function, and there is no automatic way to choose those parameters. In this paper, we propose an unsupervised feature selection by using a more reliable objective function called the least-squares quadratic mutual information (LSQMI). The benefits of LSQMI are that the parameters needed in the objective function can be chosen by cross validation, and it is less sensitive to noises and outliers that may cause the estimation of the selected criteria unreliable [26], [27]. The proposed method is described as follows. Given a set of n samples denoted as {xi}n i=1, where each sample xi consists of d features (i.e., xi = {f1, . . . , fd}), our goal here is to select the most m informative features from d features, while the redundant features should be discarded. To address this problem, we apply the LSQMI [27] as our criteria for both selecting the most m important features as well as ignoring the redundant ones. Let fj ∈Rn be the jth feature, and f\j ∈Rn×(d−1) be all features excluding fj. To select the important but not redundant features, our proposed algorithm is shown in Algorithm 1. Algorithm 1 The unsupervised feature selection with least-squares quadratic mutual information (UFSLSQMI) Input: A set of features, {f1, ..., fd}, fj ∈Rn, the number of needed features, m, and a threshold, τ. Output: A subset of selected features, S = {f ∗ 1 , . . . , f ∗ m}. 1: S ←∅ 2: for j = 1, . . . , d do 3: compute \ NQMI (Equation (2)) between fj and f\j 4: end for 5: sort features {f1, ..., fd} according to their \ NQMI in descending order resulting in {f ∗ 1 , ..., f ∗ d } 6: S ←{S ∪f ∗ 1 } 7: k ←1 8: for j = 2, . . . 3. THE PROPOSED UNSUPERVISED FEATURE SELECTION METHOD In other words, if the ratio of the current score and the previous one is higher than the given threshold τ, (line 10), the feature according to the smaller score is ignored. The algorithm is repeated until the m features (lines 8 - 17) are obtained. The performance of the proposed method depends on the parameters used for approximating the LSQMI. However, thanks to the least-squares method, we can choose the appropriate parameters by cross- validation method. Although the selected features are also depended on the threshold τ, we show you through the experiments that our objective function well defines the redundant features and the noise features. In other words, if two features are redundancy, their LSQMI scores tend to be closed to each other, and if a feature is the noise feature, the LSQMI score tends to be small. 4. RESULTS AND DISCUSSION In this section, we demonstrate the effectiveness of the proposed unsupervised feature selection. We compared the proposed method with three competitive methods, including UFSMI [9], LSFS [10], and MCFS [11]. All methods are based on the k-nearest neighbor, and here we set k = 5 for all methods as their default setting. For MCFS, there is another parameter that is the number of eigenvectors, and we set this parameter at 5 as the same reason. For the proposed method, the threshold τ is set at 0.95. The competitive methods including the proposed method rank the features according to the criteria to select the top m features. This means that the objective function mainly affect a subset of selected features. Thanks to the reliability of LSQMI, our method considers not only the importance of features but the redun- dancy also. Therefore, in the experiment setups, we firstly show you that our objective function is effective for selecting the important features as well as discarding the redundant features on the synthetic data. Secondly, we illustrate the clustering performance after feature selection using six UCI benchmark datasets to confirm the usefulness of our proposed method. 3. THE PROPOSED UNSUPERVISED FEATURE SELECTION METHOD , d do 9: if (k < m) then 10: if ( \ NQMI(f ∗ j ,f ∗ \j) \ NQMI(f ∗ j−1,f ∗ \j−1) < τ) then 11: S ←{S ∪f ∗ j } 12: k ←k + 1 13: end if 14: else 15: break 16: end if 17: end for 5: sort features {f1, ..., fd} according to their \ NQMI in descending order resulting in {f ∗ 1 , ..., f ∗ d } S {S ∪f ∗} The inputs of the algorithm are a set of features, {f1, ..., fd}, the number of needed features, m, and a threshold, τ. While the output is a subset of selected features S = {f ∗ 1 , . . . , f ∗ m}. The algorithm starts by Indonesian J Elec Eng & Comp Sci, Vol. 22, No. 3, June 2021 : 1619 – 1628 1623 Indonesian J Elec Eng & Comp Sci ISSN: 2502-4752 Ì computing the normalisation of LSQMI as (2) for all features (lines 2 - 4). Then, the features are sorted in descending order according to their score (line 5). Lastly, the features are selected according to the ranking; a feature with the highest score is firstly selected and the feature with the second high score is then selected and so on. However, the redundant features are ignored by discarding the feature that has the normalisation of LSQMI score closed to the previous one. In other words, if the ratio of the current score and the previous one is higher than the given threshold τ, (line 10), the feature according to the smaller score is ignored. The algorithm is repeated until the m features (lines 8 - 17) are obtained. computing the normalisation of LSQMI as (2) for all features (lines 2 - 4). Then, the features are sorted in descending order according to their score (line 5). Lastly, the features are selected according to the ranking; a feature with the highest score is firstly selected and the feature with the second high score is then selected and so on. However, the redundant features are ignored by discarding the feature that has the normalisation of LSQMI score closed to the previous one. 4.1. Synthetic data In the first experiment, we would like to demonstrate the reliability of the LSQMI comparing to the competitive methods. Specifically, we would like to show you that the LSQMI is more reliable for evaluating both the informative feature and the redundant features. To do so, we conducted a synthetic data of 3 clusters with 3-dimensionality illustrated in Figure 1. We generated 100 instances with standard deviation = 1 for each class. Figure 1 (a)-(c) shows that the 3rd feature is the most important for underlying clusters, while the 1st feature and the 2nd feature are redundancy, because they have the same mean. Therefore, if we would like to choose two features (i.e., m = 2), the first selected one should be the 3rd feature, and the second one may be the 1st feature or the 2nd feature. In other words, the set of selected features should not be the 1st feature and the 2nd feature, because they can capture only 2 clusters of the original data as shown in Figure 1 (a). (a) (b) (c) Figure 1. Relationship between features in the synthetic data: (a) Feature 1 vs. Feature 2, (b) Feature 1 vs. Feature 3, (c) Feature 2 vs. Feature 3 (b) (c) (a) (b) (a) (c) (b) Figure 1. Relationship between features in the synthetic data: (a) Feature 1 vs. Feature 2, (b) Feature 1 vs. Feature 3, (c) Feature 2 vs. Feature 3 Unsupervised feature selection with least-squares quadratic mutual information (Janya Sainui) 1624 Ì ISSN: 2502-4752 We ran all methods using this toy data, where each method was repeated 10 times with different random data. We found that UFSMI performs the worst, because the 3rd feature was not selected 4 times. LS missed selecting the 3rd feature 1 time, while the proposed method and MCFS always selected the 3rd feature. The average scores between each feature and the rest features obtained from each objective function are shown in Table 1, showing that our objective function, \ NQMI, tends to be more sensible than other ones. As can be seen, the \ NQMI score between the 3rd feature and the rest features is significantly highest, while the \ NQMI scores according to the 2nd feature and the 1st feature are close to each other. In contrast to other objective functions, the scores according to each feature are almost the same for all features. Indonesian J Elec Eng & Comp Sci, Vol. 22, No. 3, June 2021 : 1619 – 1628 4.1. Synthetic data Notice that the scores of LS showing in Tables 1 and 2 are the scores of (1 −Laplacian score) for each feature. Thus, as same as other objective functions, the feature with larger score is more important. Table 1. The average scores over 10 runs between the feature j and the rest of the features obtained from different objective functions using the synthetic data Objective functions feature 1 feature 2 feature 3 LS 0.9935 0.9934 0.9936 MI -9.3682 -9.3771 -9.3730 MCFS 0.7330 0.8010 0.9848 \ NQMI 0.7424 0.7404 1.9034 Table 1. The average scores over 10 runs between the feature j and the rest of the features obtained from different objective functions using the synthetic data Table 2. The average scores over 10 runs between the feature j and the rest of the features obtained from different objective functions after adding the noise feature (feature 4) Objective functions feature 1 feature 2 feature 3 feature 4 LS 0.9821 0.9818 0.9801 0.9704 MI -8.1727 -8.1980 -8.2047 -8.8783 MCFS 0.6872 0.7244 0.8226 0.9997 \ NQMI 0.3331 0.3415 0.8952 0.0167 he average scores over 10 runs between the feature j and the rest of the features obtained from different objective functions after adding the noise feature (feature 4) Table 2. The average scores over 10 runs between the feature j and the rest of the features obtained from different objective functions after adding the noise feature (feature 4) Next, we added a noise feature (the 4th feature) with mean = 0 for all clusters and standard deviation = 1 into the above toy data. We ran the experiment 10 times for each method again, and the results show that our method outperforms other methods including MCFS. Moreover, MCFS performs the worst in this experiment as it always selected the 4th feature, while the other three methods never selected the 4th feature. The average scores of each method are shown in Table 2, confirming that \ NQMI is more reliable than other objective functions. Notice that MCFS works well if the number of used eigenvectors is equal to the number of clusters; however, in an unsupervised scenario, we do not know the actual number of clusters. 4.2. UCI datasets Here, we evaluated the clustering performance after feature selection using six UCI benchmark datasets, namely Abalone, Sonar, Glass, Pima, Heart, and Cancer datasets. For each dataset, the number of clusters (c), the number of features (d), and the number of samples (n) are shown in Figure 2. For each run, we randomly chosen 90% samples from each dataset, then we performed feature selection. Finally, we performed clustering by the k-means algorithm; we ran the k-means algorithm 10 times with random initialization and chose the best solution with the minimum error rate. The clustering accuracy is evaluated. The results of each dataset are shown in Figure 2, showing the average accuracies over 10 runs at different number of seleced features, m. The results indicate that the proposed method (i.e., UFSLSQMI) almost performs better than the counterpart approaches. However, we cannot say that the proposed method is the best. Because, in practice, there is no method that works well for all data. Moreover, it is hard to explain the reasons why it works or it does not work. What we can say is that the proposed method can be an alternative for solving unsupervised feature selection problem that may work well for your data. Indonesian J Elec Eng & Comp Sci ISSN: 2502-4752 ISSN: 2502-4752 Indonesian J Elec Eng & Comp Sci Ì 1625 Ì (a) (b) (c) (d) (e) (f) Figure 2. The average of clustering accuracies over 10 runs on UCI datasets: (a) Abalone (c=10 ,d=8 , n=2493), (b) Sonar (c=2 ,d= 60, n=188), (c) Glass (c=6 ,d=9 , n= 193), (d) Pima (c= 2,d= 8, n= 692), (e) Heart (c=2 ,d=13 , n=244), (f) Cancer (c=2 ,d=9 , n=615) (b) (a) (a) (b) (c) (d) (d) (c) (e) (f) (f) (e) Figure 2. The average of clustering accuracies over 10 runs on UCI datasets: (a) Abalone (c=10 ,d=8 , n=2493), (b) Sonar (c=2 ,d= 60, n=188), (c) Glass (c=6 ,d=9 , n= 193), (d) Pima (c= 2,d= 8, n= 692), (e) Heart (c=2 ,d=13 , n=244), (f) Cancer (c=2 ,d=9 , n=615) 4.3. Discussion In the experiments, we compared the proposed unsupervised feature selection method with the other filter methods. All methods including the proposed method select the informative features from the rank of features according to the scores of the objective function. This means that the objective function plays an important role for this task. We discuss here each objective function as follows. Firstly, Laplacian score (LS) is computed by constructing of a nearest neighbor graph with the specific-defined number of nearest neighbors, k. This is the reason why the Laplacian score feature selection does not always perform well. In addition, as can be seen in the experiments on toy data (Table 1 and Table 2), the Laplacian scores of the most important feature and the noise feature are not significantly different from each other, this may cause the rank of features relating to the important features is unreliable. Unsupervised feature selection with least-squares quadratic mutual information (Janya Sainui) 1626 Ì ISSN: 2502-4752 Secondly, the mutual information (MI) is also computed based on the k-nearest neighbors graph. The results in Tables 1 and 2 demonstrate that this MI score does not describe the difference between the informative feature and the noise feature well. In other words, as same as LS, the MI scores of the important feature and the noise feature are not significantly different from each other. Thirdly, the MCFS score is also computed based on the k-nearest neighbor graph such that the suitable k effects the good score. Not only the parameter k, the MCFS score also needs the number of used eigenvectors that makes the MCFS score depend on these two parameters. As can be seen in the experiment on toy data with noise feature, the MCFS score of noise feature is unreliable; however, if we change the number of used eigenvectors to be 2 instead of 5, the MCFS score of noise feature becomes more reliable. This confirms that the MCFS score heavily depends on the number of used eigenvectors. Lastly, the proposed method utilizes the LSQMI that approximates by least-square method so that the cross-validation can be included, resulting in that the estimator of QMI is sensible. 4.3. Discussion As can be seen in Tables 1 and 2, the normalized LSQMI describe the structure of the original data as well; the LSQMI is highest if the feature is the most important as the 3rd feature of the toy data; in contrast, the LSQMI is smallest if the feature is the noise feature that provides no information about data like the 4th feature. The main drawback of the proposed method is the time complexity that is higher than other methods because of the cross-validation process. To sum up, the main problem of the filter based unsupervised feature selection methods is lacking of the ability to automatically choose the parameters included in their objective function so that the approximation of the objective function may be unreliable. Thus, we here propose to use the LSQMI for unsupervised feature selection, that is not only reliable for evaluating the important features but also for ignoring the redundant features. 5. CONCLUSION The goal of feature selection is to select a subset of features in order to reduce the dimensionality of the original data, while the structure of data should be preserved. There are two main points that are important in order to achieve this goal. Firstly, we need to select the most informative features underlying preserving the structure of the original data. Secondly, we have to remove the redundant features because they do not provide more information about data. However, the existing approaches may not select the important features because the use of unreliable objective functions. Moreover, they do not ignore the redundant features. In this paper, we focus on these two points by exploring the reliable objective function that is the most important for evaluating the informative features as well as the redundant features in an unsupervised manner. Specifically, we propose to use the least-squares quadratic mutual information (LSQMI) as the objective function to select the valuable features excluding the redundant features. The benefit of LSQMI is that the needed parameters can be chosen by cross validation resulting that the estimation of the objective function will be more reliable, while most of the other objective functions lack of the systematic way to choose their parameters. Through the experiments, we show that the LSQMI can be used to determine the important features including the redundant feature as well. Indonesian J Elec Eng & Comp Sci, Vol. 22, No. 3, June 2021 : 1619 – 1628 [5] Ma. Dash, K. Choi, P. Scheuermann, and H. Liu, ”Feature selection for clustering—a filter solution,” 2002 IEEE International Conference on Data Mining, 2002. Proceedings, 2002, pp. 115-122, doi: 10.1109/ICDM.2002.1183893. [1] S. B. 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Li, ”Unsupervised feature selection with structured graph optimiza- tion,” Proceedings of the 30th Conference on Artificial Intelligence, 2016, pp. 1302–1308, doi: 10.5555/3015812.3016004 . 5] S. S.-Fern´andez, J. A. C.-Ochoa, and J. Fco. M.-Trinidad, ”A review of unsupervised feature selectio methods,” Artificial Intelligence Review, vol. 53, pp. 907–948, 2020, doi: 10.1007/s10462-019-09682-y methods,” Artificial Intelligence Review, vol. 53, pp. 907–948, 2020, doi: 10.1007/s10462-019-09682-y. [16] S. Alelyani, J. Tang, and H. Liu, ”Feature selection for clustering: a review,” Data Cluster Algorithms Applications, pp.29-60, 2018. [16] S. Alelyani, J. Tang, and H. Liu, ”Feature selection for clustering: a review,” Data Cluster Algorithms Applications, pp.29-60, 2018. [17] M. Dash and Y. Ong, ”RELIEF-C: efficient feature selection for clustering over noisy data,” 2011 IEEE 23rd International Conference on Tools with Artificial Intelligence, 2011, pp. 869-872, doi: 10.1109/IC- TAI.2011.135. [18] V. M. Rao and V. N. Sastry, ”Unsupervised feature ranking based on representation entropy,” 2012 1st International Conference on Recent Advances in Information Technology (RAIT), 2012, pp. 421-425, doi: 10.1109/RAIT.2012.6194631. [19] J. G. Dy and C. E. Brodley, ”Feature selection for unsupervised learning,” Journal of Machine Learning Research, vol. 5, pp. 845–889, 2004. 0] P. Y. Lee, W. P. Loh, and J. F. Chin, ”Feature selection in multimedia: the state-of-the-art review,” Imag Vision Computing, vol. 67, pp. 29–42, 2017, doi: 10.1016/j.imavis.2017.09.004. 1] R. Varshavsky, A. Gottlieb, M. Linial, and D. Horn, ”Novel unsupervised feature filtering of biologic data,” Bioinformatics, vol.22, no. 14, pp. e507–e513, 2006, doi: 10.1093/bioinformatics/btl214. f pp [22] M.i Banerjee and N. R. Pal, ”Feature selection with SVD entropy: some modification and extension,” Information Sciences, vol. 264, pp. 118–134, 2014, doi: 10.1016/j.ins.2013.12.029. [23] S. S.-Fern´andez, J. F. Mart´ınez-Trinidad, and J. A. Carrasco-Ochoa, ”A new unsupervised spectral feature selection method for mixed data: a filter approach,” Pattern Recognition, vol. 72, pp. 314–326, 2017, doi: 10.1016/j.patcog.2017.07.020. [24] X. Zhu, Y. Wang, Y. Li, Y. Tan, G. Wang, and Q. Song, ”A new unsupervised feature selection algorithm using similarity-based feature clustering,” Computational Intelligence, vol. 35, no. 1, pp. 2-22, 2018, doi: 10.1111/coin.12192. [25] H. Wang, Y. Zhang, J. Zhang, T. Li, and L. Peng, ”A factor graph model for unsupervised feature selec- tion,” Information Sciences, vol. 480, pp. 144-159, 2019, doi: 10.1016/j.ins.2018.12.034. 6] K. Torkkola, ”Feature extraction by non-parametric mutual information maximization,” The Journal Machine Learning Research, vol. 3, pp. 1415-1438, 2003. g pp [27] J. Sainui and M. Indonesian J Elec Eng & Comp Sci, Vol. 22, No. 3, June 2021 : 1619 – 1628 REFERENCES Sugiyama, ”Direct approximation of quadratic mutual information and its application to dependence maximization clustering,” IEICE Transactions on Information and Systems, vol. E96-D, no. 10, pp. 2282-2285, 2013, doi: 10.1587/transinf.E96.D.2282. Unsupervised feature selection with least-squares quadratic mutual information (Janya Sainui) 1628 Ì ISSN: 2502-4752 [28] J. Sainui and M. Sugiyama, ”Unsupervised key frame selection using information theory and colour histogram difference,” International Journal of Business Intelligence and Data Mining, vol. 16, no. 3, pp. 324-344, 2020, doi: 10.1504/ijbidm.2020.106137. [28] J. Sainui and M. Sugiyama, ”Unsupervised key frame selection using information theory and colour histogram difference,” International Journal of Business Intelligence and Data Mining, vol. 16, no. 3, pp. 324-344, 2020, doi: 10.1504/ijbidm.2020.106137. [28] J. Sainui and M. Sugiyama, ”Unsupervised key frame selection using information theory and colour histogram difference,” International Journal of Business Intelligence and Data Mining, vol. 16, no. 3, pp. 324-344, 2020, doi: 10.1504/ijbidm.2020.106137. BIOGRAPHIES OF AUTHORS Janya Sainui received her BS and MS in Computer Science from Prince of Songkla University, Hat Yai, Songkhla, Thailand, in 2005 and 2009, respectively. She is currently a Lecturer in Computer Science at Prince of Songkla University, Thailand. She has conducted research on topics such as bitmap indexing, video indexing and detection, clustering and unsupervised dimension reduction. She is interested in algorithm and application of machine learning, especially, applying machine learning techniques to real world applications such as image/video processing, medical imaging, bioinformatics, as well as information retrieval. Chouvanee Srivisal is currently a Lecturer in Computer Science at Faculty of Science Prince of Songkla University, Thailand. She received her BS in Computer Science from Prince of Songkla University and MS in Information Technology from King Mongkut’s Institute of Technology Lad- krabang, Thailand in 1997 and 2002, respectively. She has interested in algorithm and application of machine learning that for conducting research on topics such as image processing and information retrieval.
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https://www.qeios.com/read/O0X1UB/pdf
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Review of: "Ancient Houses in Ben Tre City: A Multifaceted Approach to Preserve Artistic Architectural Heritage and Boost Local Tourism"
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Qeios, CC-BY 4.0 · Review, March 17, 2024 Review of: "Ancient Houses in Ben Tre City: A Multifaceted Approach to Preserve Artistic Architectural Heritage and Boost Local Tourism" Rute Matos1 Rute Matos1 Rute Matos1 1 Universidade de Evora 1 Universidade de Evora 1 Universidade de Evora Potential competing interests: No potential competing interests to declare. The topic is of great interest in terms of preserving a region's heritage and identity. The topic is of great interest in terms of preserving a region's heritage and identity. However, the structure of the article is not clear. It needs an introduction that includes: It needs an introduction that includes: - Contextualisation of the article. Why and how does it come about? - Its objectives. What is the aim of this article? - The methodology used - The methodology used - The results obtained or expected The methodology is missing. The bibliographical research could be more consistent and broader. Although there is no specific information on the Ben Tre City case, information could be used by analogy; for example, what has been done in the French neighbourhood of Ha Noi or in Hoi An city. Both are examples of heritage and identity preservation with touristic and locals utilisation, which are widely recognised and publicised. There is a lack of overall images. There is a lack of urban cartography that allows us to read the relationship between the houses and the existing urban fabric, as well as the relationship/articulation between the houses and the surrounding space and landscape. Results are lacking. If there are no results at the end, those expected at the conclusion of this article should be presented. For example, the completion of the aforementioned inventory could be a result, as well as a proposal for the use/dissemination of these houses and their ensemble. Of this landscape space. Qeios ID: O0X1UB · https://doi.org/10.32388/O0X1UB 1/1
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https://bmjopen.bmj.com/content/bmjopen/12/1/e053327.full.pdf
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Adoption and continued use of mobile contact tracing technology: multilevel explanations from a three-wave panel survey and linked data
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Strengths and limitations of this study Objective  To identify the key individual-­level (demographics, attitudes, mobility) and contextual (COVID-­19 case numbers, tiers of mobility restrictions, urban districts) determinants of adopting the NHS COVID-­19 contact tracing app and continued use overtime. Design and setting  A three-­wave panel survey conducted in England in July 2020 (background survey), November 2020 (first measure of app adoption) and March 2021 (continued use of app and new adopters) linked with official data. ►Our data captures reported behaviour at two points to assess within-­subject changes over time. ►Our results are based on a large, nationally repre- sentative sample, as opposed to the convenience and limited-­N samples of previous contact tracing studies. on October 23, 2024 by guest. P http://bmjopen.bmj.com/ 17 January 2022. Downloaded from ►Integrating demographic/structural and attitudinal explanations relating to technology acceptance with questions adopted from the results of a deliberative poll. ►Prepublication history and additional supplemental material for this paper are available online. To view these files, please visit the journal online (http://dx.doi.org/10.1136/​ bmjopen-2021-053327). Participants  N=2500 adults living in England, representative of England’s population in terms of regional distribution, age and gender (2011 census). Primary outcome  Repeated measures of self-­reported app usage. Participants  N=2500 adults living in England, representative of England’s population in terms of regional distribution, age and gender (2011 census). Primary outcome  Repeated measures of self-­reported app usage. ►Studied population is England (see section 2.3) where overall mobility is restricted in wave 3 during national lockdown, allowing for limited opportunities for app usage for example, venue check-­ins.i Received 11 May 2021 Accepted 23 December 2021 Analytical approach  Multilevel logistic regression linking a range of individual level (from survey) and contextual (from linked data) determinants to app usage. Results  We observe initial app uptake at 41%, 95% CI (0.39% to 0.43%), and a 12% drop-­out rate by March 2021, 95% CI (0.10% to 0.14%). We also found that 7% of nonusers as of wave 2 became new adopters by wave 3, 95% CI (0.05% to 0.08%). Initial uptake (or failure to use) of the app associated with social norms, privacy concerns and misinformation about third-­party data access, with those living in postal districts with restrictions on mobility less likely to use the app. Perceived lack of transparent evidence of effectiveness was associated with drop-­out of use. In addition, those who trusted the government were more likely to adopt in wave 3 as new adopters. 1Politics, Birkbeck College, University of London, London, UK 2Politics, University of Exeter, Exeter, UK 3Institute for Community Research and Development, University of Wolverhampton, Wolverhampton, UK 4School of Geography, Politics and Sociology, Newcastle University, Newcastle upon Tyne, UK 5Faculty of Laws, UCL, London, UK 6Sociology, Philosophy and Anthropology, University of Exeter, Exeter, UK Correspondence to Dr Laszlo Horvath; ​L.​Horvath@​bbk.​ac.​uk © Author(s) (or their employer(s)) 2022. Re-­use permitted under CC BY. Published by BMJ. sending users a notification of possible expo- sure to the virus, along with health advice. Public acceptance is key to efficiency: Several studies have shown that the app’s ability to suppress the epidemic depends on the level of overall uptake. An early estimate indi- cated app usage of 56%1 could have helped to avoid a second nationwide lockdown in the UK. Another study indicated 15% uptake would decrease the death toll if combined with effective human contact tracing.2 On the other hand, the rejection of contact tracing apps by some may suggest that the govern- ment failed to secure the public’s trust that is crucial for compliance with restrictions on mobility and social contact.3 4 ober 23, 2024 by guest. Protected by copyright. Current evidence about who uses contact tracing apps and why is limited in several ways. First, prior to their introduction, studies were only able to measure intention to use apps because they had not been devel- oped and rolled out.5 The studies relied on To cite: Horvath L, Banducci S, Blamire J, et al. Adoption and continued use of mobile contact tracing technology: multilevel explanations from a three-­wave panel survey and linked data. BMJ Open 2022;12:e053327. doi:10.1136/ bmjopen-2021-053327 Original research Original research Original research Open access on October 23, 2024 by guest. Protected by http://bmjopen.bmj.com/ BMJ Open: first published as 10.1136/bmjopen-2021-053327 on 17 January 2022. Downloaded from BMJ Open: first published as 10.1136/bmjopen-2021-053327 on 17 Janu Adoption and continued use of mobile contact tracing technology: multilevel explanations from a three-­wave panel survey and linked data Adoption and continued use of mobile contact tracing technology: multilevel explanations from a three-­wave panel survey and linked data http://bmjo blished as 10.1136/bmjopen-2021-053327 on 17 January 2022. Downloaded from Laszlo Horvath  ‍ ‍ ,1,2 Susan Banducci,2 Joshua Blamire,3 Cathrine Degnen,4 Oliver James,2 Andrew Jones,5 Daniel Stevens,2 Katharine Tyler6 on October 23, 2024 by guest. Protected by copyright. http://bmjopen.bmj.com/ 36/bmjopen-2021-053327 on 17 January 2022. Downloaded from 1Politics, Birkbeck College, University of London, London, UK 2Politics, University of Exeter, Exeter, UK 3Institute for Community Research and Development, University of Wolverhampton, Wolverhampton, UK 4School of Geography, Politics and Sociology, Newcastle University, Newcastle upon Tyne, UK 5Faculty of Laws, UCL, London, UK 6Sociology, Philosophy and Anthropology, University of Exeter, Exeter, UK Correspondence to Dr Laszlo Horvath; ​L.​Horvath@​bbk.​ac.​uk © Author(s) (or their employer(s)) 2022. Re-­use permitted under CC BY. Published by BMJ. Strengths and limitations of this study Conclusions  Successful uptake of the contact tracing app should be evaluated within the wider context of the UK Government’s response to the crisis. Trust in government is key to adoption of the app in wave 3 while continued use is linked to perceptions of transparent evidence. Providing clear information to address privacy concerns could increase uptake, however, the disparities in continued use among ethnic minority participants needs further investigation. ►Drawing on our findings, an ethnic minority booster sample will in the future allow us to better under- stand inequalities across and within diverse ethnic populations. Subjects, setting and data linkage Our panel vendor is ORB International. We use a sample of 2500 respondents across three waves of data collection. We consulted the vendor and planned attrition so that the first wave of surveys were completed by 5000 respondents in July 2020, the second wave by 3700 in November 2020, and the final wave by 2500 in March 2021—consisting of those who completed all three surveys. Our study of adop- tion is embedded in a larger population survey about people’s lived experience in ‘Brexit-­Covid-­19 Britain (For more information, see https://brexit-studies.org/covid-​ 19)’ thus our sample size is not determined by power calculations for this specific study. Other than compli- ance with quota sampling demographics (managed by panel vendor) and participation in all three waves, there were no exclusion criteria for this study. While the NHS COVID-­19 app is used by citizens living in England and Wales, we needed to restrict our study to England’s popu- lation on the funder’s request (We used the Revised Stan- dards for Quality Improvement Reporting Excellence (SQUIRE) reporting guidelines12). g p y We report uptake at 41% in November 2020 with a 12% drop-­out rate by March 2021, and that 7% of non-­ adopters in November 2020 had installed the app by March 2021. Of the predictors of the uptake, we find that individual-­level attitudinal measures best capture the reasons why some adopted the technology while others have not (privacy and norms). We also report concern and misinformation about third party data access among nonusers, and that trust in government was a significant predictor of new adoption in March 2021 which, we spec- ulate, could be related to the severity of the January– March 2021 wave and/or perceptions about the UK Government’s early success in its vaccination programme. Explaining continued use specifically, we highlight the role of perceived usefulness and concern about trans- parent evidence. on October 23, 2024 by guest. Protected by copyright. mjopen.bmj.com/ We provide an overview of the study design in figure 1 below. Matching the survey dates and respondents’ self-­reported postal districts (first part of postcodes), we merged COVID-­19 cases data, regional closure and restrictions data (three-­tier system overlapping with wave 2), and urban/rural neighbourhood data from external sources, as detailed below. Subjects, setting and data linkage Our data linkages are proba- bilistic as neither units of analyses across the official data constitute an exact match to postal districts, however, asking for more granular location data (eg, postal area) from our respondents would have potentially compro- mised privacy. ber 23, 2024 by guest. Protected by copyright. METHODS S j In this study, our objective is to address these limita- tions with a large-­scale multiwave study in England, drawn from a probability-­based research panel, with represen- tative sample demographics. We measured adoption of contact tracing apps first in November 2020 and again in March 2021. To explain adoption and continued use, we link data from this survey (demographics, attitudes and reported behaviour) with their postal districts’ COVID-­19 case numbers, urban versus rural majority population, as well as policy restrictions on social gatherings and mobility. Our model specification is informed by the liter- ature on Technology Acceptance particularly of health technology, trust and findings from a deliberative public forum with UK residents.10 Our predictor on views of the app in respondents’ social networks echoes additional fieldwork insights reported recently.11 INTRODUCTION As a tool in national COVID-­19 Track and Trace systems, mobile contact tracing apps automate the process of contact tracing by Horvath L, et al. BMJ Open 2022;12:e053327. doi:10.1136/bmjopen-2021-053327 1 Open access on October 23, 2024 by guest. Protected by http://bmjopen.bmj.com/ BMJ Open: first published as 10.1136/bmjopen-2021-053327 on 17 January 2022. Downloaded from experimental scenarios looking at the potential proper- ties of the apps that could influence adoption, such as data storage and sponsors.6 7 Second, of the limited obser- vational evidence available, studies have been restricted to convenience sampling which tends to overestimate adop- tion rates.8 A recent study looking at user feedback on Google Play fails to capture nonusers entirely.9 Important qualitative work has identified key areas of citizen concern (eg, transparency and the needs of vulnerable groups10 or social norms or pressure11) but the distribution of these concerns remains to be investigated at the national level. Third, studies have been limited to exploring adoption at a single time point given the relatively short time since the roll-­out of the technology in many countries. Continued use and drop-­out rates, thus, remain to be investigated. We include additional notes about our theoretical expec- tations of the relevant predictors in section 2.3. to the virus being determined locally on the users’ phones, minimising data sharing (see also ‘Third party data access’ under section 2.3 for additional details). In prac- tice, this also means that, as opposed to direct interven- tion by NHS Test and Trace (human contact tracing or receipt of a positive test result), there is ‘no legal duty’ to self-­isolate if instructed by the app (see https://faq.​ covid19.nhs.uk/article/KA-01398/ and https://www.nhs.​ uk/conditions/coronavirus-covid-19/self-isolation-and-​ treatment/if-youre-told-to-self-isolate-by-nhs-test-and-​ trace-or-the-covid-19-app/). The advice to self-­isolate and count-­down lasts for ten days after predicted exposure. Additional features of the app include routine venue check-­ins (pubs, restaurants), local public health advice, a symptom checker—encouraging continued use. http://bmjop ublished as 10.1136/bmjopen-2021-053327 on 17 January 2022. Downloaded from Contact tracing in the UK The roll-­out of the government-­backed NHS COVID-­19 app on 24 September 2020, by National Health Services (NHS) England and Wales, makes the UK a relatively late adopter of digital contact tracing, 6 months behind their first recorded use globally (Singapore) and 4 months behind the first adopters in Europe (Italy and France). It is built on a decentralised system, with potential exposure Coronavirus cases are published on the UK Govern- ment’s official website, and updated on a weekly basis (week’s end) on the Middle Layer Super Output Area (MSOA) level. While sometimes MSOAs are entirely contained within a district constituting an exact match, often a number of these overlap with a district. For simplicity, we link data from the largest overlapping 2 Horvath L, et al. BMJ Open 2022;12:e053327. doi:10.1136/bmjopen-2021-053327 p Figure 1  Research design. on October 23, 2024 by guest. Protected by copyright. http://bmjopen.bmj.com/ irst published as 10.1136/bmjopen-2021-053327 on 17 January 2022. Downloaded from http://bmjopen shed as 10.1136/bmjopen-2021-053327 on 17 January 2022. Downloaded from Figure 1  Research design. launched an NHS contact tracing app for England and Wales that will notify you if you have been in close contact with someone who has tested positive for Covid-­19. Are you using this app? MSOA in terms of population wherever there is ambiguity (For example, people living in an EX4 postcode may be counted across thirteen MSOAs but the percentage overlap ranges between one per cent (Mid Devon 10) to 100% of MSOA located within EX4 (Exeter 002) thus we merged it with the latter. We tried an alternative method of estimating case numbers by weighting the MSOA totals according to the proportion of overlap with postcode districts and this produced similar results.). Based on the responses submitted in wave 2, we split the sample for analysis in wave 3 to examine continued use separately from new adoption, see section 2.6. Predictors Tiers data were published on the UK Government’s official website when changes occurred, with restrictions applied on the local authority district (LAD) level. Postal districts were linked to LADs and a tier assigned where a postcode was situated wholly within a single tier. Where a postcode overlapped with LADs assigned to different tiers, we used the respondent’s self-­reporting of their tier. For exact question wording and additional information about these variables, see data availability statement. Demographics: wave 1 Among standard sociodemographic questions were age, gender, education level, and identification with a list of 14 ethnic minority groups (including ‘mixed’) in addition to ‘Whites’, following the recommendation by the Office of National Statistics England-­specific list (https://www.​ ons.gov.uk/methodology/classificationsandstandards/​ measuringequality/ethnicgroupnationalidentityandre​ ligion). Location is provided as the first section of the postcode (first three to four digits), which we refer to as the postal district. Official urban–rural classification data (Census 2011) is available on the more granular Output Area level that are linked to postcodes by the ONS. We aggregated these into districts comprising a number of urban and rural loca- tions, of which we took the modal category for merging with our dataset. on October 23, 2024 by guest. Protected by copyright. mjopen.bmj.com/ The linked, final dataset is publicly available, see also data availability statement.1 App attitudes: wave 2 Patient and public involvement The first set of attitudinal questions, measured on five-­ point agreement scales about the app itself tap into four aspects of an extended version of the Technology Accep- tance Model to health tech including apps and wearable devices14 (These variables are subject to missingness, see 2.4 Analytical framework and online supplemental appendix figure A1): Patients or the public were not involved in the design, conduct, or reporting plans of this research. However, a range of public impact activities connected to the broader project about inequalities linked to COVID-­19 in Britain will include a summary of this research. Third-party data access: wave 2 Third-party data access: wave 2 We asked both users and nonusers ‘Who do you think will have access to the data collected by the NHS COVID-­19 app?’ In response, they could use a check- list of up to eight items or ‘none of these.’ The parties were listed as follows: the NHS, UK Government, Local health authority, UK Police, Apple, Google, Your telecommunications provider, Your internet network provider. For simplicity, we use concern about privacy (see above) in our complex multilevel models predicting app usage and scrutinise privacy further in section 3.3 (see the Results section) using this measure separately. The app’s primary purpose is the automa- tion of contact tracing locally on phone, third-­party access is kept to the minimum by sharing anonymised data only. Apps match a list of ‘broadcast codes’ and venues encountered by the app with a list curated by public health officials showing evidence of infection.15 y p p ►The number of new cases recorded by the end of the week while the survey was in the field, available for all waves. on October 23, 2024 by guest. Prot http://bmjopen.bmj.com/ nuary 2022. Downloaded from ►The temporary local restriction tiers at the time of wave 2 coded tier 1 (medium alert), tier 2 (high alert), tier 3 (very high alert). ►The dichotomous urban location measure derived from ONS Rural-­Urban Classifications data (in which a location is classified urban if 74% or more of the resident population living in urban areas). Trust: all waves In each survey wave, we asked about general trust in government on a 0–10 scale; we predict wave 2 adoption with wave 2 trust and wave 3 continued use with wave 3 trust. We expect that the government’s ability to gain and maintain its citizens’ trust will motivate uptake of contact tracing apps.3 4 Analytical approach We combine the measures listed above in two sets of multilevel logistic regression models, first estimating adoption at wave 2, then depending on wave 2 response either continued use in wave 3 or new adoption in wave 3. In all cases, we first fit null models estimating variance of uptake across second-­level units (postal districts) as random intercepts, and continue to add individual and district-­level predictors as appropriate. We scale and mean-­centre all continuous predictors. As we observed a pattern of non-­random missingness on the attitudinal predictors, we carried out multiple imputation and pooled the estimates across five imputed datasets, see online supplemental appendix for further details (As we show in online supplemental appendix figure A1), missing data particularly on ease of use is related to non-­ adoption thus exclusion of these cases would be inappro- priate. In practice, omitting these observations had little impact on our initial uptake and new uptake models, but had an effect on the significance of three technology acceptance variables in the continued use model.). For parsimony, we analyse respondents’ perceptions of third-­ party data access by adoption in section 3.3 separately as their inclusion in the regression models would add eight additional categorical predictors relating to a similar underlying concept (privacy). Outcomes pp g ►The app’s perceived ease of use (or judgement whether it would be easy to use if respondent has not adopted the app yet). The dependent variable is adoption measured first in wave 2, shortly after the app’s roll-­out on 24 September 2020 by the Department of Health and Social Care; and again, in wave 3. We provided the following description along with a close-­ended question: ►Its perceived usefulness to slow the spread of the virus. ►Whether and how concerned respondent is about privacy when using the app. Contact tracing is a tried and tested method used to slow down the spread of infectious diseases. Contact tracing can be done by public health officials or dig- itally with mobile phone applications or wearable devices. On the 24th of September, the government ►Social norms in terms of whether people in respond- ents’ social networks think it is a good idea to use the app. pp The second set of questions expand on the above with questions adopted from the results of the qualitative work Horvath L, et al. BMJ Open 2022;12:e053327. doi:10.1136/bmjopen-2021-053327 3 Second-level (postal district) variables Second-level (postal district) variables The procedure of data linkage is described in section 2.3. District-­level characteristics such as case numbers, stricter local lockdowns or higher population density in urban and metropolitan locations may affect overall anxiety and uncertainty that can generate more compliance with health interventions. We include the following measures varying across respondents’ postal districts: Compliance: all waves Compliance: all waves Across a set of 20 questions, we asked about the ways in which respondents have been affected by the corona- virus. One of these options was ‘Have worn a face mask when out in public,’ which we use as a proxy for compli- ance with other non-­pharmaceutical public health interventions to control the spread of COVID-­19. on October 23, 2024 by guest. Protected by copyright. http://bmjopen.bmj.com/ s 10.1136/bmjopen-2021-053327 on 17 January 2022. Downloaded from ►Whether respondent needs transparent evidence that the app is indeed effective. ►Whether respondent needs further information about how the app treats and uses data. ►Whether respondent needs further information about how the needs of vulnerable groups (eg, older age) are addressed. Open access on October 23, 2024 by guest. Pro http://bmjopen.bmj.com/ BMJ Open: first published as 10.1136/bmjopen-2021-053327 on 17 January 2022. Downloaded from of app users8 on consultation with the study’s authors. The study mode was a ‘rapid online discussion’ event with a deliberative format (deliberative poll) where 28 members of the public were selected to discuss and consider a variety of viewpoints about the app while crys- tallising their own opinions. The additional questions in our survey reflect the concerns that emerged from this event (p. 4)8 and are similarly measured on five-­point scales: mobile (followed 100% or mostly complied with stay at home) and those who likely remained mobile (those who did not/could not comply in addition to those who did not need to comply). Uptake and geographical variation We observe uptake in 2020 November at 41%, 95% CI (0.39% to 0.43%). Of the initial adopters, 124 or 12% of respondents no longer said they used the app by wave 3, 95% CI (0.10% to 0.14%); while of those initially not adopting, 98 respondents or 7% reported usage by wave 3, 95% CI (0.05% to 0.08%) (including five who responded with ‘Don’t know’ in wave 2). Of those not using the app in wave 2, 36%, 95% CI (0.34% to 0.38%) reported that they did not own a suitable device (This might seem high, but given (1) that iPhone 6 and earlier as well as Android 6 (Marshmallow) and earlier phones cannot run the app (https://faq.covid19.nhs.uk/article/KA-01116/en-us); (2) the high level of misinformation we report about third party data access among nonusers and (3) that notable segments of the UK population perceive the Govern- ment’s COVID-­19 communication as ‘low’ in clarity according to recent reports,17 we think it 36% of nonusers may very well have concerns about device compatibility), 1% (16 people) that they were discouraged to use it by their employer (eg, reports in September 2020 confirmed that police officers were asked not to instal app on work phones or ignore advice on personal phones, see https:// www.bbc.co.uk/news/technology-54328644), while the rest may be linked with other reasons including what we report in Section 3.2. Although not part of our theoret- ical framework, we note that respondents who had the coronavirus were just as likely to be using the app as not using it: 2.39% (95% CI 1.61% to 3.53%) and 2.71% (95% CI 1.99% to 3.69%), respectively. By wave 3, people who had the coronavirus were users of the app in only slightly higher proportion than non-­users, 3.41% (95% CI 2.44% to 4.75%) and 2.82% (95% CI 2.10% to 3.80%), respectively, with 95% CIs. on October 23, 2024 by guest. Protected by copyright. http://bmjopen.bmj.com/ bmjopen-2021-053327 on 17 January 2022. Downloaded from Open access on October 23, 2024 by guest. Protected by http://bmjopen.bmj.com/ BMJ Open: first published as 10.1136/bmjopen-2021-053327 on 17 January 2022. Downloaded from across postcode districts, SD=0.34, shown in figure 2. This is similar to the magnitude of mobility and compliance effects and about half of the magnitude of the most influ- ential attitudinal effects. By contrast, we find no variation across districts in wave 3 either for continued use or for new adoption. We explain this by comparing the two time points in terms of mobility and social contact: while in November 2020, these districts belonged to different tiers of restrictions (less open districts in tiers 2 and 3 with lower mobility thus lower adoption), in March 2021 all districts faced similar restrictions under a national lock- down. Beyond local tiers of restrictions, we find little evidence that COVID-­19 case numbers influenced adop- tion but we found that initial enthusiasm to adopt the app was higher in urban locations. Mobility: all waves ber 23, 2024 by guest. Protected by copyright. As for behavioural predictors, we asked a set of ques- tions about stay at home orders including ‘working from home.’ This predictor draws on an influential contact study showing that high infection rates particu- larly in disadvantaged neighbourhoods were explained by mobility patterns due to these residents’ inability to work from home.16 The response options were ‘followed 100%’, ‘mostly complied’, ‘mostly not complied’, ‘was not possible to comply’ and ‘does not apply to me’. We used working from home as a proxy of more substantial and regular mobility (for work rather than recreational purposes). We dichotomised this measure so that we obtained a group of respondents who were likely not Horvath L, et al. BMJ Open 2022;12:e053327. doi:10.1136/bmjopen-2021-053327 4 Individual-level predictors W id i We provide an overview of the results visually in figure 3 below, and summarise all fitted models and list ORs in online supplemental appendix table A1. The individual-­ level predictors of initial adoption in wave 2 are drawn from a multilevel model accounting for the postal district-­ level variation as shown above. The individual-­level predic- tors of wave 3 continued use (subset of respondents who were adopters in wave 2) and of new adoption (subset of respondents who were non-­adopters in wave 2) are drawn from simpler linear models as we found no comparable variation on the postal district-­level (In both cases, the null model consisting of an intercept and random effects only, the variance component is either zero (singular) or would not reach convergence.) and thus multilevel modelling was not appropriate. on October 23, 2024 by guest. Protecte http://bmjopen.bmj.com/ nuary 2022. Downloaded from Attitudinal predictors, particularly technology accep- tance model variables, appear to be the most powerful predictors of adoption. When it comes to demographics, we find older respondents less likely to be adopters in both waves but not more likely to drop out of usage. on October 23, 2024 by guest. Protected by copyright. p://bmjopen.bmj.com/ Looking at the initial measure of adoption in wave 2, our random intercept model detects some variation Figure 2  Geographical variation of uptake percentage (left, plotted here across postal area eg, BH) and log odds and 95% CI of group-­level predictors. Figure 2  Geographical variation of uptake percentage (left, plotted here across postal area eg, BH) and log odds and 95% CI of group-­level predictors. Horvath L, et al. BMJ Open 2022;12:e053327. doi:10.1136/bmjopen-2021-053327 5 on October 23, 2024 by guest. Protected by http://bmjopen.bmj.com/ BMJ Open: first published as 10.1136/bmjopen-2021-053327 on 17 January 2022. Downloaded from http://bmjopen.bmj.com BMJ Open: first published as 10.1136/bmjopen-2021-053327 on 17 January 2022. Downloaded from Open access BMJ Open: first published as 10.1136/bmjopen-2021-053327 on 17 January 20 Figure 3  Individual-­level predictors: log odds and 95% CI. TAM, technology acceptance model. on October 23, 2024 by guest. Protected by copyright. http://bmjopen.bmj.com/ 6/bmjopen-2021-053327 on 17 January 2022. Downloaded from Figure 3  Individual-­level predictors: log odds and 95% CI. TAM, technology acceptance model. Education has an impact on new adoption in wave 3 only with respondents higher than the median education level more likely to opt into usage. Individual-level predictors W id i We find that ethnic minority respondents were somewhat less likely to be adopters and more likely to drop out of initial usage, as opposed to those identifying with the group ‘Whites’ only. The small group size (8.20% of the sample) is, however, reflected in the large uncertainty around the estimate not meeting conventional thresholds of statistical significance. The items adopted from the deliberative poll have only small impacts on initial adoption. We expected that concern about the lack of transparent evidence would predict non-­usage from the outset, but our results suggest that initial adopters of the app are more likely to think in these terms. When it comes to continued use, however, we find that those who had concern about the lack of transparent evidence were indeed more likely to drop out of usage. We find small effects regarding information on data usage with initial adopters who were more likely to miss this kind of information. We find negligible effects relating to the vulnerable groups steer. However, we find that concern about the needs of vulnerable groups is related to age and work status with retired respondents a little more likely to express concern, t(1316.4)=−3.18, p<0.01, mean difference of 0.20; and also people in the highest age group, t(1191.9)=−2.68, p<0.01, mean differ- ence of 0.13 on a five point scale. on October 23, 2024 by guest. Protected by copyright. mjopen.bmj.com/ g In terms of technology acceptance, we find that the perceived usefulness of digital contact tracing to slow the spread of the virus is not influential on initial adoption or wave 3 new adoption. When it comes to continued use, however, respondents who thought the app was useful were somewhat more likely to continue using it by wave 3. Related variables also have large effects: people concerned about privacy were less likely to adopt the app, while those who agreed that people in their social circles (family, friends, work) thought it was a good idea to use the app were also likely to adopt. See also section 3.2 for additional insights on privacy. Perceived or expected ease of use also appears important although the direction of causality is less clear; users exposed to the app may have become more confident in its usability. ober 23, 2024 by guest. Protected by copyright. Individual-level predictors W id i Of the rest of the individual-­level characteristics, we note that trust in the UK government, although not influential in the first decision to adopt was predictive of new adoption in wave 3. In terms of mobility, people not working from home were less likely to adopt in wave 2 which may be cause for concern, potentially making the 6 Horvath L, et al. BMJ Open 2022;12:e053327. doi:10.1136/bmjopen-2021-053327 6 Figure 4  Respondents’ perceptions of third-­party data access, wave 2. NHS, National Health Services. Open access on October 23, 2024 by guest. Protected by http://bmjopen.bmj.com/ BMJ Open: first published as 10.1136/bmjopen-2021-053327 on 17 January 2022. Downloaded from data collection (see https://www.adalovelaceinstitute.​ org/project/covid-19-digital-contact-tracing-tracker), strengthening our survey’s external validity. The observed drop-­out rate between waves 2 and 3 was 12%. We note that at the timing of our follow-­up survey, England was under national lockdown, thus mobility and social contact decreased overall, making the app less needed for regular use (eg, venue check-­ins). Similarly, while there was a lot more overall mobility in wave 2 during initial uptake, the linked contextual variables showed that the app was less used where there were some regional restric- tions. Notably, we found that new adoption (people not using the app in wave 2 but opting into usage in wave 3 of the survey, 7%) was facilitated by high trust in the UK government, in line with emerging literature linking trust to compliance during times of crisis.18 We speculate this is linked to the increased severity of crisis including new variants by wave 3, and perceptions about the UK govern- ment’s early success in its vaccination programme. y p g We found that attitudinal characteristics, notably social norms and privacy concerns, were powerful predictors of (non-­)adoption. With additional analysis, we also found that non-­users particularly overestimated the potential for third-­party data access including non-­health actors such as broadband and telecom providers. This contrasts with earlier experimental findings on intention to use in the UK7 which polled respondents in June 2020 thus well before the app’s roll-­out, documenting little concern about varying privacy features and or potential data breaches. We speculate these differences are, on the one hand, due to study design: In the experimental scenario, respondents were given complete and transparent infor- mation about the app’s data usage and storage settings, whereas in the field this information is likely to be more opaque, with citizens more risk-­averse. Third-party data access Further scrutinising privacy, we ask if users and non-­ users have different perceptions about who has access to the data collected by the app (We also asked about the kind of data collected by the app, but only from app adopters. The top answers were: over 60% of users correctly identified venue check-­ins; approximately 40% incorrectly identified exact location; approximately 20% correctly identified user-­provided health data and the same percentage incorrectly identified ‘contacts from phone.’). While above we found that overall ‘concern about privacy’ is a powerful predictor of non-­adoption, figure 4 below looking at third-­party data demonstrates how this concern may translate into perceptions about data access. on October 23, 2024 by guest. Protected by copyright. mjopen.bmj.com/ Among other variables, we found work-­related mobility is also associated with adoption16 which partially explains why indicators of structural inequality (eg, ethnicity) are less relevant. Yet, after controlling for mobility, ethnic minority participants are still slightly less likely to adopt, a phenomenon that might be better explained with targeted data collection with a larger pool of ethnic minority respondents. ber 23, 2024 by guest. Protected by copyright. While a plurality of both users and non-­users think the NHS has access to data collected by the app, stark differ- ences emerge when it comes to other parties such as the police or broadband providers producing a ¼–¾ split between users and non-­users. While in itself the relation- ship may not be causal, it is consistent with the model-­ based results above regarding privacy concern. Our findings contribute to the understanding of inequalities around the adoption and public acceptance of digital technologies supporting the public health response to the pandemic globally, extending beyond contract tracing to telemedicine, digital health passports or targeted public health messaging. To tap inequalities, Individual-level predictors W id i Indeed, in this study we found transparency of evidence about effective- ness was a concern that predicted drop-­out. On the other hand, the early versions of the app would have used an NHS-­centralised system as opposed to the final decen- tralised Exposure Notifications System created by Google and Apple jointly. While the latter has better overall privacy preserving characteristics, high public trust in the NHS may have mitigated concern about privacy more effectively. on October 23, 2024 by guest. Protecte http://bmjopen.bmj.com/ nuary 2022. Downloaded from Figure 4  Respondents’ perceptions of third-­party data access, wave 2. NHS, National Health Services. app less effective, but in wave 3, this effect is no longer significant. Mask wearing predicts adoption in wave 2 only, suggestive of masks becoming increasingly norma- tive and less contentious in the population as a whole. REFERENCES 1 Hinch R, Probert W, Nurtay A, et al. Effective configurations of a digital contact tracing APP: a report to NHSX, 2020. 2 Abueg M, Hinch R, Wu N. Modeling the combined effect of digital exposure notification and non-­pharmaceutical interventions on the COVID-­19 epidemic in Washington state. medRxiv 2020. 3 Lalot F, Heering MS, Rullo M, et al. The dangers of distrustful complacency: low concern and low political trust combine to undermine compliance with governmental restrictions in the emerging Covid-­19 pandemic. Group Processes & Intergroup Relations 2020;81:136843022096798. Twitter Laszlo Horvath @_lhorvath and Andrew Jones @andrewtjones79 Acknowledgements  We thank Cary Kind and The Ada Lovelace Institute for recommending questions to include from their deliberative poll. Acknowledgements  We thank Cary Kind and The Ada Lovelace Institute for recommending questions to include from their deliberative poll. 4 Mohammed A, Johnston RM, van der Linden C. Public responses to policy reversals: the case of mask usage in Canada during COVID-­19. Canadian Public Policy 2020;46:S119–26. Contributors  LH is the author responsible for the overall content as the guarantor. All authors contributed to the study concept and design, and the development of the questionnaire. LH, SB, AJ and DS have full access to the data and have performed analysis leading to the results reported in this paper. LH, SB, JB, CD, AJ, OJ, DS and KT contributed to the interpretation of results, writing and critical revisions. All authors approved the final version to be published and are accountable for all aspects of the work. y 5 Milsom L, Abeler J, Altmann SM. Survey of acceptability of app-­ based contact tracing in the UK, US, France, Germany and Italy [Internet]. OSF 2020. [ ] 6 Zhang B, Kreps S, McMurry N, et al. Americans' perceptions of privacy and surveillance in the COVID-­19 pandemic. PLoS One 2020;15:e0242652. 7 Horvath L, Banducci S, James O. Citizens’ Attitudes to Contact Tracing Apps. J Exp Polit Sci 2020;12:1–13. Funding  This research is funded by the Economic and Social Research Council as part of UK Research and Innovation’s rapid response initiative to COVID-­19. The project is entitled: ‘Identity, Inequality and the Media in Brexit-­Covid-­19-­Britain’ (Grant Ref: ES/V006320/1). Project website: https://www.brexit-studies.org/​ covid-19. 8 Panchal M, Singh S, Rodriguez-­Villegas E. Analysis of the factors affecting the adoption and compliance of the NHS COVID-­19 mobile application. medRxiv 2021. pp 9 Garousi V, Cutting D, Felderer M. CONCLUSION AND DISCUSSION Our result regarding general uptake, 41% in November 2020, is consistent with other reports that followed our Horvath L, et al. BMJ Open 2022;12:e053327. doi:10.1136/bmjopen-2021-053327 7 Open access BMJ Open: first published as 10.1136/bmjopen-2021-053327 on 17 January 2 we aimed at an explanatory model with an exhaustive set of demographic, attitudinal, behavioural and postal district-­level characteristics. 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. 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/. We acknowledge that our study is limited to a sample of English residents and thus not fully representative of app usage in both England and Wales, where the NHS COVID-­19 App was released; nor of health behaviour more generally across the UK. In England, overall mobility was restricted during wave 3 of our survey which allowed limited opportunities for app usage such as venue check-­ins (venues were closed). In addition, data collec- tion in the future would benefit from a booster sample of ethnic minority participants to better understand inequalities across diverse ethnic populations: Our results suggest there may be some ethnicity effects on adoption even after controlling for a range of other predictors, however, we lack sufficient numbers to investigate these mechanisms. http://bmjop blished as 10.1136/bmjopen-2021-053327 on 17 January 2022. Downloaded from ORCID iD Laszlo Horvath http://orcid.org/0000-0003-0606-1050 ORCID iD Laszlo Horvath http://orcid.org/0000-0003-0606-1050 ORCID iD Patient consent for publication  Not applicable. 12 Ogrinc G, Davies L, Goodman D, et al. Squire 2.0 (standards for quality improvement reporting excellence): revised publication guidelines from a detailed consensus process. BMJ Qual Saf 2016;25:986–92. Ethics approval  This study was approved by Prior to data collection, our research design received ethical approval from the University of Exeter College of Social Sciences and International Studies Ethics Committee on 16 July 2020, Certificate No. 201920-­131. Our panel vendor ORB International manages informed consent from their panel respondents. 13 Stevens D, Banducci S, Horvath L, et al. Identity, inequality, and the media in Brexit-­COVID-­19-­Britain surveys, waves 1-­3. figshare 2021. 14 Wang X, White L, Chen X. An empirical study of wearable technology acceptance in healthcare. Industrial Management & Data Systems 2015:1704–23. REFERENCES Mining user reviews of COVID contact-­tracing apps: an exploratory analysis of nine European apps. arXiv 2012:13589. Map disclaimer  The inclusion of any map (including the depiction of any boundaries therein), or of any geographic or locational reference, does not imply the expression of any opinion whatsoever on the part of BMJ concerning the legal status of any country, territory, jurisdiction or area or of its authorities. Any such expression remains solely that of the relevant source and is not endorsed by BMJ. Maps are provided without any warranty of any kind, either express or implied. 10 Ada Lovelace Institute. Confidence in a crisis? building trust in a contact tracing APP, 2020. Available: https://www.​ adalovelaceinstitute.org/wp-content/uploads/2020/08/Ada-Lovelace-​ Institute_COVID-19_Contact_Tracing_Confidence-in-a-crisis-report-​ 3.pdf on October 23, 2024 by guest. Protected by copyright. mjopen.bmj.com/ 11 Williams SN, Armitage CJ, Tampe T, et al. Public attitudes towards COVID‐19 contact tracing apps: a UK‐based focus group study. Health Expect 2021;24:377–85. Competing interests  None declared. Patient consent for publication  Not applicable. Provenance and peer review  Not commissioned; externally peer reviewed. Provenance and peer review  Not commissioned; externally peer reviewed. 15 Department of Health and Social Care. Nhs COVID-­19 APP: privacy notice. version 28 April 2021. Available: https://www.gov.uk/​ government/publications/nhs-covid-19-app-privacy-information/nhs-​ covid-19-app-privacy-notice Data availability statement  Data are available in a public, open access repository. The questionnaires, case summaries, as well as replication data are available on figshare: Stevens D, Banducci S, Horvath L, Jones A. Identity, Inequality, and the Media in Brexit-­COVID-­19-­Britain Surveys, Waves 1–3. figshare; 2021 (cited 9 May 2021). https://doi.org/10.6084/m9.figshare.14527188.V.1 y 16 Chang S, Pierson E, Koh PW, et al. Mobility network models of COVID-­19 explain inequities and inform reopening. Nature 2021;589:82–7. 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 17 Abrams D, Lalot F, Broadwood J, et al. Public perceptions of UK and local government communication about COVID-­19. Available: https:// www.belongnetwork.co.uk/wp-content/uploads/2021/08/Belong_​ PublicPerceptions_paper_V5.pdf p p p p 18 Bargain O, Aminjonov U. Trust and compliance to public health policies in times of COVID-­19. J Public Econ 2020;192:104316. 8 Horvath L, et al. BMJ Open 2022;12:e053327. doi:10.1136/bmjopen-2021-053327
https://openalex.org/W2093810977
https://figshare.com/articles/journal_contribution/PDC_Mediated_Tandem_Oxidative_8211_Wittig_Olefination/1009039/1/files/1476662.pdf
English
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PDC-Mediated Tandem Oxidative–Wittig Olefination
Synthetic communications
2,014
cc-by
2,583
2. General Procedure To a magnetically stired suspension of pyridinium dichromate (1.85 mmol), Wittig reagent (1.85 mmol) in anhyd CH2Cl2 (10 mL), alcohol (1.85 mmol) in anhyd CH2Cl2 (10 mL) was added in one portion. The reaction mixture was stirred at r.t. for 24h. Et2O (30 mL) was added to the reaction mixture and filtered. The residue was washed with ether (2 X 5 mL) and dried over anhydrous Na2SO4. The combined filtrate was then evaporated to give the crude product. Further purification by column chromatography using petroleum ether as an eluent gave the product. PDC mediated tandem oxidative-Wittig Olefination Kashinath L. Dhumaskar, Chinmay Bhat and Santosh G. Tilve* Department of Chemistry, Goa University, Taleigao Plateau, Goa 403 206, India *stilve@unigoa.ac.in Supporting Information 1. General remarks Flash chromatography was performed on silica gel (230-400 mesh). IR spectra were recorded on Shimadzu FT-IR spectrophotometer. 1H NMR (400 MHz) and 13C NMR (100 MHz) were recorded on a Bruker instrument. Chemical shift (ppm) are relative to the internal standard Me4Si (0 ppm). 3. Spectral data brown oil, yield 0.33g (99%); 1H NMR (400 MHz, CDCl3) δ = 1.252 (t, 3H, J = 7.2 Hz, CH3), 4.17 (q, 2H, J = 7.2 Hz, OCH2), 6.24 (d, 1H, J = 16 Hz, CH), 6.39 (m, 1H, Ar-H), 6.54 (d, 1H, Ar-H), 7.35 (d, 1H, J = 16 Hz, CH), 7.41 (d, 1H, Ar-H); 13C NMR (100 MHz, CDCl3); δ = 14.3 (CH3), 60.4 (OCH2), 112.2 (CH), 114.7 (CH), 115.9 (CH), 130.9 (CH), 144.7 (CH), 150.9 (Cq), 167.0 (Cq); max (thin flim); 1712, 1639, 1209 cm -1. (3E)-4-(furan-2-yl)but-3-en-2-one2 (3e). Yield, 0.27g (97%), Colourless oil; 1H NMR (400 MHz, CDCl3); δ = 2.265 (s, 3H, CH3), 6.42 (m, 1H, Ar-H), 6.55 (d, 1H, J = 16 Hz, CH), 6.59 (m, 1H, Ar-H), 7.19 (d, 1H, J = 16 Hz, CH), 7.43 (m, 1H, Ar-H); 13C NMR (100 MHz, CDCl3) δ = 27.6 (CH3), 112.5 (CH), 115.7 (CH), 124.1 (CH), 129.4 (CH), 145.0 (CH), 150.8 (Cq), 197.8 (Cq); max (thin flim); 1685, 1610, 1170 cm -1. (3E)-4-(furan-2-yl)but-3-en-2-one2 (3e). Yield, 0.27g (97%), Colourless oil; 1H NMR (400 MHz, CDCl3); δ = 2.265 (s, 3H, CH3), 6.42 (m, 1H, Ar-H), 6.55 (d, 1H, J = 16 Hz, CH), 6.59 (m, 1H, Ar-H), 7.19 (d, 1H, J = 16 Hz, CH), 7.43 (m, 1H, Ar-H); 13C NMR (100 MHz, CDCl3) δ = 27.6 (CH3), 112.5 (CH), 115.7 (CH), 124.1 (CH), 129.4 (CH), 145.0 (CH), 150.8 (Cq), 197.8 (Cq); max (thin flim); 1685, 1610, 1170 cm -1. Ethyl (2E)-4-(4-methoxyphenyl)but-2-enoate4 (3f). Yield 0.17g, (60%), Colourless oil; 1H NMR (400 MHz, CDCl3); δ = 1.20 (t, 3H, J = 7.2 Hz, CH3), 3.34 (d, 2H, CH2), 3.74 (s, 3H,OCH3), 4.15 (q, 2H, J = 7.2 Hz, OCH2), 5.76 (td, 1H, J = 16 Hz, J = 1.6 Hz, CH), 6.79 (m, 2H, Ar-H), 7.01(m, 3H, Ar-H); 13C NMR (100 MHz, CDCl3); δ = 14.3 (CH3), 37.6 (CH2), 55.2 (OCH3), 60.3 (OCH2), 114.1 (2 x CH), 122.1 (CH), 129.7 (Cq), 129.8 (2 x CH), 147.8 (CH), 158.4 (Cq), 166.6 (Cq); max (thin flim); 1716, 1604, 1174 cm -1 Ethyl (2E)-non-2-enoate1 (3g). 3. Spectral data Ethyl (2E)-3-phenylprop-2-enoate1 (3a). Yield 0.15g (90%), Colourless oil; 1H NMR (400 MHz, CDCl3); δ = 1.36 (t, 3H, J = 7.2 Hz, CH3), 4.29 (q, 2H, J = 7.2 Hz, CH2), 6.46 (d, 1H, J = 16 Hz, CH), 7.42-7.71 (m, 2H, Ar-H), 7.53-7.58 (m, 2H, Ar-H), 6.46 (d, 1H, J = 16 Hz, CH); 13C NMR (100 MHz, CDCl3); δ = 14.3 (CH3), 60.5(CH2), 118.3 (CH), 128.1 (2 x CH), 128.9 (2 x CH), 130.23 (CH), 134.5 (Cq), 144.6 (Cq), 167.0 (Cq). max (thin flim); 1705, 1637, 1203cm -1. Ethyl (2E)-3-(4-nitrophenyl)prop-2-enoate2 (3b). Yield 0.23g (82%), yellow solid; 1H NMR (400 MHz, CDCl3); δ = 1.28 (t, 3H, J = 7.2 Hz, CH3), 4.22 (q, 2H, J = 7.2 Hz, OCH2), 6.49 (d, 1H, J = 16 Hz, CH), 7.59-7.66 (m, 3H, Ar-H and CH), 8.18 (m, 2H, Ar-H), 13C NMR (100 MHz, CDCl3); δ = 14.3 (CH3), 60.0 (CH2), 122.6 (CH), 124.2 (2 x CH), 128.6 (2 x CH), 140.6 (Cq), 141.6 (CH), 148.4 (Cq), 166.2 (Cq). max (KBr); 1710, 1643, 1253 cm -1. Ethyl (2E)-3-(4-methoxyphenyl)prop-2-enoate3 (3c). Yield 0.24g (81%), Colourless oil; 1H NMR (400 MHz, CDCl3); δ = 1.25 (t, 3H, J = 7.2 Hz, CH3), 3.77 (s, 3H,OCH3), 4.17 (q, 2H, J = 7.2 Hz, OCH2), 6.24 (d, 1H, J = 16 Hz, CH), 6.83 (d, 2H, J = 8.8 Hz, Ar-H), 7.41 (d, 2H, J = 8.8 Hz, Ar-H), 7.575 (d, 1H, J = 16 Hz, CH), 13C NMR (100 MHz, CDCl3) δ = 14.3 (CH3), 55.2 (OCH3), 60.2 (CH2), 114.2 (2 x CH), 115.6 (CH), 121.3 (Cq),129.6 (2 x CH), 144.2 (CH), 161.3 (Cq), 167.4 (Cq); max (thin flim); 1707, 1602, 1170 cm -1. Ethyl (2E)-3-(4-methoxyphenyl)prop-2-enoate3 (3c). Yield 0.24g (81%), Colourless oil; 1H NMR (400 MHz, CDCl3); δ = 1.25 (t, 3H, J = 7.2 Hz, CH3), 3.77 (s, 3H,OCH3), 4.17 (q, 2H, J = 7.2 Hz, OCH2), 6.24 (d, 1H, J = 16 Hz, CH), 6.83 (d, 2H, J = 8.8 Hz, Ar-H), 7.41 (d, 2H, J = 8.8 Hz, Ar-H), 7.575 (d, 1H, J = 16 Hz, CH), 13C NMR (100 MHz, CDCl3) δ = 14.3 (CH3), 55.2 (OCH3), 60.2 (CH2), 114.2 (2 x CH), 115.6 (CH), 121.3 (Cq),129.6 (2 x CH), 144.2 (CH), 161.3 (Cq), 167.4 (Cq); max (thin flim); 1707, 1602, 1170 cm -1. Ethyl (2E)-3-(furan-2-yl)prop-2-enoate2 (3d). 3. Spectral data Ethyl (2E)-4-methylpent-2-enoate2 (3j). Yield 0.25g (66%); Colourless oil; 1H NMR (400 MHz, CDCl3); δ = 1.00 (d, 6H, J = 6.8 Hz, CH3), 1.22 (t, 3H, J = 7.2 Hz, CH3), 2.39 (m, 1H, CH), 4.12 (q, 2H, J = 7.2 Hz, CH2), 5.71 (dd, 1H, J = 16 Hz, J = 1.6 Hz, CH), 6.89 (dd, 1H, J = 16 Hz, J = 1.6 Hz, CH); 13C NMR (100 MHz, CDCl3) δ = 14.2 (CH3), 23.9 (CH), 29.8 (2 x CH3), 60.5 (OCH2), 117.9 (CH), 154.6 (CH), 167.0 (Cq); max (thin flim); 1705, 1654, 1157 cm -1. Ethyl (2E)-4-methylpent-2-enoate2 (3j). Yield 0.25g (66%); Colourless oil; 1H NMR (400 MHz, CDCl3); δ = 1.00 (d, 6H, J = 6.8 Hz, CH3), 1.22 (t, 3H, J = 7.2 Hz, CH3), 2.39 (m, 1H, CH), 4.12 (q, 2H, J = 7.2 Hz, CH2), 5.71 (dd, 1H, J = 16 Hz, J = 1.6 Hz, CH), 6.89 (dd, 1H, J = 16 Hz, J = 1.6 Hz, CH); 13C NMR (100 MHz, CDCl3) δ = 14.2 (CH3), 23.9 (CH), 29.8 (2 x CH3), 60.5 (OCH2), 117.9 (CH), 154.6 (CH), 167.0 (Cq); max (thin flim); 1705, 1654, 1157 cm -1. Ethyl (2E,4E)-5-phenylpenta-2,4-dienoate2 (3k). Yield 0.22g (73%); Colourless oil; 1H NMR (400 MHz, CDCl3); δ = 1.34 (t, 3H, J = 7.2 Hz, CH3), 4.25 (q, 2H, J = 7.2 Hz, OCH2), 6.01 (d, 1H, J = 16 Hz, CH), 6.90 (d, 1H, J = 8.4 Hz, CH), 7.3-7.50 (m, 7H, Ar-H and vinylic); 13C NMR (100 MHz, CDCl3) δ = 14.3 (CH3), 60.3 (OCH2), 121.3 (CH), 126.2 (CH), 127.4 (2 x CH), 128.8 (2 x CH), 129.0 (CH), 136.0 (Cq), 140.4 (CH), 144.6 (CH), 167.1 (Cq); max (thin flim); 1718, 1510, 1176 cm -1. Ethyl (2E)-penta-2,4-dienoate6 (3l). Yield 0.30g (70%); Colourless oil; 1H NMR (400 MHz, CDCl3); δ = 1.22 (t, 3H, J = 7.2 Hz, CH3), 4.16 (q, 2H, J = 7.2 Hz, CH2), 5.42 (d, 1H, J = 10 Hz, CH), 5.52 (dd, 1H, J = 17 Hz, 0.4 Hz, CH), 5.82 (dd, 1H, J = 15.2 Hz, 0.4 Hz, CH), 6.38 (m, 1H, CH), 7.19 (m, 1H, J = 11 Hz, CH); 13C NMR (100 MHz, CDCl3); δ = 14.2 (CH3), 60.6 (OCH2), 122.9 (CH), 125.5 (CH), 134.7 (CH), 144.7 (CH), 166.9 (Cq); max (thin flim); 1720, 1651, 1178 cm -1. Ethyl (2E)-pent-2-en-4-ynoate6 (3m). 3. Spectral data Yield 0.28g (87%), Colourless oil; 1H NMR (400 MHz, CDCl3); δ = 1.19-1.26 (m, 11H, C5H11), 1.38 (t, 3H, J = 7.2 Hz, CH3), 2.09 (m, 2H, CH2), 4.12 (q, 2H, J = 7.2 Hz, CH2), 5.73 (td, 1H, J = 16 Hz, 1.6 Hz, CH), 6.89 (td, 1H, J = 16 Hz, 6.8 Hz, CH); 13C NMR (100 MHz, CDCl3); δ = 14.04 (CH3), 14.25 (CH3), 22.54 (CH2), 27.96 (CH2), 28.99 (CH2), 31.43 (CH2), 32.19 (CH2), 60.11(CH2), 121.16 (CH), 149.5 (CH), 166.8 (Cq); max (thin flim); 1720, 1654, 1174 cm -1. Diethyl (2E,6E)-octa-2,6-dienedioate5 (3h). Yield 0.31g (62%); Colourless oil; 1H NMR (400 MHz, CDCl3); δ = 1.30 (t, 6H, J = 7.2 Hz, CH3), 2.39 (d, 4H,CH2), 4.24 (q, 4H, J = 7.2 Hz, OCH2), 5.87 (d, 1H, J = 16 Hz, CH), 6.95 (d, 1H, J = 16 Hz, CH); 13C NMR (100 MHz, CDCl3) δ = 14.2 (2 x CH3), 30.4 (2 x CH2), 60.3 (2 x OCH2), 122.3 (2 x CH), 133.6 (2 x Cq), 146.9 (2 x Cq); max (thin flim); 1720, 1654, 1182 cm -1 Diethyl (2E,4E)-hexa-2,4-dienedioate1 (3i). Yield 0.25 ( 40%); low melting solid; 1H NMR (400 MHz, CDCl3); δ = 1.32 (d, 6H, J = 6 Hz, CH3), 4.24 (t, 4H, J = 6 Hz, OCH2), 6.20 (m, 2H, CH), 7.32 (m, 2H, CH); 13C NMR (100 MHz, CDCl3) δ = 14.2 (2xCH3), 60.8 (2 x OCH2), 128.3 (2 x CH), 140.8 (2 x CH), 165.9 (2 x Cq); max (KBr); 1730, 1714, 1176 cm -1. Diethyl (2E,4E)-hexa-2,4-dienedioate1 (3i). Yield 0.25 ( 40%); low melting solid; 1H NMR (400 MHz, CDCl3); δ = 1.32 (d, 6H, J = 6 Hz, CH3), 4.24 (t, 4H, J = 6 Hz, OCH2), 6.20 (m, 2H, CH), 7.32 (m, 2H, CH); 13C NMR (100 MHz, CDCl3) δ = 14.2 (2xCH3), 60.8 (2 x OCH2), 128.3 (2 x CH), 140.8 (2 x CH), 165.9 (2 x Cq); max (KBr); 1730, 1714, 1176 cm -1. Diethyl (2E,4E)-hexa-2,4-dienedioate1 (3i). Yield 0.25 ( 40%); low melting solid; 1H NMR (400 MHz, CDCl3); δ = 1.32 (d, 6H, J = 6 Hz, CH3), 4.24 (t, 4H, J = 6 Hz, OCH2), 6.20 (m, 2H, CH), 7.32 (m, 2H, CH); 13C NMR (100 MHz, CDCl3) δ = 14.2 (2xCH3), 60.8 (2 x OCH2), 128.3 (2 x CH), 140.8 (2 x CH), 165.9 (2 x Cq); max (KBr); 1730, 1714, 1176 cm -1. 3. Spectral data Yield 0.25g (56%); Colourless oil; 1H NMR (400 MHz, CDCl3); δ = 1.35 (t, 3H, J = 7.2 Hz, CH3), 3.26 (s, 1H, CH) 4.14 (q, 2H, J = 7.2 Hz, OCH2), 6.25 (d, 1H, J = 16 Hz, CH), 6.64 (d, 1H, J = 16 Hz, CH); 13C NMR (100 MHz, CDCl3); δ = 14.2 (CH3), 60.95 (OCH2), 80.2 (Cq), 85.81 (CH), 123.9 (CH), 132.5 (CH), 164.4 (Cq); max (thin flim); 1720, 1651, 1178 cm -1. tert-Butyl 2-[(1E)-3-ethoxy-3-oxoprop-1-en-1-yl]pyrrolidine-1-carboxylate7 (3n). Yield, 0.21g ( 84%); [α]D 21=79 (c = 1 MeOH); Colourless oil; 1H NMR (400 MHz, CDCl3); δ = 1.21 (t, 3H, J = 7.2 Hz, CH3), 1.36 (s, 9H, CH3), 1.80 (m, 2H, CH2), 2.02 (m, 2H, CH2), 3.35 (t, 2H, J = 5.2 Hz, CH2), 4.12 (q, 2H, J = 7.2 Hz, OCH2), 4.352 (br, 1H, CH), 5.75 (d, 1H, J = 15.6 Hz, CH), 6.75 (dd, 1H, J = 15.6 Hz, J = 5.6 Hz, CH); 13C NMR (100 MHz, CDCl3) δ = 14.2 (CH3),22.9 (CH2), 28.2 (3 x CH3), 31.7 (CH2), 46.2 (CH2), 57.8 (CH), 60.4 (OCH2), 79.7 (Cq), 120.4 (CH), 148.5 (CH), 154.3 (Cq), 166.5 (Cq); max (thin flim); 1720, 1693, 1168 cm -1. (CH3),22.9 (CH2), 28.2 (3 x CH3), 31.7 (CH2), 46.2 (CH2), 57.8 (CH), 60.4 (OCH2), 79.7 (Cq), 120.4 (CH), 148.5 (CH), 154.3 (Cq), 166.5 (Cq); max (thin flim); 1720, 1693, 1168 cm -1. References 1. Shet, J.; Desai, V.; Tilve, S. Domino Primary Alcohol Oxidation-Wittig Reaction: Total Synthesis of ABT-418 and (E)-4-Oxonon-2-enoic Acid. Synthesis 2004, 11, 1859-1863. 2. Chintareddy, V. R.; Arkady, E.; Verkade, J. G. P[N(i-Bu)CH2CH2]3N: Nonionic Lewis Base for Promoting the 1. Shet, J.; Desai, V.; Tilve, S. Domino Primary Alcohol Oxidation-Wittig Reaction: Total Synthesis of ABT-418 and (E)-4-Oxonon-2-enoic Acid. Synthesis 2004, 11, 1859-1863. 2. Chintareddy, V. R.; Arkady, E.; Verkade, J. G. P[N(i-Bu)CH2CH2]3N: Nonionic Lewis Base for Promoting the Room-Temperature Synthesis of -Unsaturated Esters, Fluorides, Ketones, and Nitriles Using Wadsworth-Emmons Phosphonates. J. Org. Chem. 2010, 75, 7166–7174. 3. Cao, P.; Li, C-Y.; Kang, Y-B.; Xie, Z.; Sun, X-L.; Tang, Y. Ph3As-Catalyzed Wittig-Type Olefination of Aldehydes with Diazoacetate in the Presence of Na2S2O4. J. Org. Chem. 2007, 72, 6628- 6630 4. Narahashi, H.; Shimizu, I.; Yamamoto, A. Synthesis of benzylpalladium complexes through C–O bond cleavage of benzylic carboxylates: Development of a novel palladium-catalyzed benzylation of olefins. J. Organomet. Chem. 2008, 693, 283–296. 4. Narahashi, H.; Shimizu, I.; Yamamoto, A. Synthesis of benzylpalladium complexes through C–O bond cleavage of benzylic carboxylates: Development of a novel palladium-catalyzed benzylation of olefins. J. Organomet. Chem. 2008, 693, 283–296. 5. Phillips, D. J.; Pillinger, K. S.; Li, W.; Taylor, A. E.; Graham, A. E. Diol desymmetrization as an approach to the 5. Phillips, D. J.; Pillinger, K. S.; Li, W.; Taylor, A. E.; Graham, A. E. Diol desymmetrization as an approach to the synthesis of unsymmetrical dienyl diesters. Tetrahedron 2007, 63, 10528–10533. y y y , , 6. Barrett, A. G. M.; Hamprecht, D.; Ohkubo, M. Dess−Martin Periodinane Oxidation of Alcohols in the Presence of Stabilized Phosphorus Ylides: A Convenient Method for the Homologation of Alcohols via Unstable Aldehydes. J. Org. Chem. 1997, 62, 9376–9378. 7. Dieter, R. K.; Oba, G.; Chandupatla, K. R.; Topping, C. M.; Lu, K.; Watson, R. T. Reactivity and Enantioselectivity in the Reactions of Scalemic Stereogenic -(N-Carbamoyl)alkylcuprates. J. Org. Chem. 2004, 69, 3076-3086. 6. Barrett, A. G. M.; Hamprecht, D.; Ohkubo, M. Dess−Martin Periodinane Oxidation of Alcohols in the Presence of Stabilized Phosphorus Ylides: A Convenient Method for the Homologation of Alcohols via Unstable Aldehydes. J. Org. Chem. 1997, 62, 9376–9378. 6. Barrett, A. G. M.; Hamprecht, D.; Ohkubo, M. Dess−Martin Periodinane Oxidation of Alcohols in the Presence of Stabilized Phosphorus Ylides: A Convenient Method for the Homologation of Alcohols via Unstable Aldehydes. J. Org. Chem. 1997, 62, 9376–9378. 6. Barrett, A. References G. M.; Hamprecht, D.; Ohkubo, M. Dess−Martin Periodinane Oxidation of Alcohols in the Presence of Stabilized Phosphorus Ylides: A Convenient Method for the Homologation of Alcohols via Unstable Aldehydes. J. Org. Chem. 1997, 62, 9376–9378. 7. Dieter, R. K.; Oba, G.; Chandupatla, K. R.; Topping, C. M.; Lu, K.; Watson, R. T. Reactivity and Enantioselectivity in the Reactions of Scalemic Stereogenic -(N-Carbamoyl)alkylcuprates. J. Org. Chem. 2004, 69, 3076-3086. 4. Spectra 4. Spectra 4. Spectra Ethyl (2E)-3-phenylprop-2-enoate (3a). Ethyl (2E)-3-phenylprop-2-enoate (3a). COOEt COOEt O2N Ethyl (2E)-3-(4-nitrophenyl)prop-2-enoate (3b). O2N 2 Ethyl (2E)-3-(4-nitrophenyl)prop-2-enoate (3b). MeO O O   Ethyl (2E)-3-(4-methoxyphenyl)prop-2-enoate (3c). MeO Ethyl (2E)-3-(4-methoxyphenyl)prop-2-enoate (3c). O COOEt   Ethyl (2E)-3-(furan-2-yl)prop-2-enoate (3d). O   Ethyl (2E)-3-(furan-2-yl)prop-2-enoate (3d). Ethyl (2E)-3-(furan-2-yl)prop-2-enoate (3d). Ethyl (2E)-3-(furan-2-yl)prop-2-enoate (3d). O COMe   (3E)-4-(furan-2-yl)but-3-en-2-one (3e). O COMe   (3E)-4-(furan-2-yl)but-3-en-2-one (3e). (3E)-4-(furan-2-yl)but-3-en-2-one (3e). (3E)-4-(furan-2-yl)but-3-en-2-one (3e). MeO COOEt MeO   Ethyl (2E)-4-(4-methoxyphenyl)but-2-enoate (3f). MeO   Ethyl (2E)-4-(4-methoxyphenyl)but-2-enoate (3f). H13C6 COOEt Ethyl (2E)-non-2-enoate (3g). 13 6 Ethyl (2E)-non-2-enoate (3g). Ethyl (2E)-non-2-enoate (3g). COOEt EtOOC   Diethyl (2E,6E)-octa-2,6-dienedioate (3h). COOEt EtOOC   Diethyl (2E,6E)-octa-2,6-dienedioate (3h). COOEt EtOOC   Diethyl (2E,6E)-octa-2,6-dienedioate (3h). COOEt EtOOC   diethyl (2E,4E)-hexa-2,4-dienedioate (3i). COOEt EtOOC   diethyl (2E,4E)-hexa-2,4-dienedioate (3i). yl (2E,4E Ethyl (2E)-4-methylpent-2-enoate (3j). Ethyl (2E)-4-methylpent-2-enoate (3j). Ethyl (2E)-4-methylpent-2-enoate (3j). Ethyl (2E)-4-methylpent-2-enoate (3j). COOEt   Ethyl (2E,4E)-5-phenylpenta-2,4-dienoate (k). COOEt Ethyl (2E,4E)-5-phenylpenta-2,4-dienoate (k). Ethyl (2E,4E)-5-phenylpenta-2,4-dienoate (k). Ethyl (2E)-penta-2,4-dienoate (3l). Ethyl (2E)-penta-2,4-dienoate (3l). Ethyl (2E)-penta-2,4-dienoate (3l). Ethyl (2E)-pent-2-en-4-ynoate (m). Ethyl (2E)-pent-2-en-4-ynoate (m). Ethyl (2E)-pent-2-en-4-ynoate (m). Ethyl (2E)-pent-2-en-4-ynoate (m). Ethyl (2E)-pent-2-en-4-ynoate (m). N COOEt O O N COOEt O O tert-Butyl 2-[(1E)-3-ethoxy-3-oxoprop-1-en-1-yl]pyrrolidine-1-carboxylate (n). tert-Butyl 2-[(1E)-3-ethoxy-3-oxoprop-1-en-1-yl]pyrrolidine-1-carboxylate (n). tert-Butyl 2-[(1E)-3-ethoxy-3-oxoprop-1-en-1-yl]pyrrolidine-1-carboxylate (n).
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Classifying simulated wheat yield responses to changes in temperature and precipitation across a european transect
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International Crop Modelling Symposium 15-17 March 2016, Berlin 15-17 March 2016, Berlin Introduction A wide variety of dynamic crop growth simulation models have been developed over the past few decades that can differ greatly in their treatment of key processes and hence in their response to environmental conditions. Here, multi-model ensemble approaches have been adopted to quantify aspects of uncertainty in simulating yield responses to climate change (e.g. Asseng et al., 2013). We use a large ensemble of wheat models applied at sites across a European transect to compare their sensitivity to changes in climate by plotting them as impact response surfaces (IRSs; Fronzek et al., 2010). A previous paper using the same simulated yield dataset (Pirttioja et al., 2015) presented ensemble medians and inter-quartile ranges, focusing on long-term averages. This paper extends that work by classifying the responses of individual mod- els and attempting to interpret differences in response between groups of models by examining results from selected extreme years in addition to the long-term average. Classifying simulated wheat yield responses to changes in temperature and precipitation across a european transect S. Fronzek 1* – N. Pirttioja 1 – T. R. Carter 1 – M. Bindi2 – H. Hoffmann2 – T. Palosuo2 – M. Ruiz-Ramos2 – F. Tao2 – M. Trnka2 – M. Acutis2 – S. Asseng2 – P. Baranowski2 – B. Basso2 – P. Bodin2 – S. Buis2 – D. Cammarano2 – P. Deligios2 – M.-F. Destain2 – B. Dumont2 – F. Ewert2 – R. Ferrise2 – L. François2 – T. Gaiser2 – P. Hlavinka2 – I. Jacquemin2 – K. C. Kersebaum2 – C. Kollas2 – J. Krzyszczak2 – I. J. Lorite2 – J. Minet2 – M. I. Minguez2 – M. Montesino2 – M. Moriondo2 – C. Müller2 – C. Nendel2 – I. Öztürk2 – A. Perego2 – A. Rodríguez2 – A. C. Ruane2 – F. Ruget2 – M. Sanna2 – M. A. Semenov2 – C. Slawinski2 – P. Stratonovitch2 – I. Supit2 – K. Waha2 – E. Wang2 – L. Wu2 – Z. Zhao2 – R. P. Rötter2 S. Fronzek 1* – N. Pirttioja 1 – T. R. Carter 1 – M. Bindi2 – H. Hoffmann2 – T. Palosuo2 – M. Ruiz-Ramos2 – F. Tao2 – M. Trnka2 – M. Acutis2 – S. Asseng2 – P. Baranowski2 – B. Basso2 – P. Bodin2 – S. Buis2 – D. Cammarano2 – P. Deligios2 – M.-F. Destain2 – B. Dumont2 – F. Ewert2 – R. Ferrise2 – L. François2 – T. Gaiser2 – P. Hlavinka2 – I. Jacquemin2 – K. C. Kersebaum2 – C. Kollas2 – J. Krzyszczak2 – I. J. Lorite2 – J. Minet2 – M. I. Minguez2 – M. Montesino2 – M. Moriondo2 – C. Müller2 – C. Nendel2 – I. Öztürk2 – A. Perego2 – A. Rodríguez2 – A. C. Ruane2 – F. Ruget2 – M. Sanna2 – M. A. Semenov2 – C. Slawinski2 – P. Stratonovitch2 – I. Supit2 – K. Waha2 – E. Wang2 – L. Wu2 – Z. Zhao2 – R. P. Rötter2 1 Finnish Environment Institute (SYKE), 00251 Helsinki, Finland; * Corresponding author 2 Affiliations of co-authors as in Pirttioja et al., (2015) 1 Finnish Environment Institute (SYKE), 00251 Helsinki, Finland; * Corresponding author 2 Affiliations of co-authors as in Pirttioja et al., (2015) International Crop Modelling Symposium 15-17 March 2016, Berlin International Crop Modelling Symposium 15-17 March 2016, Berlin 15-17 March 2016, Berlin Results and Discussion Ensemble median responses showed declining yields with higher temperatures and decreased precipitation and yield increases with higher precipitation. However, indi- vidual models departed considerably from the average. An illustration of how respons- es are classified is given in Fig. 1, which distinguishes three patterns of winter wheat response across all three sites: (1) maximum yield at temperatures lower than the baseline, (2) stronger sensitivity to precipitation than temperature changes, and (3) large yield decreases with cooling and for strong warming. While some models were grouped into the same classes of response patterns for the different locations and crop varieties, a single factor could not be identified to explain common model responses. IRSs for anomalous weather-years showed larger model differences than for 30-year averages (e.g. in a cool year some models simulated crop failure over large parts of the IRS and others only small reductions relative to the baseline). Figure 1. Ensemble mean changes in winter wheat grain yield ( %) relative to the 1981-2010 baseline for the three dominant patterns of response identified using a hierarchical clustering approach across all study sites -2 0 2 4 6 8 -40 -20 0 20 40 Cluster 1 (n=39) Temperature change ( C) Precipitation change (%) -60 -50 -40 -30 -20 -10 0 -2 0 2 4 6 8 -40 -20 0 20 40 Cluster 2 (n=24) Temperature change ( C) Precipitation change (%) -50 -50 -40 -30 -20 -10 0 10 20 -2 0 2 4 6 8 -40 -20 0 20 40 Cluster 6 (n=4) Temperature change ( C) Precipitation change (%) -40 -40 -30 -30 -20 -20 -10 -10 0 0 10 -50 -40 -30 -20 -10 0 10 20 Figure 1. Ensemble mean changes in winter wheat grain yield ( %) relative to the 1981-2010 baseline for the three dominant patterns of response identified using a hierarchical clustering approach across all study sites Temperature change ( C) Temperature change ( C) Temperature change ( C) Conclusions At the time of writing, analysis of the modelled patterns of response were still ongoing. Preliminary results indicate that the study site is an important determinant of the posi- tioning of the response pattern for a given crop with respect to baseline climate. Dif- ferences in the shape and strength of the response pattern, especially under high-end changes and in anomalous weather-years, appear to be related to the model represen- tation of processes such as heat stress, moisture stress and vernalisation. Differences in calibration methods may also contribute to inter-model discrepancies. Acknowledgements This work is part of the FACCE-JPI Knowledge Hub MACSUR. For funding sources see Pirttioja et al., (2015). References Asseng, S., F. Ewert, C. Rosenzweig et al., (2013). Nature Climate Change 3 (9): 827–32. Fronzek, S., T.R. Carter, J. Räisänen et al., (2010). Climatic Change 99 (3): 515–534. Pirttioja, N., T.R. Carter, S. Fronzek et al., (2015). Climate Research, 65: 87–105. This work is part of the FACCE-JPI Knowledge Hub MACSUR. For funding sources see Pirttioja et al., (2015 This work is part of the FACCE-JPI Knowledge Hub MACSUR. For funding sources see Pirttioja et al., (2015). References Materials and Methods An ensemble of 26 process-based crop models was used to simulate yields of winter and spring wheat at three sites: in Finland (mainly temperature-limited), Germany (close to optimal conditions) and Spain (precipitation limited). The sensitivity of simu- lated yield to systematic increments of changes in temperature (−2 to +9°C) and pre- cipitation (−50 to +50 %) was tested by modifying values of baseline (1981 to 2010) daily weather. The results were plotted as IRSs that show the changes in yields relative to the baseline. IRSs of 30-year averages and selected extreme years were classified using a hierarchical clustering method and a second approach based on the location of the maximum yield and strength of the model response. IRSs were classified and com- pared to aspects of model performance, structure and genealogy (indicating the de- velopment history and relationships among some of the models). 64 nternational Crop Modelling Symposium 15-17 March 2016, Berlin References 65
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Interactions Increase Forager Availability and Activity in Harvester Ants
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RESEARCH ARTICLE Data Availability Statement: All relevant data are within the paper and its Supporting Information files. Data Availability Statement: All relevant data are within the paper and its Supporting Information files. Funding: Field work for EP in 2012 was funded by a Stanford UAR Student Small Grant, and field work for JQ in 2013 was funded by a Stanford UAR Student Major Grant (https://undergrad.stanford.edu/ opportunities/research/get-funded/apply-student- grants/grant-types). Other expenses were funded by the grant DMG received from the National Institutes of Health (grant number R01GM105024, http://www. nih.gov/). The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript. OPEN ACCESS Citation: Pless E, Queirolo J, Pinter-Wollman N, Crow S, Allen K, Mathur MB, et al. (2015) Interactions Increase Forager Availability and Activity in Harvester Ants. PLoS ONE 10(11): e0141971. doi:10.1371/ journal.pone.0141971 Evlyn Pless1☯¤a*, Jovel Queirolo1☯¤b, Noa Pinter-Wollman2, Sam Crow3, Kelsey Allen4, Maya B. Mathur5, Deborah M. Gordon1 1 Department of Biology, Stanford University, Stanford, California, United States of America, 2 BioCircuits Institute, University of California, San Diego, La Jolla, California, United States of America, 3 Department of Computer Science and Engineering, University of Washington, Seattle, Washington, United States of America, 4 Department of Brain and Cognitive Sciences, Massachusetts Institute of Technology, Cambridge, Massachusetts, United States of America, 5 Quantitative Sciences Unit, Stanford University, Stanford, California, United States of America a1111 ☯These authors contributed equally to this work. ¤a Current address: Department of Ecology and Evolution, Yale University, New Haven, Connecticut, United States of America ¤b Current address: Stanford Teacher Education Program, Stanford University, Stanford, California, United States of America * evlyn.pless@yale.edu ☯These authors contributed equally to this work. ¤a Current address: Department of Ecology and Evolution, Yale University, New Haven, Connecticut, United States of America ¤b Current address: Stanford Teacher Education Program, Stanford University, Stanford, California, United States of America * evlyn.pless@yale.edu ☯These authors contributed equally to this work. ¤a Current address: Department of Ecology and Evolution, Yale University, New Haven, Connecticut, United States of America ¤b Current address: Stanford Teacher Education Program, Stanford University, Stanford, California, United States of America * evlyn.pless@yale.edu ¤a Current address: Department of Ecology and Evolution, Yale University, New Haven, Connecticut, United States of America ¤b Current address: Stanford Teacher Education Program, Stanford University, Stanford, California, United States of America * l l @ l d Abstract Social insect colonies use interactions among workers to regulate collective behavior. Har- vester ant foragers interact in a chamber just inside the nest entrance, here called the 'entrance chamber'. Previous studies of the activation of foragers in red harvester ants show that an outgoing forager inside the nest experiences an increase in brief antennal con- tacts before it leaves the nest to forage. Here we compare the interaction rate experienced by foragers that left the nest and ants that did not. We found that ants in the entrance cham- ber that leave the nest to forage experienced more interactions than ants that descend to the deeper nest without foraging. Additionally, we found that the availability of foragers in the entrance chamber is associated with the rate of forager return. An increase in the rate of forager return leads to an increase in the rate at which ants descend to the deeper nest, which then stimulates more ants to ascend into the entrance chamber. Thus a higher rate of forager return leads to more available foragers in the entrance chamber. The highest den- sity of interactions occurs near the nest entrance and the entrances of the tunnels from the entrance chamber to the deeper nest. Local interactions with returning foragers regulate both the activation of waiting foragers and the number of foragers available to be activated. Editor: Nicolas Chaline, Universidade de São paulo, BRAZIL Received: June 4, 2015 Accepted: October 15, 2015 Published: November 5, 2015 Copyright: © 2015 Pless 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. Interactions Regulate Foraging in Harvester Ants [2,3]. Social insect colonies offer a compelling example of collective behavior, as they operate without any central control [4]. Competing Interests: The authors have declared that no competing interests exist. In social insect colonies, individual workers use local interactions to perform and regulate collective behavior [4,5], such as nest construction [6,7], nest relocation [8,9,10], and foraging [11,12,13,14,15]. Many studies suggest that wasps, honeybees, and ants all use interaction rate to activate foraging [14,15, 16, 17]. In wasps, foragers may be activated by the biting of incom- ing foragers and other nestmates, and foragers are bitten more frequently than other wasps [14]. In honeybees, forager arrival and interactions inside the hive [15], such as the waggle dance [18] and vibration signals [19], are associated with an increase in outgoing foraging [15,19], while another 'stop signal’ may counter the waggle dance [18]. Local encounters also play an important role for the organization of foraging in ants, for exam- ple to respond to the intensity of crowding [20,21,22]. Ants appear to use the rate of antennal con- tact as an indication of local density [23,24,25] and adjust recruitment and trail networks accordingly [20,21,22]. For example, Lasius niger ants downregulate the production of recruitment signals [22] and bifurcate their trail during crowding to maintain a high rate of food return [20]. It appears that interactions also regulate the availability of foragers. For example, interac- tions among honeybees affect hormonal factors that determine the onset of foraging and there- fore the number of bees available to forage [26]. Depleting a colony of the older foragers induces an early onset of foraging in younger bees [26]. Removal experiments in ants also sug- gest that the availability of foragers depends on interactions, because the removal of foragers leads other ants to switch tasks and become foragers [27, 28]. Red harvester ants (Pogonomyrmex barbatus) use interaction rates to decide which task to perform, including whether and when to forage [12,13,29]. Interactions between harvester ants are tactile and chemical and take the form of brief antennal contacts [30,31]. During an anten- nal contact, one ant assesses the cuticular hydrocarbon profile of the other, which reveals an ant’s task and whether it is carrying food [30,31,32]. Foraging poses trade-offs for seed-eating ants in the desert. Ants must spend water to gain water because foraging in the desert heat can lead to desiccation, and they obtain some water by metabolizing the fats in the seeds they col- lect [33,34]. In addition, colonies compete with their neighbors for foraging area [35,36]. How well a colony manages these trade-offs influences its reproductive success [37]. In harvester ants, returning foragers come into the nest entrance through a tunnel and into a chamber just inside the entrance that we call the 'entrance chamber' (Fig 1). In previous work we referred to the 'vestibule' as the area just inside the nest entrance [13], but in this study we excavated more deeply to observe ants in the full chamber at the end of the entrance tunnel. Foragers deposit the food they collected in this chamber or carry it down tunnels leading from the entrance chamber to the deeper nest. The sizes and shapes of tunnels and chambers vary among colonies, and these differences probably influence the rate and location of interactions. Nest structure affects ant movement [38], which affects the rate of interaction [39,40]. In turn, rate of interaction can influence traffic flow [41,42]. Here we consider three questions about the regulation of foraging through interaction rate in harvester ants: Introduction A fundamental question in the study of animal behavior and other networks is how simple individual behaviors add up to complex collective behaviors [1]. Distributed networks, includ- ing those found in natural populations, are regulated using feedback based on local interactions 1 / 18 PLOS ONE | DOI:10.1371/journal.pone.0141971 November 5, 2015 Question 1: Do outgoing foragers experience a different interaction rate from other ants? Studies on harvester ants have shown that interactions with successful returning foragers stim- ulate foraging activity [12,43]. The chemical odors of both a returning forager and the seed it is carrying are required in combination to stimulate foraging [32]. A previous study showed that outgoing foragers experience a substantial increase in interactions 3–8 seconds before they 2 / 18 PLOS ONE | DOI:10.1371/journal.pone.0141971 November 5, 2015 Interactions Regulate Foraging in Harvester Ants Fig 1. Diagram of the entrance chamber. A schematic vertical cross section of a typical P. barbatus ant nest. The solid line indicates the ground surface, and the dashed line indicates where a horizontal section was made to observe activity inside the nest. Interactions occur in the entrance chamber, connected to the outside by the entrance tunnel, with tunnels leading to the deeper nest. Fig 1. Diagram of the entrance chamber. A schematic vertical cross section of a typical P. barbatus ant nest. The solid line indicates the ground surface, and the dashed line indicates where a horizontal section was made to observe activity inside the nest. Interactions occur in the entrance chamber, connected to the outside by the entrance tunnel, with tunnels leading to the deeper nest. doi:10.1371/journal.pone.0141971.g001 doi:10.1371/journal.pone.0141971.g001 choose to leave the nest [13]. However, this study examined the interaction rates only of the foragers that left the nest. Here we test whether ants that left the nest to forage experienced a higher interaction rate than ants that did not leave the nest to forage and instead descended from the entrance chamber to the deeper nest without foraging. Question 2: What regulates the number of outgoing foragers available in the entrance chamber? Previous work on harvester ants shows that while foragers inside the nest respond to interac- tions within seconds, the pool of ants available to become foragers is regulated on the order of minutes [13]. Inhibiting forager return for more than four minutes leads to a decrease in the number of foragers inside the nest entrance that are available to forage (S1 Movie) [13]. Here we examine how the rate of returning foragers regulates the number of ants in the entrance chamber available to forage. We hypothesized that the descent into the deeper nest of returning foragers, or other ants transporting food within the nest, influences the rate at which ants ascend from the deeper nest to the entrance chamber to become available to forage. We manipulated the rate of forager return to determine the effect on the rate at which ants ascend from the deeper nest, and return to the deeper nest, thus regulating the numbers in the entrance chamber available to forage. Question 3: What determines the spatial distribution of interactions? Previous work shows that an ant’s interaction frequency depends on its path shape and loca- tion [35,38,39,40,41,42,44,45]. By influencing interactions, the shape and size of the entrance chamber, which varies among colonies, probably affects the regulation of foraging in harvester ants. Previous studies show that most interactions occur at the entrances of the tunnels from the entrance chamber to the deeper nest [13]. We asked how manipulating the rate of incoming foragers would change the spatial distribution and frequency of interactions in the nest. Materials and Methods Data were collected over the course of one week in August 2012 and two weeks in August 2013 at the site of a long-term study in Rodeo, New Mexico [37]. Prof. Gordon’s long-term study 3 / 18 PLOS ONE | DOI:10.1371/journal.pone.0141971 November 5, 2015 Interactions Regulate Foraging in Harvester Ants site is owned by Stanford University, and no permission was required to work on the site. This study did not involve any endangered or protected species. Nests of P. barbatus have an entrance approximately 2cm in diameter, which leads to one or more entrance tunnels approximately 5cm long. The entrance tunnels lead into an entrance chamber approximately 8cm wide, and from this chamber, tunnels descend into the deeper nest (Fig 1). To observe interactions inside the entrance chamber, we excavated the soil above the entrance chamber, as in Pinter-Wollman et al. [13]. We positioned a rectangular piece of ply- wood (23cm x 28cm) such that one of the short edges of the wood was directly over the nest entrance. The rest of the wood was positioned over the entrance tunnel and entrance chamber. We drew an outline around the wood, removed the wood, and dug out approximately 4cm of soil from the rectangular area for all films made in 2012 and approximately 9cm of soil for films made in 2013. The area exposed in 2012 corresponds to the area inside the nest entrance described as the 'vestibule' in Pinter-Wollman et al. [13]. The area we exposed in the 2013 observations described here included more of the entrance chamber than in 2012. Here we refer to both as the 'entrance chamber'. We covered the area with the wood overnight so that the ants would acclimate to their new nest ceiling. Each morning between 6am and 11am, we removed the wood and covered the excavated area with a rectangular glass sheet (23cm x 28cm) for filming (Fig 2). Previous studies show that the incoming light does not disturb the ants [13]. In 2012 we filmed three colonies for three consecutive days, and in 2013 we filmed six colo- nies over a period of 10 days (S1 Table). We converted the films into JPEGs (30 frames/second) using Adobe Premiere Elements 7.0 in 2012 and Java software that we developed in 2013. PLOS ONE | DOI:10.1371/journal.pone.0141971 November 5, 2015 Materials and Methods We observed the films of ants in the entrance chamber, which included all area covered by the glass sheet and all area immediately around the glass window which was depressed at the same level as the rest of the ant chamber. When observing the films for each of our analyses, we used the following definitions for cat- egorizing ant activities: 1. Outgoing foragers: ants that leave the entrance chamber and go outside not carrying anything 2. Returning foragers: ants that come from outside of the nest and enter the nest (Observations were made at a time when most ants outside the nest were foragers, but it is possible that some of the ants considered to be returning foragers were ants of another task that did not carry a food item.) 3. Descending ants: ants that were first observed in the entrance chamber and go down a tun- nel into the deeper nest 4. Ascending ants: ants that were in the deeper nest, come up a tunnel, and emerge into the entrance chamber 5. Nest maintenance workers: ants carrying dirt or debris inside the nest To test whether outgoing foragers experience a different interaction rate from other ants (Question 1), we made 3 sets of measurements from the films made in 2012. First, we deter- mined what percentage of the ants that ascended from the deeper nest into the entrance cham- ber (‘ascending ants’) later performed each of the following: outgoing foraging, descending into the deeper nest, or nest maintenance. The focal ants for this analysis were chosen by selecting the first ant to emerge from the deeper nest at five-second intervals. Second, we observed out- going foragers and recorded the time and location of all their interactions, in the form of brief PLOS ONE | DOI:10.1371/journal.pone.0141971 November 5, 2015 4 / 18 Interactions Regulate Foraging in Harvester Ants Fig 2. Labeled entrance chamber. A photograph of the entrance chamber of colony N_4 covered with the window used during filming. The tunnels to the deeper nest are labeled, as is the trail of returning and outgoing foragers. doi:10 1371/journal pone 0141971 g002 Fig 2. Labeled entrance chamber. A photograph of the entrance chamber of colony N_4 covered with the window deeper nest are labeled, as is the trail of returning and outgoing foragers. Fig 2. Labeled entrance chamber. PLOS ONE | DOI:10.1371/journal.pone.0141971 November 5, 2015 Interactions Regulate Foraging in Harvester Ants Fig 3. Comparison of activity distribution by colony. Each bar shows the mean proportion of ascending ants that foraged (black), did nest maintenance (white), or ascended into the nest entrance but then descended back into the deeper nest without leaving the nest to forage (grey), over the course of three days. Error bars show standard errors of the mean. Fig 3. Comparison of activity distribution by colony. Each bar shows the mean proportion of ascending ants that foraged (black), did nest maintenance (white), or ascended into the nest entrance but then descended back into the deeper nest without leaving the nest to forage (grey), over the course of three days. Error bars show standard errors of the mean. doi:10.1371/journal.pone.0141971.g003 obstruction, and ants that did not perform any of the indicated behavior until the end of the film were excluded from analysis. Out of 147 possible focal ants per colony (pooled over the three days), 16 were excluded from the analysis of N_2, 21 from the analysis of N_4, and 9 from the analysis of N_13 (S1 Dataset). To record the interactions of outgoing foraging and descending ants, we analyzed the JPEGs from the films using a custom written MATLAB script as in Pinter-Wollman et al. [13] (code provided in S1 Appendix). We observed ten outgoing foragers and four or five descending ants per film, the first foraging and descending ants in the film segment that were clearly visible, from when the ant entered the entrance chamber from the deeper nest to when she either left the entrance chamber or descended to the deeper nest. (Five descending ants were selected for each film except for the film of N_2 on August 15th, which showed only four descending ants.) We recorded the time each ant entered and left the entrance chamber, and the time and loca- tion of each interaction of the focal ant. Interactions were recorded when the focal ant made antennal contact with the head or body of another ant, and the location of the interaction was defined as the point in between the head of the first ant and the place of contact on the second ant. Our analysis did not examine interactions with other outgoing foragers, because previous work showed that only successful returning foragers stimulated foraging [43]. PLOS ONE | DOI:10.1371/journal.pone.0141971 November 5, 2015 Materials and Methods A photograph of the entrance chamber of colony N_4 covered with the window used during filming. The tunnels to the deeper nest are labeled, as is the trail of returning and outgoing foragers. doi:10.1371/journal.pone.0141971.g002 doi:10.1371/journal.pone.0141971.g002 doi:10.1371/journal.pone.0141971.g002 antennal contacts, before leaving the entrance chamber. Third, we recorded the time and loca- tions of the interactions of descending ants. The first clearly visible foraging and descending ants from each video were chosen as the focal ants for the second and third analyses. To determine the sample size of outgoing foragers, we considered the possibility that if the proportion of foragers were extremely different in the three colonies observed, a given sample size would not be equally representative in all three colonies. We found that the proportions foraging in the three colonies were not significantly different (ANOVA, F2,6 = 3.854; p = 0.08) (Fig 3) (S1 Dataset). Therefore, we decided to measure interaction rates in the same number of foragers and descending ants in all three colonies, rather than choose different sample sizes for each colony. To determine what percentage of the ants that ascended from the deeper nest into the entrance chamber were outgoing foragers, descending ants, or nest maintenance workers within our 5–7 minutes of film, we observed randomly selected ants in each of the nine films (three per colony for three colonies taken over the course of three days, S1 Table). Every five seconds for the first four minutes of film, the first ant to ascend from the entrance tunnel was tracked until it left the nest, performed nest maintenance by moving dirt or debris around the nest entrance, or descended to the deeper nest. Ants that were lost from view due to glare or an 5 / 18 PLOS ONE | DOI:10.1371/journal.pone.0141971 November 5, 2015 3. rates of ascending ants and descending ants 3. rates of ascending ants and descending ants We tested for cross-correlations in each of the three combinations of variables listed above, to determine whether the two variables tended to be correlated in time, and if so, with what lag. For example, if an ant in the entrance chamber often descended to the deeper nest 10 seconds after a returning forager came in, then there would be a positive correlation at a lag of 10 sec- onds in the rates of returning foragers and descending ants. To normalize each time series for differences in numbers of ants, we subtracted from each point the moving average of each time series using a time window that was half the duration of each film (S2 Table). The timescale of the smoothing window was minutes (median 7 minutes), whereas observed lags were on the order of seconds (median ~15 seconds), and thus the results were probably not affected by this normalization procedure. To identify significant cross-correlations, indicating a time dependence for two rates, we calculated the likelihood of a correlation for each possible lag time, relative to the null hypothe- sis of no time dependence. We report lag times that were statistically significant at p < 0.01. In addition, we ran 1000 simulations in which the time of each event (ant returning, ascending, or descending) was randomly assigned from a uniform distribution covering the average length of the videos. We then compared the proportion of significant cross correlations in the simulated data with that proportion in the observed data. A smaller proportion of significant events in the simulated data compared with the observed indicates that the observed correlations were not found by chance. We next investigated the spatial distribution of interactions (Question 3). Using Java software we developed, we split the films into JPEG frames (at 30 frames per sec- ond) and marked the location in the entrance chamber of each interaction of the ants we tracked. The locations of interactions were recorded in two instantaneous images, one at the start of the removal of returning foragers, and the other immediately after forager removals ended 3–5 minutes later. To illustrate the spatial pattern of interactions, we used a 2D Gaussian kernel density esti- mator in R package MASS to produce 13 pairs of utilization distribution maps for the 13 trials with forager removals [46]. An outgoing for- ager was identified as an ant that moved directly toward the outside of the nest and immedi- ately left the nest as soon as it reached the edge of the entrance chamber. We used a linear multilevel model with normal errors, fit via REML using R’s lme4 package, to determine if the rate of interactions and time spent in the entrance chamber differed between outgoing foragers and descending ants [46]. To account for colony and day effects, ant 6 / 18 PLOS ONE | DOI:10.1371/journal.pone.0141971 November 5, 2015 Interactions Regulate Foraging in Harvester Ants activity was a fixed effect with colony and day as random effects with normally distributed error terms. To investigate what regulates the number of outgoing foragers available in the entrance chamber (Question 2), we filmed the entrance chamber in the morning when the ants were for- aging and relatively undisturbed for about 6 minutes in 2013 (S1 Table). To test how changes in the rate of forager return influence the rate at which ants ascend into and descend from the entrance chamber, we manipulated forager return rate. Returning foragers were removed for 3–5 minutes (depending on the amount of time needed to collect most of the returning forag- ers) and kept in a plastic box (and returned to the nest after observations were completed), as in Gordon et al. [12]. We observed and filmed behavior inside the nest during and after forager removals. We performed removal experiments in 13 out of the 16 trials (S1 Table). We counted from the films the numbers of returning and outgoing foragers at the nest entrance and the number of ascending and descending ants at all tunnel entrances. We used MATLAB to test for cross-correlations (p < 0.01) among: 1. rates of returning foragers and ascending ants 2. rates of returning foragers and descending ants PLOS ONE | DOI:10.1371/journal.pone.0141971 November 5, 2015 1. Interaction rates of outgoing foragers The mean (±SD) time outgoing foragers were tracked was 7.51 (±7.43) seconds (range 0.401 to 36.6) and the mean time descending ants were tracked was 16.0 (±10.8) seconds (range 2.91 to 47.6). Although sampling times varied widely, the distribution was similar across each colony (S1 Fig). The mean (±SD) interaction rate for outgoing foragers was 1.47 (±1.09) interactions per sec- ond, while the mean interaction rate for descending ants was 0.83 (±0.587) interactions per sec- ond (S2 Dataset). Taking into account differences in colony- and day-specific interaction rates, the interaction rates of foragers were significantly higher than those of descending ants (linear multilevel model, b = 0.64; 95% CI: 0.33, 0.95; p < 0.001). (The coefficient (b) represents the model-based estimate for the increase in interaction rate between foragers and descending ants.) Foragers also spent a shorter time in the entrance chamber than ants that eventually descended to the deeper nest (linear multilevel model with normal errors, b = -8.55; 95% CI: 11.65, -5.44; p<0.001). For illustrative purposes, interaction rates and time in the entrance chamber by colony (pooling across all three days) are shown in Fig 4. The three colonies differed in the interaction rates of outgoing foragers (ANOVA, F2,27 = 8.42; p = 0.0004) and descending ants (ANOVA, F2,11 = 4.83; p = 0.0002), and all differed in pairwise tests except colonies N_2 and N_13 (N_2 vs N_13 Tukey multiple comparison of means, p = 0.622 for outgoing foragers, p = 0.92 for descending). The three days also differed in the interaction rates of outgoing foragers (ANOVA, F2,27 = 5.64; p = 0.005) and descending ants (ANOVA, F2,11 = 12.21; p <0.0001), due to a difference between August 13 and August 14 (Tukey multiple comparisons of means, p = 0.0033 for outgoing foragers, p < 0.0001 for descending ants). Within the 5–7 minutes of each of our films, the proportion of ascending ants that left the nest ranged from 0.31–0.89, (mean (±SD) 0.59 (±0.18)) (Fig 3) (S1 Dataset). The proportion of ascending ants that performed nest maintenance, carrying soil or debris out of or around the nest, ranged from 0.00–0.083 (mean (±SD) 0.067 (±0.048)), and the proportion that returned to the deeper nest ranged from 0.056–0.65 (mean (±SD) 0.34 (±0.19)) (Fig 3). 1. Interaction rates of outgoing foragers In all colonies and days, significantly more ascending ants left the nest (Student’s t-test, p<0.0001) or returned to the deeper nest (Student’s t-test, p = 0.0036) than did nest maintenance. In seven of the nine colonies and days, more ants left the nest than returned to the deeper nest without foraging. 3. rates of ascending ants and descending ants These maps show the spatial density of interactions in the entrance chamber at the start and at the end of the forager removals. 7 / 18 PLOS ONE | DOI:10.1371/journal.pone.0141971 November 5, 2015 Interactions Regulate Foraging in Harvester Ants PLOS ONE | DOI:10.1371/journal.pone.0141971 November 5, 2015 2. Availability of foragers in entrance chamber There was evidence of time-dependence among the rates at which ants returned to the nest, ascended from the deeper nest to the entrance chamber, and descended from the entrance chamber to the deeper nest (S3 Dataset). We found significant cross-correlations between the rates of returning foragers and ascending ants, returning foragers and descending ants, and ascending ants and descending ants in some of the trials, when removals were performed, for each of the 3 colonies. (Table 1; Cross-correlation, p < 0.01). The cross-correlation between rates of returning foragers and ascending ants was significant in one out of three trials for col- ony 367 and one out of two trials for colony 229 (2/5 trials) (Table 1). The cross-correlation between rates of returning foragers and descending ants was significant in one out of three tri- als for colony N_5, two out of three trials for colony 367, one out of one trial for colony 25, and one out of one trials for colony 242 (5/7 trials) (Table 1). The cross-correlation between rates of ascending ants and descending ants was significant in two out of three trials for colony N_5, 8 / 18 PLOS ONE | DOI:10.1371/journal.pone.0141971 November 5, 2015 Interactions Regulate Foraging in Harvester Ants Table 1. Lag times found after removals were performed. The lag values in seconds between returning foragers and ascending ants, returning foragers and descending ants, and ascending ants and descending ants. Mean and standard deviation across all colonies and days also shown. For cross-correla- tions in which more than one lag time was detected, all significant lags are reported. A “—” indicates that no significant lags were found. Colony name Date filmed Lag times between returning foragers and ascending (seconds) Lag times between returning foragers and descending ants (seconds) Lag times between ascending and descending ants (seconds) N_5 8-17- 2013 - 13 - N_5 8-18- 2013 - - 5 N_5 8-20- 2013 - - 30, 68 367 8-18- 2013 - 6, 26 - 367 8-20- 2013 11, 61 2, 17, 45 13, 29, 69 367 8-21- 2013 - - - 868 8-19- 2013 - - 43 868 8-20- 2013 - - 4 868 8-21- 2013 - - - 25 8-24- 2013 - 22 - 229 8-24- 2013 - - - 229 8-26- 2013 58 - 62, 68 242 8-25- 2013 - 13 - Mean 43.33 18 39.1 Standard Deviation 28.04 13.42 26.59 doi:10.1371/journal.pone.0141971.t001 cross-correlations: colony 367 on August 20, 2013. There were two statistically significant lag times found between the returning foragers and ascending ants, three for returning foragers and descending ants, and three for ascending ants and descending ants. Fig 5 shows all the sta- tistically significant cross correlations from this trial. The rate at which ants descended into the deeper nest depended significantly on the rate of returning foragers. Of the three relationships considered, the shortest lag time was between for- agers returning and ants descending into the deeper nest. The mean (±SD) lag, 18.00 (±13.42) seconds, between returning foragers and descending ants was significantly shorter than the lag between ascending ants and descending ants, 39.10 (±26.59) seconds (Student’s t-test, p = 0.048) (Fig 6). It was also significantly shorter than the lag between returning foragers and ascending ants, 43.33 (±28.04) seconds (Student’s t-test, p = 0.032) (Fig 6). Some colonies showed significant lag times between more than one of the tested relationships, while other col- onies had only one (Table 1). Interactions Regulate Foraging in Harvester Ants Fig 4. Comparison of interaction rate and time in entrance chamber in outgoing foragers and descending ants. A) Each bar shows the mean brief antennal contacts, in interactions per second, of ants in the entrance chamber. B) Each bar shows the mean time in seconds that ants spent in entrance chamber. Black: ants that subsequently left the nest to forage (outgoing foragers); White: ants that descended from the entrance chamber deeper nest (descending ants). Error bars show standard errors of the mean. doi:10.1371/journal.pone.0141971.g004 Fig 4. Comparison of interaction rate and time in entrance chamber in outgoing foragers and descending ants. A) Each bar shows the mean rate of brief antennal contacts, in interactions per second, of ants in the entrance chamber. B) Each bar shows the mean time in seconds that ants spent in the entrance chamber. Black: ants that subsequently left the nest to forage (outgoing foragers); White: ants that descended from the entrance chamber to the deeper nest (descending ants). Error bars show standard errors of the mean. doi:10.1371/journal.pone.0141971.g004 doi:10.1371/journal.pone.0141971.g004 one out of three trials for colony 367, two out of three trials for colony 868, and one out of two trials for colony 229 (9/11 trials) (Table 1). The simulations in which ascending, descending, and returning ant times were chosen from a uniform distribution produced only 76/1000 sig- nificant cross correlations, a smaller proportion than any of the observed cross correlations (0.076 for the randomization simulations compared with 0.4, 0.71, and 0.82 for the observed data). There was one removal trial that produced statistically significant lag times for all three PLOS ONE | DOI:10.1371/journal.pone.0141971 November 5, 2015 PLOS ONE | DOI:10.1371/journal.pone.0141971 November 5, 2015 9 / 18 doi:10.1371/journal.pone.0141971.t001 3. Spatial distribution of interactions Considering each of the thirteen films individually, eight films showed significantly fewer interactions after removals than before removals: colony 367 on 8- 20-13 and 8-21-13, colony 868 on 8-19-13 and 8-21-13, colony 25 on 8-24-13, colony 229 on 8-24-13 and 8-26-13, and colony 242 on 8-25-13 (Binomial test, p < 0.05) (Fig 7). 8) (S4 Dataset, S1 Movie). Considering each of the thirteen films individually, eight films showed significantly fewer interactions after removals than before removals: colony 367 on 8- 20-13 and 8-21-13, colony 868 on 8-19-13 and 8-21-13, colony 25 on 8-24-13, colony 229 on 8-24-13 and 8-26-13, and colony 242 on 8-25-13 (Binomial test, p < 0.05) (Fig 7). The interaction utilization distribution maps show that the highest number of interactions occurs at the entrances to the tunnels to the deeper nest, both before and after removals (Fig 8) (S4 Dataset). 3. Spatial distribution of interactions Several minutes after the rate of returning foragers was experimentally reduced, there were sig- nificantly fewer ant interactions in the entrance chamber (Paired t-test, p = 0.003) (Figs 7 and 10 / 18 PLOS ONE | DOI:10.1371/journal.pone.0141971 November 5, 2015 Interactions Regulate Foraging in Harvester Ants Fig 5. Comparison of significant cross-correlations in lag times for a single colony. All lag times are from colony 367 on August 20, 2013, the only trial that showed significant lag times for all three cross- correlations. Each bar shows the lag of a significant cross-correlation between 1) rate of forager return and rate at which ants ascended to the entrance chamber from the deeper nest, 2) rate of forager return and rate at which ants descended to the deeper nest, and 3) rate at which ants ascended to the entrance chamber and rate at which ants descended to the deeper nest. Fig 5. Comparison of significant cross-correlations in lag times for a single colony. All lag times are from colony 367 on August 20, 2013, the only trial that showed significant lag times for all three cross- correlations. Each bar shows the lag of a significant cross-correlation between 1) rate of forager return and rate at which ants ascended to the entrance chamber from the deeper nest, 2) rate of forager return and rate at which ants descended to the deeper nest, and 3) rate at which ants ascended to the entrance chamber and rate at which ants descended to the deeper nest. Fig 5. Comparison of significant cross-correlations in lag times for a single colony. All lag times are from colony 367 on August 20, 2013, the only trial that showed significant lag times for all three cross- correlations. Each bar shows the lag of a significant cross-correlation between 1) rate of forager return and rate at which ants ascended to the entrance chamber from the deeper nest, 2) rate of forager return and rate at which ants descended to the deeper nest, and 3) rate at which ants ascended to the entrance chamber and rate at which ants descended to the deeper nest. doi:10.1371/journal.pone.0141971.g005 8) (S4 Dataset, S1 Movie). PLOS ONE | DOI:10.1371/journal.pone.0141971 November 5, 2015 Interactions Regulate Foraging in Harvester Ants Fig 6. Comparison of time lags between ant activities. Each bar shows the duration in seconds of the lag between two rates, with data pooled across six colonies and eight days: 1) rate of forager return and rate at which ants ascended to the entrance chamber from the deeper nest, 2) rate of forager return and rate at which ants descended to the deeper nest from the entrance chamber, and 3) rate at which ants ascended to the entrance chamber from the deeper nest and rate at which ants descended to the deeper nest from the entrance chamber. The figure shows lag values only for the cross-correlations that were statistically significant. Error bars show standard errors of the mean. Numbers above bars signify sample sizes. Fig 6. Comparison of time lags between ant activities. Each bar shows the duration in seconds of the lag between two rates, with data pooled across six colonies and eight days: 1) rate of forager return and rate at which ants ascended to the entrance chamber from the deeper nest, 2) rate of forager return and rate at which ants descended to the deeper nest from the entrance chamber, and 3) rate at which ants ascended to the entrance chamber from the deeper nest and rate at which ants descended to the deeper nest from the entrance chamber. The figure shows lag values only for the cross-correlations that were statistically significant. Error bars show standard errors of the mean. Numbers above bars signify sample sizes. doi:10.1371/journal.pone.0141971.g006 interactions per sec). Ants that left the nest to forage also spent significantly less time in the entrance chamber (mean 7.51 sec) than those that descended to the deeper nest (mean 16.03 sec). Our results show that during the period of foraging activity, the rate of forager return helps to regulate the rate at which foragers become available to forage. Most of the ants in the entrance chamber are foragers: of the ants that came from the deeper nest into the entrance chamber, most left the nest to forage. Almost half the ants that did not forage went back down into the deeper nest (Fig 2). The regulation of the number of ants in the entrance chamber allows the colony to adjust forager availability quickly in response to changing conditions. PLOS ONE | DOI:10.1371/journal.pone.0141971 November 5, 2015 Discussion Ants in the entrance chamber that leave the nest to forage have experienced more interactions, in the form of brief antennal contacts, than ants that descend to the deeper nest without forag- ing. The availability of foragers in the entrance chamber is regulated by another process that determines the flow of ants into and out of the entrance chamber from the deeper nest. This movement of ants in and out of the entrance chamber from the deeper nest, which determines the availability of foragers, is associated with the rate of forager return. The more frequently that returning foragers come in, the more frequently ants descend from the entrance chamber to the deeper nest, and in turn, the more frequently foragers ascend to the entrance chamber from the deeper nest. In this way, higher rates of forager return lead to the presence of more available foragers, as previously observed [13]. Interactions occur at highest density in the nest entrance and the entrances of the tunnels leading to the deeper nest. Our result supports previous work showing that ants in the entrance chamber determine whether to forage based on the rate of interactions with returning, successful foragers [11,12,32] and showed that the rate of forager return is correlated with the rate of interactions inside the nest [13]. Here we show directly that the ants that left the entrance chamber to forage had experienced a significantly higher rate of interactions (mean 1.47 interactions per sec) than ants that descended from the entrance chamber to the deeper nest without foraging (mean 0.83 PLOS ONE | DOI:10.1371/journal.pone.0141971 November 5, 2015 11 / 18 When food availability is high, foragers find food quickly, leading to a high rate of forager return [47]. The number of ants in the entrance chamber depends on the rate of forager return [13]. The rate of forager return is linked with a short lag (mean 18 seconds) to the rate at which ants descend to the deeper nest (Table 1). The returning foragers sometimes deposit their food in the entrance chamber for other ants to carry to the lower nest, but in the exposed entrance chambers that we filmed, more often returning foragers descended still carrying the food items they brought back (more 12 / 18 PLOS ONE | DOI:10.1371/journal.pone.0141971 November 5, 2015 Interactions Regulate Foraging in Harvester Ants Fig 7. Comparison of number of interactions in the entrance chamber before and after removals. Black: the number of interactions observed from one film frame at the start of forager removals; White: the number of interactions observed from one film frame 3–5 minutes after removal of returning foragers was completed. doi 10 1371/jo rnal pone 0141971 g007 Fig 7. Comparison of number of interactions in the entrance chamber before and after removals. Black: the number of interactions observed from one film frame at the start of forager removals; White: the number of interactions observed from one film frame 3–5 minutes after removal of returning foragers was completed. doi:10.1371/journal.pone.0141971.g007 doi:10.1371/journal.pone.0141971.g007 discussion below). The rate at which foragers ascend is linked with a significantly longer lag (mean 38.25 seconds) to the rate at which ants descend (Table 1). When the rate of forager return slows, indicating low food availability, the number of available foragers in the entrance chamber also slows [13]. The number of interactions in the entrance chamber nest decreases with low forager return (Figs 7 and 8), further decreasing the probability ants will leave the nest to forage (Fig 4A). The process that regulates the numbers in the entrance chamber is very rapid; most ants in the entrance chamber either forage or descend in less than 20 seconds. The rapid turnover of ants entering and leaving the entrance chamber may help to limit the number of waiting ants in the entrance chamber [13], who could slow the rate of interactions with incoming, successful foragers. The different lags between rates of forager return, ascending ants, and descending ants may be due to different processes. PLOS ONE | DOI:10.1371/journal.pone.0141971 November 5, 2015 Interactions Regulate Foraging in Harvester Ants Fig 8. Spatial distribution of interactions. Each interaction utilization distribution map shows the location of interactions, brief antennal cont ants, that were observed from instantaneous images. The left figure of each pair shows interactions at the very beginning of an experiment tha the rate of incoming foragers, and the right figure shows interactions 3–5 minutes after the experiment was complete. White shows the areas o interaction density for each pair of heat maps, yellow shows areas with some interactions, and red shows areas with no interactions. The tunn is indicated with a T and the nest entrance/exit is indicated with an N. The scales for size and color are relative to each pair of maps. doi:10.1371/journal.pone.0141971.g008 Fig 8. Spatial distribution of interactions. Each interaction utilization distribution map shows the location of interactions, brief antennal contacts between ants, that were observed from instantaneous images. The left figure of each pair shows interactions at the very beginning of an experiment that decreased the rate of incoming foragers, and the right figure shows interactions 3–5 minutes after the experiment was complete. White shows the areas of highest interaction density for each pair of heat maps, yellow shows areas with some interactions, and red shows areas with no interactions. The tunnel entrance/ex is indicated with a T and the nest entrance/exit is indicated with an N. The scales for size and color are relative to each pair of maps. Fig 8. Spatial distribution of interactions. Each interaction utilization distribution map shows the location of interactions, brief antennal contacts between ants, that were observed from instantaneous images. The left figure of each pair shows interactions at the very beginning of an experiment that decreased the rate of incoming foragers, and the right figure shows interactions 3–5 minutes after the experiment was complete. White shows the areas of highest interaction density for each pair of heat maps, yellow shows areas with some interactions, and red shows areas with no interactions. The tunnel entrance/exit is indicated with a T and the nest entrance/exit is indicated with an N. The scales for size and color are relative to each pair of maps. Fig 8. Spatial distribution of interactions. Each interaction utilization distribution map shows the location of interactions, brief antennal contacts between ants, that were observed from instantaneous images. For example, the lag times between ascending ants and descend- ing ants were approximately factors of one another in two cases (30 and 68 seconds for colony N_5 on 8-20-13; and 13, 29, and 69 seconds for colony 367 on 8-20-13). This suggests a period- icity in the behavior of ascending into and descending from the entrance chamber. It is inter- esting to note that the lag between returning foragers and ascending ants tends to be larger than both the lags between returning foragers and descending ants and between ascending and descending ants. This is what we would expect if the same ants return from foraging, descend to the deeper nest, drop off their food, and then ascend back to the entrance chamber to be available to leave on the next foraging trip. More work is needed to determine how often returning foragers carry their food into the deeper nest, or instead deposit it in the entrance chamber for other ants to transport down to the seed chambers in the deeper nest. A number of factors influence the spatial distribution and frequency of interactions. Har- vester ant colonies vary consistently from year to year in foraging activity [12,37]. These PLOS ONE | DOI:10.1371/journal.pone.0141971 November 5, 2015 13 / 18 The left figure of each pair shows interactions at the very beginning of an experiment that decreased the rate of incoming foragers, and the right figure shows interactions 3–5 minutes after the experiment was complete. White shows the areas of highest interaction density for each pair of heat maps, yellow shows areas with some interactions, and red shows areas with no interactions. The tunnel entrance/exit is indicated with a T and the nest entrance/exit is indicated with an N. The scales for size and color are relative to each pair of maps. doi:10.1371/journal.pone.0141971.g008 doi:10.1371/journal.pone.0141971.g008 variations may arise from colony-specific differences in how interaction rates affect the forag- ing decisions of ants. The three colonies filmed in 2013 differed in interaction rates, and inter- action rates differed between two of the days (August 14th and August 15th). Colony differences in interaction rate could be related to variation in nest structure [38,41,42]. Nests vary in the size and shape of the entrance chamber and in the number of tunnels leading from the chamber to the deeper nest (Fig 8). This variation may lead to differences among colonies PLOS ONE | DOI:10.1371/journal.pone.0141971 November 5, 2015 PLOS ONE | DOI:10.1371/journal.pone.0141971 November 5, 2015 14 / 18 Interactions Regulate Foraging in Harvester Ants in the timing of interactions and in the relation between the rate of forager return and of the flow of ants into the entrance chamber. In another species of harvester ant, the rate of recruit- ment to food increased with the number of tunnels leading from the entrance chamber to the deeper nest [48]. Our heat maps of interaction rate (Fig 8) show that interactions in the entrance chamber tend to occur at the exit to the surface and at the entrances to tunnels leading down to the deeper nest. Conclusions Individual workers in social insect colonies respond to rates of interaction with other workers. Collectively, this allows a colony to regulate its behavior and respond to changing conditions. Here we show that interactions regulate both the activation of foragers and the availability of foragers to be activated. Variation among harvester ant colonies in the regulation of foraging is associated with variation in reproductive success [37]. Investigating the allocation of effort in ant colonies contributes to a fundamental question in biology: how local interactions produce the collective behavior of the whole system. Supporting Information S1 Appendix. Matlab script for recording information about ant interactions. This is a Matlab function that opens up a stack of JPEG images and allows a user to move through the images and ‘click’ anywhere on any image to record x-y coordinates of events in the frame. The output is a csv file with 6 columns: xy coordinates of the click, time = frame/image number, ant ID, ant type, and activity—both of which are pre-defined by the user, see below. Copyright (C) 2015 Noa Pinter-Wollman. (PDF) S1 Dataset. 2012 Ant Activity Data. We observed ants in two minutes of each of the nine films to determine what percentage of the ants that ascended from the deeper nest into the entrance chamber were later outgoing foragers, descending ants, or nest maintenance workers. Every five seconds, the first ant to ascend from the entrance tunnel was tracked until it left the nest, performed nest maintenance by moving dirt or debris around the nest entrance, or descended to the deeper nest. This dataset shows the activity type of each of the tracked ants. (PDF) S2 Dataset. 2012 Ant Interactions Data. We analyzed the JPEGs from the films using a cus- tom written MATLAB script as in Pinter-Wollman et al. [13] (code provided in S1 Appendix). We observed ten outgoing foragers and four or five descending ants per film, the first foraging and descending ants in the video segment that were clearly visible. This dataset shows the time each ant entered and left the entrance chamber, and the time and location of each interaction of the focal ants. (PDF) S2 Dataset. 2012 Ant Interactions Data. We analyzed the JPEGs from the films using a cus- tom written MATLAB script as in Pinter-Wollman et al. [13] (code provided in S1 Appendix). We observed ten outgoing foragers and four or five descending ants per film, the first foraging and descending ants in the video segment that were clearly visible. This dataset shows the time each ant entered and left the entrance chamber, and the time and location of each interaction of the focal ants. (PDF) S3 Dataset. 2013 Correlation Data. We observed and filmed behavior inside the nest during and after forager removals. This dataset shows our counts made from the films of the numbers of returning and outgoing foragers at the nest entrance and the number of ascending and descending ants at all tunnel entrances. Acknowledgments We thank LeAnn Howard, Andrew Merrell, Stefan Popp, Jack Rasiel, Wallis Robinson, Brett Salazar, Daniel Washington, and Charlotte Wayne for assistance in the field. We are grateful to Luke Frishkoff, Aaron Goodman, and Lane McIntosh for help with coding and plotting data. Mark Goldman provided very helpful discussion and advice. We thank Tomer Czackes and an anonymous reviewer whose comments greatly improved the manuscript. Author Contributions Conceived and designed the experiments: EP JQ NP-W DMG. Performed the experiments: EP JQ. Analyzed the data: EP JQ NP-W SC KA MBM DMG. Contributed reagents/materials/anal- ysis tools: NP-W SC KA. Wrote the paper: EP JQ NP-W DMG. Supporting Information (ZIP) S3 Dataset. 2013 Correlation Data. We observed and filmed behavior inside the nest during and after forager removals. This dataset shows our counts made from the films of the numbers of returning and outgoing foragers at the nest entrance and the number of ascending and descending ants at all tunnel entrances. (ZIP) S4 Dataset. 2013 Heat Maps Data. After splitting the 2013 videos into JPEG frames, we marked the location in the entrance chamber of each interaction of the ants we tracked. This dataset shows the locations of interactions from two instantaneous images, one at the start of S4 Dataset. 2013 Heat Maps Data. After splitting the 2013 videos into JPEG frames, we marked the location in the entrance chamber of each interaction of the ants we tracked. This dataset shows the locations of interactions from two instantaneous images, one at the start of PLOS ONE | DOI:10.1371/journal.pone.0141971 November 5, 2015 15 / 18 Interactions Regulate Foraging in Harvester Ants the removal of returning foragers, and the other immediately after forager removals ended 3–5 minutes later. (PDF) the removal of returning foragers, and the other immediately after forager removals ended 3–5 minutes later. (PDF) S1 Fig. Sampling time distribution histograms. These histograms show the distribution of times that foraging and descending ants filmed in 2012 remained in the entrance chamber. (TIF) S1 Fig. Sampling time distribution histograms. These histograms show the distribution of times that foraging and descending ants filmed in 2012 remained in the entrance chamber. (TIF) S1 Movie. Interactions over time for a single colony. The attached clip shows the location of interactions, in the form of brief antennal contacts between ants, in colony 367 on August 20, 2013. Removal of returning foragers began at 00:37 and ended at 05:41. There is an observable difference between 00:37 seconds and 2:37. By 2:37, as the rate of forager return decreases, the ants are no longer as spread out in the entrance chamber, and interactions occur only at the interaction hotspots (Fig 8), the entrance to tunnels to the deeper nest and the exit from the chamber to outside the nest. The circular metal device is a thermometer and humidity sensor. Some of the ants were painted previously for a different experiment. (MP4) S1 Table. Red harvester ant colonies filmed in August 2012 and August 2013. (DOCX) S1 Table. Red harvester ant colonies filmed in August 2012 and August 2013. (DOCX) PLOS ONE | DOI:10.1371/journal.pone.0141971 November 5, 2015 References 1. Sumpter DJT. Collective Animal Behavior. Princeton, New Jersey: Princeton University Press; 2010. 1. Sumpter DJT. Collective Animal Behavior. Princeton, New Jersey: Princeton University Press; 2010. 2. Navlakha S, Bar-Joseph Z. Distributed information processing in biological and computational systems. Commun. ACM. 2014; 58:94–102. 3. 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Städte und Orte 555 Linke, Juden, „Zigeuner“, Zwangsarbeiter, Homosexuelle und andere so genannte „Volksschädlinge“ geprägt; Folterungen und willkürliche Exekutionen in großer Zahl waren an der Tagesordnung. Betroffene haben ihn beschrieben als „fauchenden Zwerg und zappelnden Sadisten mit kreischender Stimme“. Bei Kriegsende kam er als Kriegsverbrecher in US-Untersuchungshaft, wo er 1946 seinem Leben selbst ein Ende setzte. Der leidvolle Lebensweg des unbeugsamen Widerständlers Hans Gasparitsch (1918 – 2002) wird von Peter Poguntke behandelt. Gasparitsch wuchs im proletarischen Milieu des Stuttgarter Ostens auf, einer Hochburg von Sozialisten und Kommunisten. Mit wacher Sorge beobachteten er und sein Freundeskreis den Aufstieg des Nationalsozialismus. Die Machtergreifung Hitlers war für Gasparitsch ein Signal zum offenen Widerstand durch das Anbringen nazifeindlicher Parolen im öffentlichen Raum. Er wurde gefasst, womit ein über zehnjähriger lebensgefährlicher Leidensweg durch Gefängnisse und Konzentrationslager begann, welcher erst im April 1945 im KZ Buchenwald endete. Auch nach der Befreiung setzte Gasparitsch sein reges politisches Engagement im Sinn seiner sozialistisch-kommunistischen Überzeugung in vielfältiger Weise fort. In der jungen DDR holte er das Abitur nach und studierte Journalismus und Bauingenieurwesen. Den letztgenannten Beruf übte er, nun wieder oft auch im Westen, bis 1980 aus. Erst 2000 wurde er mit einem Bundesverdienstkreuz gewürdigt. Herausgeber und Autoren haben den flüssig zu lesenden Sammelband bewusst genau siebzig Jahre nach Zusammenbruch und Befreiung vom Nazi-Regime publiziert. Ein wisHelmut Gerber senschaftlicher Apparat rundet das Buch ab. Ernst Schmidt, Hohenlohe Waldenburg. Heimatgeschichtliches Lesebuch, überarb. Fassung 2016, kommentiert und hg. vom Waldenburger Kreis für Kulturgeschichte, zum Gedenken an Ernst Schmidt (Veröffentlichungen zur Ortsgeschichte und Heimatkunde in Württembergisch Franken, Bd. 26), Waldenburg 2016. 346 S. mit 83 meist farb. Abb. € 16,– Ernst Schmidt (1874 – 1952), in Waldenburg geboren, war bis 1937 Postbeamter im württembergischen Staats- und später im Reichsdienst. Auch im Ruhestand arbeitete er während des 2. Weltkrieges eine Zeit lang als kaufmännischer Angestellter in einer Bedarfsgüter für den Krieg herstellenden Fabrik. Gegen Kriegsende nach Hohenlohe zurückgekehrt, widmete sich Ernst Schmidt ganz der Geschichte seiner Heimatstadt Waldenburg, die durch den Krieg schwer zerstört worden war. Zunächst entstanden die für seine Kinder und Geschwister abgefassten persönlichen Jugenderinnerungen und seit Winter 1944/45 das heimatgeschichtliche Lesebuch, das er 1951 im Manuskript abschloss. Schmidt stellte ihm die Widmung „Meiner leidgeprüften Heimatstadt und dem angestammten Fürstenhaus Hohenlohe-Waldenburg“ (Titelseite) voran. Die Arbeit wurde jedoch nie gedruckt, sondern stand nur als maschinenschriftliches Manuskript mit einer „Auflage“ von ca. 300 Stück für einen heimatgeschichtlich interessierten Leserkreis zur Verfügung. Dank des Engagements von ehrenamtlich engagierten Mitgliedern des Waldenburger Kreises für Kulturgeschichte konnte der Wunsch nach Veröffentlichung nach 45 Jahren realisiert werden. Die Arbeit Schmidts wurde sorgfältig überarbeitet, etwas aktualisiert und liegt nun als gedrucktes Buch vor. Erläuterungen und Ergänzungen sowie Anmerkungen ergänzen den ursprünglichen Text, wobei immer deutlich bleibt, was vom Autor und was von den Bearbeitern stammt. Im Druck bleibt sogar die Seitenverteilung der Original­ Zeitschrift für Württembergische Landesgeschichte 76 (2017) © Kommission für geschichtliche Landeskunde in Baden-Württemberg und Württembergischer Geschichts- und Altertumsverein e.V. ISSN 0044-3786 556 Buchbesprechungen fassung erkennbar, um das Auffinden von Zitaten zu ermöglichen. Durch Beigabe zahl­ reicher Abbildungen illustrierten die Bearbeiter den Text und lockerten ihn dadurch auf. Sie fügten im Anhang des Buches die Jugenderinnerungen Schmidts „Bilderbuch aus meiner Jugendzeit“ (S. 296 – 311) und einen Auszug aus dem Schriftwechsel 1945 bis 1953 zur ­Entstehung des Lesebuches bei (S. 312 – 331). Am Schluss befindet sich eine Stammtafel der Familie Schmidt (vom 18. bis 20. Jh.); ein Personenindex, ein Ortsindex sowie ein „Stichwortverzeichnis“ erleichtern dem Leser die Orientierung. Somit wurde die Arbeit eines Waldenburger Hobbyhistorikers durch ein gedrucktes Buch einem größeren Leserkreis zugänglich gemacht. Es handelt sich um eine Ehrung für Ernst Schmidt, der durch die zurückhaltende Redaktion seines Textes und durch die Bei­ fügung der – persönlichen – Jugenderinnerungen und die Stammtafel seiner Familie sehr in den Mittelpunkt gerät. Das „Heimatgeschichtliche Lesebuch“ richtet sich an Leser, die sich – wie Ernst Schmidt – der Region Hohenlohe und ganz besonders der Stadt Waldenburg eng verbunden fühlen und daher auch an ihrer Geschichte besonders interessiert sind. Peter Schiffer Markus Josef Maier, Würzburg zur Zeit des Fürstbischofs Julius Echter von Mespelbrunn (1570 – 1617). Neue Beiträge zu Baugeschichte und Stadtbild (Veröffentlichungen des Stadtarchivs Würzburg, Bd. 20), Würzburg: Verlag Ferdinand Schöningh 2016. XVII, 579 S., 80 Abb. Mit CD-ROM. ISBN 978-3-87717-857-7. € 39,90 Stadtbilder verändern sich. Trotz der intensiven Bautätigkeit in den letzten Jahrhunderten und auch der massiven Zerstörung ihrer Bausubstanz im Bombenangriff vom 16. März 1945 zum Trotz ist das Wirken des Fürstbischofs Julius Echter als Bauherr für das Erscheinungsbild der Stadt Würzburg bis heute prägend geblieben. Der Bautätigkeit in der fürstbischöflichen Residenzstadt in den Jahrzehnten um 1600 geht Markus Josef Maier in seiner von Stephan Kummer betreuten Dissertationsschrift nach. Dabei geht er höchst kenntnisreich und mit sehr viel Sinn für Details vor, ohne sich darin zu verlieren. Die vorliegende Studie kann an einige ältere Forschungs- und Überblickswerke anknüpfen. Indem Markus Josef Maier städtische Bestände sowie die Akten des Domkapitels und einzelner Stifte zu zentralen Ausgangspunkten seiner Forschung macht, kann er nicht nur die Begrenzungen der Überlieferung in den fürstlichen Unterlagen sprengen, sondern auch eine weite Perspektive auf die gesamte Bautätigkeit in der Stadt während der langen Herrschaftszeit Julius Echters einnehmen. Außerdem hat er sich eine Vielzahl von Bilddokumenten erschlossen, die er kompakt gebunden in der Mitte des Bandes zur Illustration – allerdings mit sehr knappen Bildunterschriften – präsentiert. Maiers Fragestellung bezieht sich auf der Basis dieser Quellenlage und -auswahl nicht nur auf die fürstbischöfliche Bautätigkeit, sondern auf eine umfängliche Erfassung des Stadtbildes von Würzburg und seinen Wandlungen in der Echterzeit – bis hin zu Fragen der Pflasterung und Abwasserbeseitigung (S. 45). Die eigentliche Darstellung beginnt mit einem höchst suggestiven, aber anschaulichen fiktiven Rundgang durch die Stadt Würzburg vor 1570, der auf guter Quellenkenntnis basiert. Dieser geht über eine detaillierte Aufnahme aller Bautätigkeiten zur Zeit Julius Echters hinaus, weil er die Vorstellungskraft und Phantasie des Lesers anregt. Dabei gilt, dass sich der Autor in seinen Schilderungen auf belegbare Einzelheiten konzentriert. Zugleich gewährt die Darstellung einen Einblick in die Dynamik des Untersuchungszeitraums. Diese Zeitschrift für Württembergische Landesgeschichte 76 (2017) © Kommission für geschichtliche Landeskunde in Baden-Württemberg und Württembergischer Geschichts- und Altertumsverein e.V. ISSN 0044-3786
https://openalex.org/W2989903755
http://www.scielo.br/pdf/pat/v49/1983-4063-pat-49-e55628.pdf
English
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Behavioral aspects of Helicoverpa armigera in the cotton vegetative phase1
Pesquisa Agropecuária Tropical
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RESUMO Aspectos comportamentais de Helicoverpa armigera na fase vegetativa do algodoeiro Helicoverpa armigera (Hübner) is part of an important complex of insects-pests that attack the cotton crop. This study aimed to identify the preferential plant parts for the oviposition of moths, as well as the movement and feeding behavior of caterpillars, in the vegetative phase of the cotton cultivar BRS 369 RF. Plants emitting floral buds were used to study the oviposition behavior of moths. For this, a completely randomized design, with three treatments (adaxial and abaxial sides of the leaf and floral buds) and twenty replications (plants in pots), was used. In order to evaluate the behavior of the caterpillars in the cotton vegetative phase, another 3 × 5 factorial design, with nine replications, was used. The treatments were three ages (first, second and third instars) combined with five behaviors (walking, resting, waving, eating and drop-off). In this trial, the period of the day when caterpillars are more active was also evaluated. It was observed that H. armigera moths prefer to lay their eggs on the adaxial leaf side and on the last two nodes of the plant, in a descending direction. The adaxial leaf side is also the part of the plant preferred by caterpillars for walking, eating and resting, being this last behavior the most frequent in all instars. In general, the caterpillars walk more in the morning. Helicoverpa armigera (Hübner) faz parte de um importante complexo de insetos-pragas que atacam a cultura do algodoeiro. Objetivou-se identificar partes da planta preferenciais para a oviposição das mariposas, bem como os comportamentos de movimentação e alimentação das lagartas, na fase vegetativa da cultivar de algodão BRS 369 RF. Plantas emitindo botões florais foram utilizadas para estudar o comportamento de oviposição das mariposas. Para isso, utilizou-se delineamento inteiramente casualizado, com três tratamentos (faces adaxial e abaxial da folha e botões florais) e vinte repetições (plantas em vasos). Para avaliar o comportamento das lagartas na fase vegetativa do algodoeiro, adotou-se outro delineamento fatorial 3 × 5, com nove repetições. Os tratamentos foram três idades (1º, 2º e 3º ínstares) combinadas com cinco comportamentos (caminhar, repousar, acenar, comer e cair). Neste ensaio, o período do dia em que as lagartas são mais ativas também foi avaliado. Constatou-se que as mariposas de H. armigera preferem ovipositar na face adaxial da folha e nos dois últimos nós da planta, em direção descendente. 1. Received: Oct. 30, 2018. Accepted: Apr. 17, 2019. Published: Oct. 15, 2019. DOI: 10.1590/1983-40632019v4955628. 2. Empresa Brasileira de Pesquisa Agropecuária (Embrapa Algodão), Campina Grande, PB, Brasil. E-mail/ORCID: sandra.rodrigues@embrapa.br/0000-0001-9043-4694. 3. Universidade Federal de Mato Grosso, Instituto de Ciências Agrárias e Ambientais, Sinop, MT, Brasil. E-mail/ORCID: nubiadocarmo@hotmail.com/0000-0001-9923-4437, janadenadai@gmail.com/0000-0002-1013-5414. 4. Empresa Brasileira de Pesquisa Agropecuária (Embrapa Agrossilvipastoril), Sinop, MT, Brasil. E-mail/ORCID: rafael.pitta@embrapa.br/0000-0002-8503-7106. e-ISSN 1983-4063 - www.agro.ufg.br/pat - Pesq. Agropec. Trop., Goiânia, v. 49, e55628, 2019 Research Article RESUMO A face adaxial da folha também é a parte da planta preferida pelas lagartas para caminhar, comer e repousar, sendo este último comportamento o mais frequente em todos os ínstares. Em geral, as lagartas caminham mais no período matutino. PALAVRAS-CHAVE: Gossypium hirsutum, Heliothinae, etologia. KEYWORDS: Gossypium hirsutum, Heliothinae, ethology. PALAVRAS-CHAVE: Gossypium hirsutum, Heliothinae, etologia. MATERIAL AND METHODS The experiments were carried out at the Embrapa Agrossilvipastoril (Sinop, Mato Grosso state, Brazil), in 2017. The moths and caterpillars used were obtained from those reared in laboratory, fed with an artificial diet proposed by Parra (2001). The use of an artificial diet removed the possibility of alterations in the adult behavior due to host plant experience in the larval stage. Seeds of the BRS 369 RF cotton cultivar (not Bt) were sown into 5 L pots and kept in a greenhouse, and the cotton plant received the necessary cultural treatment for its development. l Heliothinae moths prefer to feed and oviposit on plants that contain flowers and nectar (Cunninghan & Zalucki 2014). The females oviposit in cotton at night and eggs are laid single (Topper 1987, Ahmad 2004). The attack may begin in the vegetative phase, with small caterpillars consuming the leaves (Pascua & Pascua 2002), and then later in the reproductive phase, with larger caterpillars feeding on flower buds, flowers and bolls (Gomes et al. 2017). Experimental evidence for learning of H. armigera has demonstrated that a previous experience with a host species increases the relative attractiveness of that host (Cunningham et al. 1998). Therefore, preferences for different hosts displayed by adult moths which have encountered a host species may be vastly different from the preferences of moths without any host experience (Cunningham et al. 1999). When the plants began to emit flower buds, they were then taken to the laboratory and housed in a cage (1 m × 1 m × 1 m), in groups of five plants, and kept in a controlled environment (25 ± 1 ºC and 60 ± 1 % RH), with a 13-h scotophase, since oviposition occurs at night (Topper 1987). The moths used had 72 h of emergence and were previously mated and fed with a honey solution. At 6 p.m., the moths were released into the cages and remained in the plants for 13 h. The ratio used was one moth to each plant and, in the following morning, the number of eggs deposited on the adaxial and abaxial surface of the leaves and floral buds were counted, as well as the vertical distribution of the eggs on the plants. The main method for controlling this pest has been to use synthetic insecticides, what is often performed inadequately (Costa et al. 2018). INTRODUCTION Mato Grosso state is the largest producer, with 777.8 thousand hectares of cotton, corresponding to 66.1 % of the area planted in the country. The projected cotton seed production for this state is 3,213.9 thousand tons, 27.1 % higher than in the 2016/2017 harvest, with a yield of 4,132 kg ha-1 (Conab 2018). Brazil is among the largest cotton producers and the planted area in the 2017/2018 crop season was 1,176.1 thousand hectares, with an expected production of 4,947,000 tons of cotton seed and 1,979.4 thousand tons of fiber. The Central-West region is responsible for 70.4 % of this production, where the The crop is attacked by a complex of insects- pests capable of infesting all the phenological plant S. M. M. Rodrigues et al. (2019) 2 phases (Santos 2015), and its production may be significantly reduced if control measures are not adopted at the right time. Within this complex is Helicoverpa armigera (Hübner) (Lepidoptera: Noctuidae), a pest detected in Brazil in 2013, in soybean and cotton crops (Czepak et al. 2013). This pest is important worldwide, because it has a wide geographic distribution, polyphagous behavior and high biotic potential (Zalucki et al. 1986, Behere et al. 2013, Silva et al. 2018). the moths’ preferential oviposition plant sites in the reproductive phase of the cotton crop, as well as the caterpillars’ behavior of the 1st, 2nd and 3rd instars, in the vegetative phase of the cotton crop. e-ISSN 1983-4063 - www.agro.ufg.br/pat - Pesq. Agropec. Trop., Goiânia, v. 49, e55628, 2019 MATERIAL AND METHODS The crawling, resting, waving, feeding and drop-off behaviors were observed regarding the frequency of use of the different plant sites. Waving, according to Johnson & Zalucki (2005), is when the caterpillar has its front legs raised and shakes its head from side to side, and this indicates a search behavior. The observations were made with the aid of a pocket magnifying glass (40x magnification) and performed every 30 min. Furthermore, the period of the day when the largest larval movement occurs and which plant region is preferred by the caterpillars to carry out the five behaviors were also evaluated. The adaxial surface (18.7 eggs) was the most preferred, followed by the abaxial surface (13.5 eggs) and the floral bud (3.4 eggs) (Table 1), similarly to the results observed by Butter & Singh (1996), in cotton. It was found that the eggs present on the adaxial surface of the leaf were generally on the leaf veins which have trichomes. It is known that the plant morphological and chemical characteristics are involved in choosing the oviposition site (Navasero & Ramaswamy 1991). Sites with trichomes are probably chosen by H. armigera to oviposite (Zalucki et al. 1986, Luong et al. 2016) because these structures may hinder the movement of natural enemies when searching for their prey or hosts, resulting in protection for the pests’ progeny (Vendramim 2002). if A significant difference was observed between the nodes, regarding the vertical distribution of the eggs in the plant. All nodes presented leaves with the presence of eggs, except the node one. The largest ovipositions occurred at nodes 8 (6.2 eggs) and 9 (6.5 eggs), corresponding to the nodes at the top of the plant (Figure 1). Its preference for the upper third of the plant is closely related to its food habit, since it mainly attacks flower buds and cotton flowers that are concentrated in the middle and upper thirds of the plant. Olsen & Daly (2000) also verified this fact in cotton and Santos et al. (2017) in soybean, a behavior also found in Chloridea virescens and H. zea Heliothinae (Torres & Ruberson 2006). To study the caterpillar behavior in the cotton vegetative phase, other completely randomized design, with nine replications, was used. MATERIAL AND METHODS The continuous use of insecticides has selected populations of pests which are resistant to various molecules (Mota-Sanches & Wise 2018). Another factor related to the ineffectiveness of insecticides is the pest location in parts of the plant which hinder the deposition of insecticide drops, thus compromising the minimum amount of active ingredient to cause the target death (Costa et al. 2018). In order to perform both sampling and application of insecticides efficiently, it is necessary to know the habits of the target insect. Considering that caterpillars in the first instars are more vulnerable to insecticides, knowing the behavior of the pest in these stages is fundamental to assist in the decision-making on managing this pest. Such information will enable detecting how best to sample the insect in the field, as well as in what period of the day it will be most exposed to the applied chemicals. To study the oviposition behavior of moths, a completely randomized design, with three treatments (adaxial and abaxial leaf surface and floral buds) and twenty replications (plants in pots), was used. For the statistical analysis, the data were transformed by √(x+1), to correct the heterogeneity of the variances before being submitted to the analysis of variance (Anova), and the means were compared by the Scott-Knott test when there were significant treatment effects (α = 0.05). The number of nodes constituted the treatments in the vertical distribution of the eggs on the plant. The nodes refer to the place where the branch goes out from the stem and were counted upwards, being located just above the cotyledon node. The data were analyzed with the same aforementioned procedure. The separation of the caterpillars by instar was done by observing the replacement of the cephalic capsule. Plants ranging between V4 and V5 were Due to the importance of H. armigera in the Brazilian cotton crop, this research aimed to study Behavioral aspects of Helicoverpa armigera in the cotton vegetative phase 3 used to evaluate the behavior in the vegetative phase, according to the scale by Marur & Ruano (2001). The first evaluation was carried out 20 min after distributing the caterpillars on the leaves. The caterpillars remained on the plants for 12 h, from 6 a.m. to 6 p.m., and the ratio used was one caterpillar per plant, with no replacement or disposal of caterpillars. MATERIAL AND METHODS First, the treatments were assumed in a 3 x 5 factorial scheme, in which three ages (1st, 2nd and 3rd instars) were combined with five behaviors (crawling, resting, waving, feeding and drop-off). To evaluate the period of the day when caterpillars are more active, the treatments assumed a 3 x 2 factorial, combining the three instars with two periods of the day (morning and evening). Similarly, to identify the plant region preferred by the caterpillars, another 3 x 5 factorial was assumed, in which the treatments were the three ages combined with five plant parts (adaxial surface, abaxial surface, leaflet, stem and petiole). Data analyses were the same as described before, using transformations as √(x+0.5) (for count data) or arcoseno√(x+0.5) (for percentage data) to correct the heterogeneity of the variances. Regarding the behaviors of the caterpillars in the cotton vegetative phase, no difference (p > 0.01) among the H. armigera instars was observed (Figure 2). However, differences were observed (p < 0.01) when analyzing the behavior within each instar. During the evaluation period of each instar, it was observed that the caterpillars often presented alternating behaviors. In the three studied instars, it was noticed that the caterpillars most frequent behavior was to rest, and this differed from the others. RESULTS AND DISCUSSION Table 1. Means and standard errors (SE) for number of eggs per plant of Helicoverpa armigera moths in cotton plants. 1 Original data. 2 Means followed by the same letter do not differ by the Scott-Knott test at 5 % of significance [for the analysis, data were transformed to √(x+1); F-test. = 18.93; p < 0.001; d.f. = 37]. Treatment Mean ± SE1,2 Adaxial leaf surface 18.7 ± 2.13 a Abaxial leaf surface 13.5 ± 2.38 b Floral bud 3.4 ± 0.68 c CV (%) 33.04 Table 1. Means and standard errors (SE) for number of eggs per plant of Helicoverpa armigera moths in cotton plants. The moths oviposited on the adaxial and abaxial leaf surfaces and floral bud (Table 1). It was found a significant difference between treatments by the Scott-Knott test (p = 0.05). Even without a prior experience and not knowing a cotton plant, moths oviposited in all parts of the evaluated plants. Cunnighan et al. (1999) reported that the inexperience with the host plant may influence the selection of hosts by the moth, but this did not occur in this research. 1 Original data. 2 Means followed by the same letter do not differ by the Scott-Knott test at 5 % of significance [for the analysis, data were transformed to √(x+1); F-test. = 18.93; p < 0.001; d.f. = 37]. e-ISSN 1983-4063 - www.agro.ufg.br/pat - Pesq. Agropec. Trop., Goiânia, v. 49, e55628, 2019 4 S. M. M. Rodrig 0 2 4 6 8 10 1 2 3 4 5 6 7 8 9 Eggs (nº mean ± SE) Nodes of cotton plant b b b b b b a a Figure 1. Means and standard errors (SE) for number of eggs of Helicoverpa armigera moths in different nodes of cotton plants. Means followed by the same letter do not differ by the Scott-Knott test at 1% of probability. S. M. M. Rodrigues et al. (2019) 4 0 2 4 6 8 10 1 2 3 4 5 6 7 8 9 Eggs (nº mean ± SE) Nodes of cotton plant b b b b b b a a of finding food with higher nutritional quality, as well as natural enemies (Johnson & Zalucki 2005). Control tactics based on the period of day that the caterpillar moves the most may be established, what increases the probability of reaching the target pest, since it will be more exposed. RESULTS AND DISCUSSION 2 Means followed by the same lowercase letter in the column, or upper case in the row, do not differ from each other by the F-test at 5 % of significance. Instar Period Mean1,2 Morning Evening First 7.6 ± 2.78 8.0 ± 2.21 7.8 ± 1.73a Second 12.4 ± 3.23 5.8 ± 1.78 9.1 ± 1.96a Third 7.6 ± 2.05 4.4 ± 2.05 6.0 ± 1.46a Mean 9.2 ± 1.58 A 6.1 ± 1.16 B CV (%) = 51.81 Table 2. Means and standard errors (SE) for crawling frequency (%) of Helicoverpa armigera larvae, at two periods of the day, on cotton plants. Table 2. Means and standard errors (SE) for crawling frequency (%) of Helicoverpa armigera larvae, at two periods of the day, on cotton plants. Table 2. Means and standard errors (SE) for crawling frequency (%) of Helicoverpa armigera larvae, at two periods of the day, on cotton plants. 1 Original values. 2 Means followed by the same lowercase letter in the column, or upper case in the row, do not differ from each other by the F-test at 5 % of significance. Instar Period Mean1,2 Morning Evening First 7.6 ± 2.78 8.0 ± 2.21 7.8 ± 1.73a Second 12.4 ± 3.23 5.8 ± 1.78 9.1 ± 1.96a Third 7.6 ± 2.05 4.4 ± 2.05 6.0 ± 1.46a Mean 9.2 ± 1.58 A 6.1 ± 1.16 B CV (%) = 51.81 Figure 2. Behavior of Helicoverpa armigera larvae on cotton plants by instar. Mean frequency followed by the same letter, within each instar, do not differ by the Scott- Knott test at 1 % of probability. The bar above each column is the standard error of the respective mean. 1 Original values. 2 Means followed by the same lowercase letter in the column, or upper case in the row, do not differ from each other by the F-test at 5 % of significance. 0 1 2 3 4 5 adaxial surface abaxial surface leaflet stem petiole Frequency (nº) plant part 1st instar 2nd instar 3rd instar a b b b b a b b b a b c c 0 1 2 3 4 5 adaxial surface abaxial surface leaflet stem petiole Frequency (nº) plant part 1st instar 2nd instar 3rd instar a b c a a b a a c bf Figure 3. RESULTS AND DISCUSSION Preference of Helicoverpa armigera larvae to crawling in cotton plant parts, in the vegetative phase, by instar. Mean frequency followed by the same letter, within each instar, do not differ by the Scott-Knott test at 1 % of probability. The bar above each column is the standard error of the respective mean. 0 1 2 3 4 5 adaxial surface abaxial surface leaflet stem petiole Frequency (nº) plant part 1st instar 2nd instar 3rd instar a b b b b a b b b a b c c 0 1 2 3 4 5 adaxial surface abaxial surface leaflet stem petiole Frequency (nº) plant part 1st instar 2nd instar 3rd instar a b c a a b a a c bf The fact that resting occurs more often is possibly related to the digestive process of caterpillars. The crawling and feeding behaviors were similar for the three evaluated instars. Johnson & Zalucki (2005, 2007) obtained results similar to those of the present study, when they evaluated the behavior of H. armigera in the vegetative stage of Vigna radiata L. However, the first and third instars in this research differed for the waving and drop-off behaviors on the plant, with waving being more frequent than drop-off. The fact that resting occurs more often is possibly related to the digestive process of caterpillars. The crawling and feeding behaviors were similar for the three evaluated instars. Johnson & Zalucki (2005, 2007) obtained results similar to those of the present study, when they evaluated the behavior of H. armigera in the vegetative stage of Vigna radiata L. However, the first and third instars in this research differed for the waving and drop-off behaviors on the plant, with waving being more frequent than drop-off. Regarding the period of the day when the caterpillar moved the most, an absence of interaction between period and instars in all the evaluated behaviors was observed, except for the crawling variable, since it was verified that all instars move more in the morning (Table 2). The caterpillars movement on the plant may increase the probability Figure 3. Preference of Helicoverpa armigera larvae to crawling in cotton plant parts, in the vegetative phase, by instar. Mean frequency followed by the same letter, within each instar, do not differ by the Scott-Knott test at 1 % of probability. The bar above each column is the standard error of the respective mean. RESULTS AND DISCUSSION During the evaluation period in the instars, it was observed that the caterpillars moved in different regions of the plants, and a generally similar behavior pattern was observed in the analysis of these regions (Figures 3, 4 and 5). First-instar caterpillars always preferred the adaxial surface for crawling (Figure 3), resting (Figure 4), waving (Figure 5) and feeding (Figure 6), differing from other sites on the plant. For the acts of crawling and waving, there was no significant difference between the abaxial surface, leaflet, stem and petiole, while, for resting and feeding, the abaxial surface of the leaf differed from the rest of the treatments. Figure 1. Means and standard errors (SE) for number of eggs of Helicoverpa armigera moths in different nodes of cotton plants. Means followed by the same letter do not differ by the Scott-Knott test at 1% of probability. 0 1 2 3 4 5 crawling resting waving feeding drop off Frequency (nº) behavior 1st instar 2nd instar 3rd instar a a a b b b b b b c c c d c d - Figure 2. Behavior of Helicoverpa armigera larvae on cotton plants by instar. Mean frequency followed by the same letter, within each instar, do not differ by the Scott- Knott test at 1 % of probability. The bar above each column is the standard error of the respective mean. 0 1 2 3 4 5 crawling resting waving feeding drop off Frequency (nº) behavior 1st instar 2nd instar 3rd instar a a a b b b b b b c c c d c d - 0 1 2 3 4 5 adaxial surface abaxial surface leaflet stem petiole Frequency (nº) plant part 1st instar 2nd instar 3rd instar a b b b b a b b b a b c c 0 1 2 3 4 5 adaxial surface abaxial surface leaflet stem petiole Frequency (nº) plant part 1st instar 2nd instar 3rd instar a b c a a b a a c bf the rest of the treatments. Figure 3. Preference of Helicoverpa armigera larvae to crawling in cotton plant parts, in the vegetative phase, by instar. Mean frequency followed by the same letter, within each instar, do not differ by the Scott-Knott test at 1 % of probability. The bar above each column is the standard error of the respective mean. 1 Original values. Behavioral aspects of Helicoverpa armigera in the cotton vegetative phase Behavioral aspects of Helicoverpa armigera in the cotton vegetative phase 5 Figure 4. Preference of Helicoverpa armigera larvae to resting in cotton plant parts, in the vegetative phase, by instar. Mean frequency followed by the same letter, within each instar, do not differ by the Scott-Knott test at 1 % of probability. The bar above each column is the standard error of the respective mean. 0 2 4 6 8 10 12 adaxial surfaceabaxial surface leaflet stem petiole Frequency (nº) plant part 1st instar 2nd instar 3rd instar a b c c b a a b c c a b c 0 1 2 3 4 5 adaxial surface abaxial surface leaflet stem petiole Frequency (nº) plant part 1st instar 2nd instar 3rd instar a b c a a b a a c b 0 2 4 6 8 10 12 adaxial surfaceabaxial surface leaflet stem petiole Frequency (nº) plant part 1st instar 2nd instar 3rd instar a b c c b a a b c c a b c 0 1 2 3 4 5 adaxial surface abaxial surface leaflet stem petiole Frequency (nº) plant part 1st instar 2nd instar 3rd instar a b c a a b a a c b When evaluating the second-instar caterpillars for crawling (Figure 3) and feeding (Figure 6), it was noted that the preferred region was also the adaxial surface of the leaf. For the acts of resting (Figure 4) and waving (Figure 5), there was no significant difference when comparing the adaxial with the abaxial surface, but these behaviors differed from the others. In relation to the third-instar caterpillars, the adaxial surface of the leaf is again observed for crawling (Figure 3), resting (Figure 4) and feeding (Figure 6), while the caterpillars preferred the adaxial and abaxial surfaces, regarding the waving behavior (Figure 5).i Figure 4. Preference of Helicoverpa armigera larvae to resting in cotton plant parts, in the vegetative phase, by instar. Mean frequency followed by the same letter, within each instar, do not differ by the Scott-Knott test at 1 % of probability. The bar above each column is the standard error of the respective mean. The caterpillars of the first three instars generally preferred the adaxial surface of the cotton leaf to move, rest and eat. This preference may be associated with a positive phototropism, which is a common behavior in lepidoptera (Perkins et al. 2008). Behavioral aspects of Helicoverpa armigera in the cotton vegetative phase Johnson & Zalucki (2007) also reported that third-instar H. armigera fed more frequently on exposed areas of mature V. radiata leaves. However, this result differs from that obtained by Cribb et al. (2010), who verified first-instar H. armigera caterpillars searching for closed spaces and positioning themselves on the abaxial surface of pea leaves, when there was light. The divergence between the results obtained in this research and those of other authors may be related to the fact that the feeding site chosen by a caterpillar is influenced by factors such as plant architecture, light intensity, nutritional value, presence of trichomes and wax, microclimate and volatiles (Navasero & Ramaswamy 1991, Cribb et al. 2010, Perkins et al. 2013). Costa et al. (2018) reported that the highest deposition of insecticides was observed in the upper third of cotton plants, when they used 140 L ha-1 and 194 L ha-1 flow rates. As the larvae of these instars are often on the adaxial surface and prefer to move in the morning, this indicates that they will be exposed to sprays. 0 1 2 3 4 5 adaxial surface abaxial surface leaflet stem petiole Frequency (nº) plant part 1st instar 2nd instar 3rd instar a b b b a a b a a 0 1 2 3 4 5 adaxial surface abaxial surface leaflet stem petiole Frequency (nº) plant part 1st instar 2nd instar 3rd instar a b b b a a b a a Figure 5. Preference of Helicoverpa armigera larvae to waving in cotton plant parts, in the vegetative phase, by instar. Mean frequency followed by the same letter, within each instar, do not differ by the Scott-Knott test at 1 % of probability. The bar above each column is the standard error of the respective mean. Figure 5. Preference of Helicoverpa armigera larvae to waving in cotton plant parts, in the vegetative phase, by instar. Mean frequency followed by the same letter, within each instar, do not differ by the Scott-Knott test at 1 % of probability. The bar above each column is the standard error of the respective mean. Behavioral aspects of Helicoverpa armigera in the cotton vegetative phase 0 1 2 3 4 5 adaxial surface abaxial surface leaflet stem petiole Frequency (nº) plant part 1st instar 2nd instar 3rd instar a b c a a b a a c b 0 1 2 3 4 5 adaxial surface abaxial surface leaflet stem petiole Frequency (nº) plant part 1st instar 2nd instar 3rd instar a b c a a b a a c b Figure 6. Preference of Helicoverpa armigera larvae to feeding in cotton plant parts, in the vegetative phase, by instar. Mean frequency followed by the same letter, within each instar, do not differ by the Scott-Knott test at 1 % of probability. The bar above each column is the standard error of the respective mean. RESULTS AND DISCUSSION e-ISSN 1983-4063 - www.agro.ufg.br/pat - Pesq. Agropec. Trop., Goiânia, v. 49, e55628, 2019 REFERENCES JOHNSON, M. L.; ZALUCKI, M. P. Feeding and foraging behaviour of a generalist caterpillar: are third instars just bigger versions of firsts? Bulletin of Entomological Research, v. 97, n. 1, p. 81-88, 2007. AHMAD, M. A comprehensive review on cotton bollworm, Helicoverpa armigera (Hübner). Journal of Agricultural Research, v. 42, n. 1, p. 73-106, 2004. JOHNSON, M. L.; ZALUCKI, M. P. Foraging behavior of Helicoverpa armigera first instar larvae on crop plants of different developmental stages. Journal of Applied Entomology, v. 129, n. 5, p. 239-245, 2005. BEHERE, G. T.; TAY, W. T.; RUSSEL, D. A.; KRANTHI, K. R.; BATTERHAM, P. Population genetic structure of the cotton bollworm Helicoverpa armigera (Hübner) (Lepidoptera: Noctuidae) in India as inferred from EPIC PCR DNA Markers. Plos One, v. 8, n. 1, e53448, 2013. LUONG, T. T. A.; DOWNES, S. 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E.; PERINA, F. J.; WADT, L. G.; CHAIM, A. Aspectos da tecnologia de aplicação para o manejo de lepidópteros-praga. Luís Eduardo Magalhães: Fundação de Apoio à Pesquisa e Desenvolvimento do Oeste Baiano, 2018. (Boletim técnico, 5). NAVASERO, R. C.; RAMASWAMY, S. B. Morphology of leaf surface trichomes and its influence on egglaying by Heliothis virescens. Crop Science, v. 31, n. 2, p. 342- 353, 1991. CRIBB, B. W.; HANAN, J.; ZALUCKI, M. P.; PERKINS, L. E. Effects of plant microenvironment on movement of Helicoverpa armigera (Hübner) larvae and the relationship to a hierarchy of stimuli. Arthropod-Plant Interactions, v. 4, n. 1, p. 165-173, 2010. OLSEN, K. M.; DALY, J. C. CONCLUSIONS 1. Helicoverpa armigera moths prefer to oviposit on the adaxial leaf surface and in the upper third of the cotton canopy; Figure 6. Preference of Helicoverpa armigera larvae to feeding in cotton plant parts, in the vegetative phase, by instar. Mean frequency followed by the same letter, within each instar, do not differ by the Scott-Knott test at 1 % of probability. The bar above each column is the standard error of the respective mean. 2. H. armigera caterpillars crawl more in the morning, and the adaxial cotton leaf surface is also the plant region preferred by caterpillars for crawling, feeding and resting, regardless of the age (first, second or third instars). e-ISSN 1983-4063 - www.agro.ufg.br/pat - Pesq. Agropec. Trop., Goiânia, v. 49, e55628, 2019 S. M. M. Rodrigues et al. (2019) 6 REFERENCES Plant-toxin interactions in transgenic Bt cotton and their effect on mortality of Helicoverpa armigera (Lepidoptera: Noctuidae). Journal of Economic Entomology, v. 93, n. 4, p. 1293-1299, 2000. CUNNINGHAM, J. P.; ZALUCKI, M. P. Understanding Heliothine (Lepidoptera: Heliothinae) pests: what is a host plant? Journal of Economic Entomology, v. 107, n. 3, p. 881-896, 2014. PARRA, J. R. P. Técnicas de criação de insetos para programas de controle biológico. Piracicaba: FEALQ, 2001. CUNNINGHAN, J. P.; WEST, S. A.; WRIGHT, D. J. Learning in the nectar foraging behavior of Helicoverpa armigera. Ecological Entomology, v. 23, n. 4, p. 363-369, 1998. PASCUA, L. T.; PASCUA, E. M. The distribution and movement of cotton bollworm, Helicoverpa armigera Hübner (Lepidoptera: Noctuidae) larvae on cotton. Philippine Journal of Science, v. 131, n. 2, p. 91-98, 2002. CUNNINGHAN, J. P.; ZALUCKI, M. P.; WEST, S. A. Learning in Helicoverpa armigera (Lepidoptera: Noctuidae): a new look at the behaviour and control of a polyphagous pest. Bulletin of Entomological Research, v. 89, n. 3, p. 201-207, 1999. PERKINS, L. E.; CRIBB, B. W.; BREWER, P. B.; HANAN, J.; GRANT, M.; TORRES, M.; ZALUCKI, M. P. Generalist insects behave in a jasmonate-dependent manner on their host plants, leaving induced areas quickly and staying longer on distant parts. Proceedings of the Royal Society B, v. 280, n. 1756, 2013. Available at: http://rspb. royalsocietypublishing.org/content/280/1756/20122646. Access on: 21 Sep. 2018. CZEPAK, C.; ALBERNAZ, K. C.; VIVAN, L. M.; GUIMARÃES, H. O.; CARVALHAIS, T. Primeiro registro de ocorrência de Helicoverpa armigera (Hübner) (Lepidoptera: Noctuidae) no Brasil. Pesquisa Agropecuária Tropical, v. 43, n. 1, p. 110-113, 2013. PERKINS, L. E.; CRIBB, B. W.; HANAN, J.; GLAZE, E.; BEVERIDGE, C.; ZALUCKI, M. P. Where to from here?: the mechanisms enabling the movement of first instar caterpillars on whole plants using Helicoverpa armigera (Hübner). Arthropod-Plant Interactions, v. 2, n. 1, p. 197-207, 2008. GOMES, E. S.; SANTOS, V.; ÁVILA, C. J. Biology and fertility life table of Helicoverpa armigera (Lepidoptera: Noctuidae) in different hosts. Entomological Science, v. 20, n. 1, p. 419-426, 2017. e-ISSN 1983-4063 - www.agro.ufg.br/pat - Pesq. Agropec. Trop., Goiânia, v. 49, e55628, 2019 7 Behavioral aspects of Helicoverpa armigera in the cotton vegetative phase SANTOS, W. J. Manejo das pragas do algodão com destaque para o Cerrado brasileiro. In: FREIRE, E. C. (ed.). Algodão no Cerrado do Brasil. Brasília, DF: Abrapa, 2015. p. 267-347. TORRES, J. B.; RUBERSON, J. R. e-ISSN 1983-4063 - www.agro.ufg.br/pat - Pesq. Agropec. Trop., Goiânia, v. 49, e55628, 2019 REFERENCES Spatial and temporal dynamics of oviposition behavior of bollworm and three of its predators in Bt and non-Bt cotton fields. Entomologia Experimentalis et Applicata, v. 120, n. 1, p. 11-22, 2006. SILVA, I. F.; BALDIN, E. L. L.; SPECHT, A.; SOSA- GÓMEZ, D. R.; ROQUE-SPECHT, V. R.; MORANDO, R.; PAULA-MORAES, S. V. Biotic potential and life table of Helicoverpa armigera (Hübner) (Lepidoptera: Noctuidae) from three Brazilian regions. Neotropical Entomology, v. 47, n. 3, p. 344-351, 2018. VENDRAMIM, J. D. O controle biológico e a resistência de plantas. In: PARRA. J. R. P.; BOTELHO, P. S. M.; CORRÊA-FERREIRA, B. S.; BENTO, J. M. S. (ed.). Controle biológico no Brasil: parasitoides e predadores. São Paulo: Manole, 2002. p. 511-528. TOPPER, C. P. The dynamics of the adult population of Heliothis armigera (Hübner) (Lepidoptera: Noctuidae) within the Sudan Gezira in relation to cropping pattern and pest control on cotton. Bulletin of Entomological Research, v. 77, n. 3, p. 525-539, 1987. ZALUCKI, M. P.; DAGLISH, G.; FIREMPONG, S.; TWINE, P. The biology and ecology of Heliothis armigera (Hübner) and H. punctigera Wallengren (Lepidoptera: Noctuidae) in Australia: what do we know? Australian Journal of Zoology, v. 34, n. 6, p. 779-814, 1986.
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Diagnosing Polyparasitism in a High-Prevalence Setting in Beira, Mozambique: Detection of Intestinal Parasites in Fecal Samples by Microscopy and Real-Time PCR
PLoS neglected tropical diseases
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RESEARCH ARTICLE Background Many different intestinal parasite species can co-occur in the same population. However, classic diagnostic tools can only frame a particular group of intestinal parasite species. Hence, one or two tests do not suffice to provide a complete picture of infecting parasite spe- cies in a given population. The present study investigated intestinal parasitic infections in Beira, Mozambique, i.e. in the informal settlement of Inhamudima. Diagnostic accuracy of five classical microscopy techniques and real-time PCR for the detection of a broad spec- trum of parasites was compared. Editor: William Evan Secor, Centers for Disease Control and Prevention, UNITED STATES Control and Prevention, UNITED STATES Received: June 26, 2016 Accepted: January 9, 2017 Published: January 23, 2017 Copyright: © 2017 Meurs 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. OPEN ACCESS Citation: Meurs L, Polderman AM, Vinkeles Melchers NVS, Brienen EAT, Verweij JJ, Groosjohan B, et al. (2017) Diagnosing Polyparasitism in a High-Prevalence Setting in Beira, Mozambique: Detection of Intestinal Parasites in Fecal Samples by Microscopy and Real-Time PCR. PLoS Negl Trop Dis 11(1): e0005310. doi:10.1371/journal.pntd.0005310 Diagnosing Polyparasitism in a High- Prevalence Setting in Beira, Mozambique: Detection of Intestinal Parasites in Fecal Samples by Microscopy and Real-Time PCR Lynn Meurs1,2, Anton M. Polderman1, Natalie V. S. Vinkeles Melchers1¤a, Eric A. T. Brienen1, Jaco J. Verweij1¤b, Bernhard Groosjohan3, Felisberto Mendes3, Manito Mechendura3, Dagmar H. Hepp1¤c, Marijke C. C. Langenberg1, Rosanne Edelenbosch1¤d, Katja Polman2, Lisette van Lieshout1* Lynn Meurs1,2, Anton M. Polderman1, Natalie V. S. Vinkeles Melchers1¤a, Eric A. T. Brienen1, Jaco J. Verweij1¤b, Bernhard Groosjohan3, Felisberto Mendes3, Manito Mechendura3, Dagmar H. Hepp1¤c, Marijke C. C. Langenberg1, Rosanne Edelenbosch1¤d, Katja Polman2, Lisette van Lieshout1* a1111111111 a1111111111 a1111111111 a1111111111 a1111111111 a1111111111 a1111111111 a1111111111 a1111111111 a1111111111 1 Department of Parasitology, Leiden University Medical Center, Leiden, The Netherlands, 2 Department of Biomedical Sciences, Institute of Tropical Medicine, Antwerp, Belgium, 3 Faculty of Health Science, Catholic University of Mozambique, Beira, Mozambique ¤a Current address: Department of Public Health, University Medical Center Rotterdam (ErasmusMC), Rotterdam, The Netherlands ¤a Current address: Department of Public Health, University Medical Center Rotterdam (ErasmusMC), Rotterdam, The Netherlands ¤b Current address: Laboratory for Medical Microbiology and Immunology, St. Elisabeth Hospital, Tilburg, The Netherlands ¤c Current address: Neurology Department, VU University Medical Center, Amsterdam, The Netherlands ¤d Current address: Rathenau Institute, The Hague, The Netherlands * E.A.van_Lieshout@lumc.nl ¤a Current address: Department of Public Health, University Medical Center Rotterdam (ErasmusMC), Rotterdam, The Netherlands ¤b Current address: Laboratory for Medical Microbiology and Immunology, St. Elisabeth Hospital, Tilburg, The Netherlands ¤c Current address: Neurology Department, VU University Medical Center, Amsterdam, The Netherlands ¤d Current address: Rathenau Institute, The Hague, The Netherlands * E.A.van_Lieshout@lumc.nl ¤b Current address: Laboratory for Medical Microbiology and Immunology, St. Elisabeth Hospital, Tilburg, The Netherlands Methodology/Principal Findings A cross-sectional population-based survey was performed. One stool sample per participant (n = 303) was examined by direct smear, formal-ether concentration (FEC), Kato smear, Baermann method, coproculture and real-time PCR. We found that virtually all people (96%) harbored at least one helminth, and that almost half (49%) harbored three helminths or more. Remarkably, Strongyloides stercoralis infections were widespread with a preva- lence of 48%, and Ancylostoma spp. prevalence was higher than that of Necator ameri- canus (25% versus 15%), the hookworm species that is often assumed to prevail in East- Africa. Among the microscopic techniques, FEC was able to detect the broadest spectrum of parasite species. However, FEC also missed a considerable number of infections, notably Data Availability Statement: All relevant data are within the paper and its Supporting Information files. Data Availability Statement: All relevant data are within the paper and its Supporting Information files. Funding: Leiden University Fund (LUF; www.luf.nl/ en), the Jo Keur Fund (https://www.lumc.nl/sub/ 9500/att/131107035332416.pdf) and the LUSTRA scholarship (http://hum.leiden.edu/ internationalisation/outgoing-students/outgoing- exchange-students/news/lustra-scholarship- internationalisation/outgoing-students/outgoing- exchange-students/news/lustra-scholarship- 1 / 18 PLOS Neglected Tropical Diseases | DOI:10.1371/journal.pntd.0005310 January 23, 2017 Conclusions/Significance We showed intestinal parasites—especially helminths—to be omnipresent in Inhamudima, Beira. However, it is a challenge to achieve high diagnostic sensitivity for all species. Classi- cal techniques such as FEC are useful for the detection of some intestinal helminth species, but they lack sensitivity for other parasite species. PCR can detect intestinal parasites more accurately but is generally not feasible in resource-poor settings, at least not in peripheral labs. Hence, there is a need for a more field-friendly, sensitive approach for on-the-spot diagnosis of parasitic infections. Competing Interests: The authors have declared that no competing interests exist. Diagnosis of Intestinal Parasites in Mozambique S. stercoralis, Schistosoma mansoni and G. intestinalis. PCR outperformed microscopy in terms of sensitivity and range of parasite species detected. applications.html) were awarded to MCCL, DHH and RE. The PCR analyses at Leiden University Medical Center were partly supported by the Prof. Dr. P.C. Flu-Foundation. The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript. Author Summary In populations living in adverse conditions due to poverty, a wide variety of intestinal par- asite infections can be observed. These infections are usually diagnosed by stool micros- copy, but can be easily missed if the procedures used are inaccurate or performed in a suboptimal way. In the present study, we investigated the prevalence of intestinal parasite infections in an informal settlement in Beira, Mozambique. We also compared the diag- nostic performance of five different microscopic techniques and real-time PCR for the detection of DNA of five helminth and five protozoa species. For this purpose, a single stool sample was collected from members of 63 households, evenly distributed within the settlement. Parasitic infections were found to be highly abundant in the 303 examined samples. Virtually all individuals were found to be infected with at least one helminth spe- cies and a majority of the stools showed multiple parasites. None of the classical micro- scopic techniques was suitable to detect all different pathogenic species. Hence, the outcome of several microscopy procedures had to be combined to get a complete picture. We found PCR to be the most accurate diagnostic approach, even in this hyper-endemic setting. However, DNA detection is a high-tech technology, generally not applicable in resource-poor settings. PLOS Neglected Tropical Diseases | DOI:10.1371/journal.pntd.0005310 January 23, 2017 Diagnosis of Intestinal Parasites in Mozambique In comparison to helminth infections, less is known about intestinal protozoan infections. They have been associated with persistent diarrhea in developing countries [8–10], and can cause severe morbidity, especially in immunocompromised individuals [11]. Hundreds of mil- lions of people may be affected by intestinal protozoa annually [12;13]. Yet, there are no reli- able estimates of the global burden of disease [14–16]. This lack of knowledge is due to the fact that intestinal protozoa are difficult to diagnose. Also, some diagnostic techniques cannot dis- tinguish pathogenic from non-pathogenic species (i.e. Entamoeba histolytica versus the other Entamoeba spp.). For some species there is no consensus on their pathogenicity (e.g. Blastocys- tis), while for others, disease only develops in certain infected individuals but not in all (e.g. Giardia intestinalis). Loss of microscopic skills in many clinical laboratories and the general lack of awareness on protozoon infections further add to these difficulties. The diagnosis of intestinal parasites typically relies on the microscopic detection of egg, lar- val, trophozoite, cyst, and/or oocyst life stages in human feces samples [17;18]. The sensitivity of stool microscopy is generally low, and for a reliable diagnosis it is important to choose the appropriate microscopic technique [19]. For example, relatively simple techniques such as the direct smear are known to detect high A. lumbricoides loads while underestimating the pres- ence of other helminths such as Schistosoma mansoni [20]. Ideally, the technique with the high- est diagnostic accuracy for the parasite of interest should be selected. In practice however, this is difficult to achieve since many different parasite species may occur in a given population, or even in a single individual, and resources are generally limited in countries where most of these infections are endemic, so not all appropriate microscopic techniques can be used. In the past decade, alternative diagnostic procedures have become available, such as the detection of parasite DNA in stool samples using real-time PCR [21]. The disadvantage of PCR, however, is that—in contrast to microscopy—it needs a high-tech laboratory, which is even more of a chal- lenge for diagnostic laboratories within endemic countries. Relatively little is known about the distribution of intestinal parasites in Mozambique [22;23]. The present study was initiated because a local hospital noticed many cases of diarrhea in one of the informal settlements (‘bairro’) in Beira, Mozambique. Given the sanitary condi- tions in the study area, intestinal parasites were suspected to be the cause of these complaints. However, diagnostic methods that were being used in the hospital at that time were not ade- quate to detect these infections. Hence, the aim of this study was 1) to investigate which intesti- nal parasite species are most common in this area, and 2) to compare diagnostic accuracy between different microscopic techniques and real-time PCR for these intestinal parasitic infections. Five commonly used microscopic techniques were applied and evaluated, i.e. direct smear, formal-ether concentration (FEC), Kato smear, Baermann method, and coproculture, for the detection of a uniquely broad spectrum of intestinal parasites: from intestinal helminths like Strongyloides stercoralis, Ancylostoma spp., Necator americanus, A. lumbricoides, T. tri- chiura and Schistosoma spp. blood flukes, to pathogenic intestinal protozoa such as G. intesti- nalis, E. histolytica, the coccidium Cystoisospora belli and the microsporidia Enterocytozoon bieneusi and Encephalitozoon spp. Microscopy and real-time PCR results were compared to one another and to composite reference standards (CRSs). Introduction Intestinal parasitic infections are among the most prevalent infections in humans in low- and middle-income countries. They can be largely categorized into two groups, i.e. helminthic and protozoan infections. Intestinal parasitic infections can cause significant morbidity. Especially children—who are generally more prone to heavy worm burdens—suffer from the sequelae of intestinal parasitic infections, such as diarrhea, malabsorption and anemia [1;2]. The most important intestinal helminths, both in terms of abundance and disease burden, are soil-transmitted helminths (STHs) such as hookworms, Ascaris lumbricoides, and Trichuris trichiura [3]. It is estimated that STHs infect more than two billion people or more than a third of the world’s population [4]. Also, the Schistosoma spp. blood flukes are of great public health importance, with more than 250 million people infected worldwide [5;6], and an estimated global disease burden of 4.0 million disability-adjusted life years (DALYs) [7]. 2 / 18 PLOS Neglected Tropical Diseases | DOI:10.1371/journal.pntd.0005310 January 23, 2017 were infected according to microscopy were offered treatment following standard clinical practice at the local hospital. Samples were anonymized for further data analysis. were infected according to microscopy were offered treatment following standard clinical practice at the local hospital. Samples were anonymized for further data analysis. Diagnosis of Intestinal Parasites in Mozambique Materials and Methods Ethics statement Approval to perform the study was obtained from the Beira Committee of Medical Ethics, Mozambique and the study proposal was filed by the Committee of Medical Ethics of the Lei- den University Medical Centre (reference number CI5.151/NV/ib). Prior to the study, written informed consent was obtained from the head of participating households. Individuals who 3 / 18 PLOS Neglected Tropical Diseases | DOI:10.1371/journal.pntd.0005310 January 23, 2017 PLOS Neglected Tropical Diseases | DOI:10.1371/journal.pntd.0005310 January 23, 2017 Diagnosis of Intestinal Parasites in Mozambique Microscopy Fecal samples were collected from all participating household members on a door-to-door basis, 0-18h after production of the samples, and examined in Beira within 24h after collection. Three well trained microscopists performed the laboratory procedures, and on average not more than eight stool samples were processed per day to ensure high quality microscopic results. Multiple approaches were used for the detection of cysts and oocysts of the protozoa, and eggs and larvae of the helminths (Table 1). Microscopic techniques included direct smear, FEC, Kato thick smear, Baermann method, and charcoal plate coproculture [17]. Table 1. Diagnostic methods performed and definitions of composite reference standards. Parasite species Direct smear FEC Kato smear Baermann Coproculture PCR STHs Strongyloides stercoralis + + - + + + Hookworm + + + - + + a Ascaris lumbricoides + + + - - + Trichuris trichiura + + + - - - Schistosoma mansoni b + + + c - - + d Pathogenic protozoa Giardia intestinalis + + - - - + Entamoeba histolytica complex e + + - - - + Cryptosporidium parvum/C. hominis f - - - - - + g Enterocytozoon bieneusi - - - - - + g Encephalitozoon spp. - - - - - + g Cystoisospora belli f + + - - - - Cyclospora cayetanensis f - - - - - - Non-pathogenic protozoa Entamoeba coli + + - - - - Entamoeba hartmanni + + - - - - Iodamoeba bu¨tschlii + + - - - - Endolimax nana + + - - - - Chilomastix mesnili + + - - - - Blastocystis h + + - - - - Dientamoeba fragilis h - - - - - - FEC, formal-ether concentration For each parasite species, the composite reference standard (CRS) is based on the combined findings of the test(s) indicated with a plus sign (+). The minus sign (-) indicates the tests that were not applicable for a given parasite species. The latter tests were not included in the respective CRSs. a In contrast to the microscopic techniques, the PCR differentiates between the hookworm species Ancylostoma spp. and Necator americanus. b In addition to the microscopic methods mentioned, glycerin sedimentation was carried out [17]. However, this was stopped after the first 100 samples because it was very labor-intensive and did not detect any additional S. mansoni cases. Microscopy d For each parasite species, the composite reference standard (CRS) is based on the combined findings of the test(s) indicated with a plus sign (+). The minus sign (-) indicates the tests that were not applicable for a given parasite species. The latter tests were not included in the respective CRSs. a In contrast to the microscopic techniques, the PCR differentiates between the hookworm species Ancylostoma spp. and Necator americanus. b In addition to the microscopic methods mentioned, glycerin sedimentation was carried out [17]. However, this was stopped after the first 100 samples because it was very labor-intensive and did not detect any additional S. mansoni cases. c The time interval between preparation of the Kato smear and the examination of the slides was 30–60 minutes and therefore suboptimal for the detection of S. mansoni. d d The PCR detects DNA of both S. mansoni and S. haematobium. e Entamoeba histolytica cannot be differentiated microscopically from E. dispar and the other Entamoeba spp. of the E. histolytica complex: E. bangladeshi, E. ecuadoriensis, E. moshkovskii, and E. nutalli [24–26]. The PCR was specific for E. histolytica. Consequently, this parasite was not included in the analysis on diagnostic accuracy. e Entamoeba histolytica cannot be differentiated microscopically from E. dispar and the other Entamoeba spp. of the E. histolytica complex: E. bangladeshi, E. ecuadoriensis, E. moshkovskii, and E. nutalli [24–26]. The PCR was specific for E. histolytica. Consequently, this parasite was not included in the analysis on diagnostic accuracy. f The microscopic methods used—direct smear and FEC—are known to be inadequate for these protozoan infections. Hence, the modified Ziehl-Neelsen staining [17] was performed but stopped after the first 201 samples because it was labor-intensive and Cryptosporidium was detected in three samples only, while C. belli and C. cayetanensis were absent in this subsample. Because of incompleteness the modified Ziehl-Neelsen staining, data was not included in the CRS. f The microscopic methods used—direct smear and FEC—are known to be inadequate for these protozoan infections. Hence, the modified Ziehl-Neelsen staining [17] was performed but stopped after the first 201 samples because it was labor-intensive and Cryptosporidium was detected in three samples only, while C. belli and C. cayetanensis were absent in this subsample. Because of incompleteness the modified Ziehl-Neelsen staining, data was not included in the CRS. Study population The study was performed in Inhamudima (E34.86˚, S19.84˚), an informal settlement in the city of Beira, Mozambique, and was conducted on request of the local hospital and faculty of medicine. The area of Inhamudima is frequently flooded and is not connected to a sewage sys- tem. The rainy season lasts from October to March. The study was performed between June and August 2007. A geographical map of this area was prepared and households and roads were annotated. In order to obtain a random and geographically evenly distributed sample of households and a logistically feasible sample size, a grid with 75 x 75 meter quadrants was superimposed on this map and the household that was closest to each of the intersections was selected. In this way, all participants of in total 63 households were approached to participate. In the field, these houses were located using handheld GPS devices. Infants (younger than one year) and people who did not provide sufficient fecal material for all procedures were excluded from the study. Fig 1 shows that 303 out of the 399 individuals that had given informed consent provided suffi- cient fecal material for inclusion into the study (i.e. participation rate of 76%). Initially, urine samples were also collected for detection of Schistosoma haematobium (by urine filtration on one 10ml urine sample). Because of the relatively low numbers of S. haema- tobium cases however, collection of urine samples was stopped to focus on the diagnosis of intestinal parasites. Fig 1. Flow diagram of the selection of the study population and diagnostic procedures. doi:10 1371/journal pntd 0005310 g001 Fig 1. Flow diagram of the selection of the study population and diagnostic procedures. doi:10.1371/journal.pntd.0005310.g001 doi:10.1371/journal.pntd.0005310.g001 4 / 18 PLOS Neglected Tropical Diseases | DOI:10.1371/journal.pntd.0005310 January 23, 2017 doi:10.1371/journal.pntd.0005310.t001 Microscopy - - - - - + g Cystoisospora belli f + + - - - - Cyclospora cayetanensis f - - - - - - Non-pathogenic protozoa Entamoeba coli + + - - - - Entamoeba hartmanni + + - - - - Iodamoeba bu¨tschlii + + - - - - Endolimax nana + + - - - - Chilomastix mesnili + + - - - - Blastocystis h + + - - - - Dientamoeba fragilis h - - - - - - FEC, formal-ether concentration For each parasite species, the composite reference standard (CRS) is based on the combined findings of the test(s) indicated with a plus sign (+). The minus sign (-) indicates the tests that were not applicable for a given parasite species. The latter tests were not included in the respective CRSs. a In contrast to the microscopic techniques, the PCR differentiates between the hookworm species Ancylostoma spp. and Necator americanus. b In addition to the microscopic methods mentioned, glycerin sedimentation was carried out [17]. However, this was stopped after the first 100 samples because it was very labor-intensive and did not detect any additional S. mansoni cases. c The time interval between preparation of the Kato smear and the examination of the slides was 30–60 minutes and therefore suboptimal for the detection of S. mansoni. d The PCR detects DNA of both S. mansoni and S. haematobium. Table 1. Diagnostic methods performed and definitions of composite reference standards. FEC, formal-ether concentration FEC, formal ether concentration For each parasite species, the composite reference standard (CRS) is based on the combined findings of the test(s) indicated with a plus sign (+). The minus sign (-) indicates the tests that were not applicable for a given parasite species. The latter tests were not included in the respective CRSs. a In contrast to the microscopic techniques, the PCR differentiates between the hookworm species Ancylostoma spp. and Necator americanus. b In addition to the microscopic methods mentioned, glycerin sedimentation was carried out [17]. However, this was stopped after the first 100 samples because it was very labor-intensive and did not detect any additional S. mansoni cases. c The time interval between preparation of the Kato smear and the examination of the slides was 30–60 minutes and therefore suboptimal for the detection of S. mansoni. Microscopy c The time interval between preparation of the Kato smear and the examination of the slides was 30–60 minutes and therefore suboptimal for the detection of S. mansoni. d The PCR detects DNA of both S mansoni and S haematobium Table 1. Diagnostic methods performed and definitions of composite reference standards. Parasite species Direct smear FEC Kato smear Baermann Coproculture PCR STHs Strongyloides stercoralis + + - + + + Hookworm + + + - + + a Ascaris lumbricoides + + + - - + Trichuris trichiura + + + - - - Schistosoma mansoni b + + + c - - + d Pathogenic protozoa Giardia intestinalis + + - - - + Entamoeba histolytica complex e + + - - - + Cryptosporidium parvum/C. hominis f - - - - - + g Enterocytozoon bieneusi - - - - - + g Encephalitozoon spp. - - - - - + g Cystoisospora belli f + + - - - - Cyclospora cayetanensis f - - - - - - Non-pathogenic protozoa Entamoeba coli + + - - - - Entamoeba hartmanni + + - - - - Iodamoeba bu¨tschlii + + - - - - Endolimax nana + + - - - - Chilomastix mesnili + + - - - - Blastocystis h + + - - - - Dientamoeba fragilis h - - - - - - Table 1. Diagnostic methods performed and definitions of composite reference standards. Parasite species Direct smear FEC Kato smear Baermann Coproculture PCR STHs Strongyloides stercoralis + + - + + + Hookworm + + + - + + a Ascaris lumbricoides + + + - - + Trichuris trichiura + + + - - - Schistosoma mansoni b + + + c - - + d Pathogenic protozoa Giardia intestinalis + + - - - + Entamoeba histolytica complex e + + - - - + Cryptosporidium parvum/C. hominis f - - - - - + g Enterocytozoon bieneusi - - - - - + g Encephalitozoon spp. Diagnosis of Intestinal Parasites in Mozambique For the direct smear, ~2mg of feces was mixed with normal saline on a microscopy slide and examined for helminth eggs. Another ~2mg of feces was mixed with a drop of iodine and examined for protozoan cysts [17]. For FEC, the fecal parasite concentrator (FPC, Evergreen) was used. One gram of fecal material was thoroughly mixed with 8 ml of 10% formalin. An FPC strainer with 15 ml tube was attached to the tube containing this mixture. After having fil- tered the suspension into the empty tube, 3 ml of ether was added to the filtrate. This mixture was then shaken vigorously for 1 minute and centrifuged at 500 x g for 2 minutes. A thick, unstained wet mount of the sediment was used for the detection of helminth eggs and larvae. For protozoan cysts, a thin, iodine-stained wet mount of the sediment was used. The Kato smear—also known as Kato-Katz smear—consisted of a single slide of fecal mate- rial [18;29;30]. A 25 mg template was placed on the microscopy slide and filled with sieved (~300 μm pore size) fecal material. Upon removal of the template, the sample was covered with a cellophane slip soaked with glycerol and water (1:1). The sample was flattened by press- ing it onto an even surface, and examined 30–60 minutes after preparation. For the Baermann method, fecal material (~4g) was placed on a layer of 2 hydrophilic gauze bandages. The gauze was folded into a pouch by attaching the four perforated corners of the gauze to a stick. Subsequently, the pouch was placed in a 50 ml tube filled with tap water for 3h in such a way that the pouch lightly touched the water. Most of the water was decanted and the remaining sediment was left to stand for 2 hours before being examined for nematode larvae. For coproculture, the classical charcoal culture procedure was used [31]. Approximately 2g of fecal material was homogenized, mixed 1:1 with vermiculite, and placed on a filter paper on a plastic platform in a petri dish. Tap water was added to wet the filter paper and the petri dish was covered. After incubation at room temperature for 7 days, the water was collected in a tube and left standing for 2h. The sediment was examined for nematode larvae. For the Baermann method as well as for coproculture, two microscopy slides were pre- pared, each with 100μl of the sediment. A drop of iodine was added if moving larvae were detected, enabling identification and quantification of the larvae. Microscopy g These protozoa cannot be detected by any of the microscopic techniques used on all samples, and were therefore not included in the analysis on diagnostic accuracy. h As yet, the classification of Blastocystis and D. fragilis as pathogenic or non-pathogenic species remains controversial [27;28]. PLOS Neglected Tropical Diseases | DOI:10.1371/journal.pntd.0005310 January 23, 2017 5 / 18 PLOS Neglected Tropical Diseases | DOI:10.1371/journal.pntd.0005310 January 23, 2017 Analysis IBM SPSS 22.0 (IBM Corp.) and Microsoft Excel 14.0 (Microsoft Corp.) were used for statisti- cal analyses. GraphPad Prism 5 (GraphPad Software, Inc.) was used to prepare graphs. There is no gold standard for the detection of individual intestinal parasite species. Although microscopic techniques are known to lack sensitivity, they are, just as the PCR, sup- posed to be 100% specific [44]. We therefore combined the results of several diagnostic meth- ods into a composite reference standard (CRS) [45;46]. The CRS was defined in such a way that it was negative if none of the diagnostic methods detected the parasite of interest, and pos- itive if one or more methods detected the parasite. Table 1 shows how the CRS was defined for the different parasite species. Infection prevalence was based on the CRS unless stated otherwise, and 95% Wald confi- dence intervals were calculated for this parameter. For sensitivities of the different diagnostic methods, the Wilson score method without continuity correction was used to calculate 95% confidence intervals [47]. Differences between test sensitivities were considered statistically significant if there was no overlap of their confidence intervals. The independent samples Mann-Whitney U test was used to determine whether differences in Ct values between micros- copy-positives and -negatives were statistically significant. Characteristics of the study population The study population (n = 303) consisted of 144 (48%) males and 159 (52%) females with a median age of 17 years (range 1 to 72). These people were derived from 63 households. Per household 1 to 11 subjects participated (median of 4 subjects). Diagnosis of Intestinal Parasites in Mozambique an additional PCR as described by Obeng et al. [38–41]. Protozoa (E. histolytica, G. intestinalis, and C. parvum/C. hominis) DNA was detected by multiplex HGC-PCR [42]. Microsporidial (E. bieneusi and Encephalitozoon spp.) DNA was detected in another multiplex PCR described by Verweij et al. [43]. Negative and positive control samples were included in each PCR run. The PCR output from this system consisted of a cycle-threshold (Ct) value, representing the amplification cycle in which the level of fluorescent signal exceeded the background fluorescence. Hence, low Ct values correspond to high parasite-specific DNA loads in the sample tested, and vice versa. The maximum Ct value was set at 50 indicating that DNA was not detected in the sample after 50 cycles of amplification. The Ct values of the internal Phocine Herpes Virus 1 control were within the expected range (Ct value between 30 and 33) for all samples, indicating that there was no evidence of inhibition of amplification in any of these samples. Real-time PCR In Beira, an aliquot (~1g) of each stool sample was sieved and mixed with 3 volumes of 96% ethanol for preservation and shipment to Leiden, the Netherlands [32]. Here, the samples were stored at -20˚C until detection and quantification of parasite DNA loads by real-time PCR. DNA isolation, amplification and detection were performed blinded to previous microscopic results. For DNA isolation, 250μl of feces suspension was centrifuged and the pellet was washed with phosphate-buffered saline, resuspended in 200μl of 2% polyvinylpolypyrolidone (Sigma) and heated for 10 minutes at 100˚C [32;33]. After sodiumdodecyl sulfate-proteinase K treat- ment (2h at 55˚C), DNA was isolated using QIAamp Spin Columns/Mini Kit (Qiagen, Ger- many). In each sample, a fixed amount of Phocine Herpes Virus 1 was included within the isolation lysis buffer as an internal control [34]. In total, 10 PCR targets were included and 5 μl DNA was used in each real-time PCR. Amplification generally comprised of 15’ at 95˚C followed by 50 cycles of 15” at 95˚C, 30” at 60˚C, and 30” at 72˚C. Parasite-specific primers and probes were used for amplification of sequences, according to previously published protocols. Hookworm DNA (Ancylostoma spp. and N. americanus) was detected by one multiplex PCR described by Verweij et al. [35]. Ascaris lumbricoides [36] and S. stercoralis [37] DNA was detected in separate PCRs instead of in a multiplex format combined with other helminth targets. Schistosomal DNA was detected in 6 / 18 PLOS Neglected Tropical Diseases | DOI:10.1371/journal.pntd.0005310 January 23, 2017 Prevalence of intestinal parasitic infections in the study population according to different diagnostic methods. Whiskers indicate 95% confidence intervals of the observed prevalence. Percentages are based on observations in 303 individuals. (A) Prevalence of helminth infections. Strongyloides stercoralis infection was not determined (ND) in Kato smears, hookworm was not determined by the Baermann method, while A. lumbricoides, T. trichiura, and S. mansoni were not determined by the Baermann method or coproculture. Trichuris trichiura was not determined by PCR either, and the composite reference standard (CRS) for this infection was consequently based on microscopic results only. (B) Prevalence of intestinal protozoan infections. Feces were examined by both microscopy and PCR for G. intestinalis, and E. histolytica complex spp. (one observation was missing for PCR, and consequently for the CRS). Only PCR data was used for C. parvum/C. hominis (one observation missing) and for, E. bieneusi and Encephalitozoon spp. (two observations missing).* While microscopy cannot differentiate between the pathogenic species Entamoeba histolytica and the nonpathogenic species of the E. histolytica complex, PCR is specific for the pathogenic species (E. histolytica). Fig 2. Prevalence of intestinal parasitic infections in the study population according to different diagnostic methods. Whiskers indicate 95% confidence intervals of the observed prevalence. Percentages are based on observations in 303 individuals. (A) Prevalence of helminth infections. Strongyloides stercoralis infection was not determined (ND) in Kato smears, hookworm was not determined by the Baermann method, while A. lumbricoides, T. trichiura, and S. mansoni were not determined by the Baermann method or coproculture. Trichuris trichiura was not determined by PCR either, and the composite reference standard (CRS) for this infection was consequently based on microscopic results only. (B) Prevalence of intestinal protozoan infections. Feces were examined by both microscopy and PCR for G. intestinalis, and E. histolytica complex spp. (one observation was missing for PCR, and consequently for the CRS). Only PCR data was used for C. parvum/C. hominis (one observation missing) and for, E. bieneusi and Encephalitozoon spp. (two observations missing).* While microscopy cannot differentiate between the pathogenic species Entamoeba histolytica and the nonpathogenic species of the E. histolytica complex, PCR is specific for the pathogenic species (E. histolytica). population harbored three or more different helminth species (Fig 3). Up to five helminth infections were found in one individual. The prevalence of infections with pathogenic protozoa is shown in Fig 2B. doi:10.1371/journal.pntd.0005310.g002 Infection prevalence STH infections were widespread with a prevalence of 93%, 56%, 48% and 38% for T. trichiura, A. lumbricoides, S. stercoralis and hookworm, respectively (Fig 2A). PCR indicated that Ancy- lostoma spp. was the most abundant hookworm: 25% of the population (75/303) harbored Ancylostoma spp. while N. americanus was detected in 15% (46/303) of the population. Mixed Ancylostoma spp. and N. americanus infections were observed in 5% (15/303) of the popula- tion. The prevalence of S. mansoni was 10.9%. Within the population of Inhamudima, 96% (292/303) of individuals were found to harbor at least one of the following helminths in their stool sample: S. stercoralis, Ancylostoma spp., N. americanus, A. lumbricoides, T. trichiura, S. mansoni, and 49% (147/303) of the total 7 / 18 PLOS Neglected Tropical Diseases | DOI:10.1371/journal.pntd.0005310 January 23, 2017 Diagnosis of Intestinal Parasites in Mozambique Fig 2. Prevalence of intestinal parasitic infections in the study population according to different diagnostic methods. Whiskers indicate 95% confidence intervals of the observed prevalence. Percentages are based on observations in 303 individuals. (A) Prevalence of helminth infections. Strongyloides stercoralis infection was not determined (ND) in Kato smears, hookworm was not determined by the Baermann method, while A. lumbricoides, T. trichiura, and S. mansoni were not determined by the Baermann method or coproculture. Trichuris trichiura was not determined by PCR either, and the composite reference standard (CRS) for this infection was consequently based on microscopic results only. (B) Prevalence of intestinal protozoan infections. Feces were examined by both microscopy and PCR for G. intestinalis, and E. histolytica complex spp. (one observation was missing for PCR, and consequently for the CRS). Only PCR data was used for C. parvum/C. hominis (one observation missing) and for, E. bieneusi and Encephalitozoon spp. (two observations missing).* While microscopy cannot differentiate between the pathogenic species Entamoeba histolytica and the nonpathogenic species of the E. histolytica complex, PCR is specific for the pathogenic species (E. hi t l ti ) Fig 2. Prevalence of intestinal parasitic infections in the study population according to different diagnostic methods. Whiskers indicate 95% confidence intervals of the observed prevalence. Percentages are based on observations in 303 individuals. (A) Prevalence of helminth infections. Strongyloides stercoralis infection was not determined (ND) in Kato smears, hookworm was not determined by the Baermann method, while A. lumbricoides, T. trichiura, and S. mansoni were not determined by the Baermann method or coproculture. Trichuris trichiura was not determined by PCR either, and the composite reference standard (CRS) for this infection was consequently based on microscopic results only. (B) Prevalence of intestinal protozoan infections. Feces were examined by both microscopy and PCR for G. intestinalis, and E. histolytica complex spp. (one observation was missing for PCR, and consequently for the CRS). Only PCR data was used for C. parvum/C. hominis (one observation missing) and for, E. bieneusi and Encephalitozoon spp. (two observations missing).* While microscopy cannot differentiate between the pathogenic species Entamoeba histolytica and the nonpathogenic species of the E. histolytica complex, PCR is specific for the pathogenic species (E. histolytica) valence of intestinal parasitic infections in the study population according to different diagnostic methods. Fig 2. Prevalence of intestinal parasitic infections in the study population according to differ Fig 2. The pathogenic protozoan Cystoisospora belli was not detected in this population. Giardia intestinalis was the 8 / 18 PLOS Neglected Tropical Diseases | DOI:10.1371/journal.pntd.0005310 January 23, 2017 Diagnosis of Intestinal Parasites in Mozambique Fig 3. Number of helminth species found per person. Prevalence of infection is based on the composite reference standard for S. stercoralis, A. lumbricoides, T. trichiura, and S. mansoni, and on PCR for hookworm —Ancylostoma spp. and N. americanus (n = 303). doi:10.1371/journal.pntd.0005310.g003 Fig 3. Number of helminth species found per person. Prevalence of infection is based on the composite reference standard for S. stercoralis, A. lumbricoides, T. trichiura, and S. mansoni, and on PCR for hookworm —Ancylostoma spp. and N. americanus (n = 303). Fig 3. Number of helminth species found per person. Prevalence of infection is based on the composite reference standard for S. stercoralis, A. lumbricoides, T. trichiura, and S. mansoni, and on PCR for hookworm —Ancylostoma spp. and N. americanus (n = 303). doi:10.1371/journal.pntd.0005310.g003 doi:10.1371/journal.pntd.0005310.g003 most common pathogenic protozoan with a prevalence of 37% (113/302). Microsporidia were found in 9% (28/301) and mainly consisted of E. bieneusi infections (27/28 cases). Cryptospo- ridium parvum/C. hominis was found by PCR in 2% (6/302) of the study population. All showed a Ct value higher than 30 (median Ct 37.6) and none of these 6 overlapped with the 3 samples positive in the Ziehl-Neelsen staining (legend f, Table 1). The prevalence of E. histoly- tica complex spp. infections was 10% (30/303) based on microscopy. However, PCR showed that only 10% (3/30) of these infections involved E. histolytica, i.e. the pathogenic species. Combining the findings of pathogenic protozoa with the detection of helminths, 98% (294/ 300) of the tested inhabitants of Inhamudima were found to be infected with at least one intes- tinal parasite species. The prevalence of non-pathogenic protozoa varied between 4% and 34% for the different species, with a prevalence of 34% (102/303) for Entamoeba coli, 26% (80/303) for Endolimax nana, 21% (63/302) for Blastocystis, 9% (26/303) for Entamoeba hartmanni, 8% (24/303) for Chilomastix mesnili and a prevalence of 4% (13/303) for Iodamoeba bütschlii. PLOS Neglected Tropical Diseases | DOI:10.1371/journal.pntd.0005310 January 23, 2017 Microscopy versus PCR The sensitivity of PCR for the detection of each of the parasite species tested was higher than that of any of the microscopic techniques applied (Fig 4). This difference was statistically sig- nificant for all microscopic methods used for S. stercoralis, hookworm, S. mansoni, and G. intestinalis, and for the direct smear for the detection of A. lumbricoides. For some species, the sensitivity of the best microscopic technique was manifold lower than that of PCR. For exam- ple, the estimated sensitivity for the detection of S. mansoni was 12% for Kato smear versus 97% for the detection of Schistosoma DNA in feces via PCR. For G. intestinalis, sensitivity of FEC was 21% while that of PCR was 99%. In addition, in only one of the three PCR-positive E. histolytica samples, E. histolytica complex spp. cysts could be detected by microscopy (FEC). The sensitivity of PCR for the detection of each of the parasite species tested was higher than that of any of the microscopic techniques applied (Fig 4). This difference was statistically sig- nificant for all microscopic methods used for S. stercoralis, hookworm, S. mansoni, and G. intestinalis, and for the direct smear for the detection of A. lumbricoides. For some species, the sensitivity of the best microscopic technique was manifold lower than that of PCR. For exam- ple, the estimated sensitivity for the detection of S. mansoni was 12% for Kato smear versus 97% for the detection of Schistosoma DNA in feces via PCR. For G. intestinalis, sensitivity of FEC was 21% while that of PCR was 99%. In addition, in only one of the three PCR-positive E. histolytica samples, E. histolytica complex spp. cysts could be detected by microscopy (FEC). Fig 5 shows that, for each parasite species, PCR-positive but microscopy-negative samples had significantly lower DNA loads (i.e. higher Ct values) than PCR-positive samples that were also microscopy-positive. Although observed less frequently, some microscopy-positive sam- ples could not be confirmed by PCR. In most of these samples, only few parasites were detected by microscopy (see Table 2). Fig 5 shows that, for each parasite species, PCR-positive but microscopy-negative samples had significantly lower DNA loads (i.e. higher Ct values) than PCR-positive samples that were also microscopy-positive. Although observed less frequently, some microscopy-positive sam- ples could not be confirmed by PCR. In most of these samples, only few parasites were detected by microscopy (see Table 2). Diagnosis of Intestinal Parasites in Mozambique Fig 4. Sensitivities of the different diagnostic methods for the detection of intestinal parasitic infections. Whiskers indicate 95% confidence intervals of observed sensitivities (n = 303). Strongyloides stercoralis infection was not determined (ND) in Kato smears, hookworm was not determined by the Baermann method, while A. lumbricoides, T. trichiura, and S. mansoni were not determined by the Baermann method or coproculture. Trichuris trichiura was not determined by PCR, and for this species the sensitivity was therefore based on microscopic results only. Giardia intestinalis was not determined by Kato smear, the Baermann method or coproculture (one observation missing). d i 10 1371/j l td 0005310 004 Fig 4. Sensitivities of the different diagnostic methods for the detection of intestinal parasitic infections. Whiskers indicate 95% confidence intervals of observed sensitivities (n = 303). Strongyloides stercoralis infection was not determined (ND) in Kato smears, hookworm was not determined by the Baermann method, while A. lumbricoides, T. trichiura, and S. mansoni were not determined by the Baermann method or coproculture. Trichuris trichiura was not determined by PCR, and for this species the sensitivity was therefore based on microscopic results only. Giardia intestinalis was not determined by Kato smear, the Baermann method or coproculture (one observation missing). doi:10.1371/journal.pntd.0005310.g004 Comparison of microscopic techniques Diagnostic sensitivity was estimated for the different microscopic techniques and for each of the parasite species (Fig 4). FEC and Kato smear had the highest sensitivities for the detection of each of the helminths, except for S. stercoralis. The direct smear was inferior to these two methods for the detection of S. stercoralis, hookworm, T. trichiura and S. mansoni. Similarly, the direct smear was inferior to FEC for the detection of G. intestinalis. The Baermann method and coproculture showed the highest sensitivities for S. stercoralis (48% and 57%, respectively), while the direct smear and FEC showed equally low sensitivities (18% and 25%, respectively). The sensitivity of coproculture for the detection of S. stercoralis and hookworm (57% and 77%, respectively) tended to be higher than the sensitivity of the other microscopic techniques (up to 48% and 69%, respectively). Moreover, the sensitivity of helminth detection increased upon combination of multiple microscopic methods. 9 / 18 PLOS Neglected Tropical Diseases | DOI:10.1371/journal.pntd.0005310 January 23, 2017 Discussion The present study was initiated because a local hospital noticed many cases of diarrhea in Inhamudima. Given the adverse living conditions in the study area, intestinal parasites were suspected to be the cause of these complaints. However, diagnostic methods that were being used in the hospital at that time were not adequate. Clinical records thus far suggested that there were only low numbers of intestinal parasite infections in the area. The first aim of the present study was to obtain a comprehensive overview of the intestinal helminth and proto- zoan infections that occur in the informal settlement of Inhamudima in Beira. 10 / 18 PLOS Neglected Tropical Diseases | DOI:10.1371/journal.pntd.0005310 January 23, 2017 Diagnosis of Intestinal Parasites in Mozambique Fig 5. Ct values in PCR-positives: microscopy-negative versus -positive samples. Total number of PCR-positives per species is indicated between brackets. ‘-’ indicates microscopy-negative and ‘+’ microscopy-positive samples. Horizontal lines indicate median Ct values. Differences in Ct values between microscopy-positive and–negative samples were all significant p0.007, a microscopy cannot differentiate the two hookworm species. doi:10.1371/journal.pntd.0005310.g005 Fig 5. Ct values in PCR-positives: microscopy-negative versus -positive samples. Total number of PCR-positives per species is indicated between brackets. ‘-’ indicates microscopy-negative and ‘+’ microscopy-positive samples. Horizontal lines indicate median Ct values. Differences in Ct values between microscopy-positive and–negative samples were all significant p0.007, a microscopy cannot differentiate the two hookworm species. doi:10.1371/journal.pntd.0005310.g005 The major strength of the present study is the unprecedentedly large panel of different diag- nostic techniques used [48]. This diagnostic rigor resulted in high quality data regarding a uniquely wide spectrum of intestinal parasite species, including helminths as well as protozoa. Prevalences observed here are thus likely to approach the true prevalence of individual para- sitic infections as well as co-infections [49], in contrast to other prevalence studies where gen- erally only one microscopic technique is used. This approach led to several important observations concerning the presence of intestinal parasite infections in the target population, such as the remarkably high levels of S. stercoralis, and high parasite infection levels in general, as well as a high degree of polyparasitism. g g p yp According to Scha¨r et al., only 44% of African countries have prevalence data on S. stercora- lis, and a majority of these studies is based on inadequate diagnostic techniques [50]. In Mozambique, so far, only Mandomando et al. reported on S. stercoralis. pp a One case was detected by coproculture as well as the Baermann method. In addition to stool, Baermann medium and coproculture medium were analyzed by PCR in the 7 Baermann-positives and 3/5 coproculture-positives, respectively. These samples also tested negative. b One case was detected by FEC Kato smear as well as coproculture Discussion A prevalence of 1.1% was reported in under-five-year-olds with diarrhea referred to a district hospital in Southern Mozambique, but only direct smear was used which is ineffective for the detection of this parasite [51]. As a result, S. stercoralis prevalences were assumed to be low, i.e. 6.2% prevalence in Mozambique [50]. Our results (prevalence of 48%) indicate that this may be a gross Table 2. Discordance between microscopy and PCR in microscopy-positives. FEC, Formal ether concentration; NA, Not applicable a One case was detected by coproculture as well as the Baermann method. In addition to stool, Baermann medium and coproculture medium were analyzed by PCR in the 7 Baermann-positives and 3/5 coproculture-positives, respectively. These samples also tested negative. b One case was detected by FEC, Kato smear as well as coproculture. c In addition, 3 S. haematobium eggs were observed d P it b d t i d i tit ti l doi:10.1371/journal.pntd.0005310.t002 , ; , pp a One case was detected by coproculture as well as the Baermann method. In addition to stool, Baermann medium and coproculture medium were analyzed by PCR in the 7 Baermann-positives and 3/5 coproculture-positives, respectively. These samples also tested negative. b One case was detected by FEC, Kato smear as well as coproculture. c In addition, 3 S. haematobium eggs were observed d Parasite numbers were determined semi-quantitatively. doi:10 1371/journal pntd 0005310 t002 Diagnosis of Intestinal Parasites in Mozambique underestimation of the nationwide prevalence and once more illustrate how often strongyloi- diasis is overlooked due to poor diagnostics. Another important observation was that Ancylostoma spp. was more prevalent than N. americanus (25% versus 15%, respectively). This is in line with the studies described by Gold- smid [52], as well as a more recent study in pre-school children living in a rural area in Malawi where Ancylostoma spp. prevailed over N. americanus [53]. Despite of these reports, it is often assumed that only N. americanus is endemic in East Africa [54]. High levels of intestinal parasitic infections as observed in the present study are not excep- tional in urban informal settlements. More than 20 years ago, it was already predicted that ongoing urbanization in the developing world would increase levels of intestinal parasites in areas like Inhamudima [55]. In such adverse living conditions, widespread environmental con- tamination is likely to occur for parasites such as A. lumbricoides, T. trichiura, and G. intestina- lis [55]. The present observations confirm this idea as these three parasites ranked in the top four of most prevalent parasites in Inhamudima, with a prevalence as high as 93% for T. tri- chiura. Augusto et al. (2009) also noted a particularly high STH prevalence in (peri-)urban areas of Mozambique (including Beira, although these were not explicitly reported), and these prevalences were higher than those reported in older studies [22]. The authors hypothesized that the increase of STH infections in Mozambican informal settlements was due to rural- urban migration, low levels of socioeconomic development, and overcrowding together with the deterioration of water supply and sewage systems after independence [22]. In addition to determining infection levels of intestinal parasites in the inhabitants of Inha- mudima, this study aimed to compare diagnostic accuracy between five microscopic tech- niques that are commonly used for the detection of intestinal parasites. The common microscopic techniques direct smear, FEC, Kato smear, Baermann method, and coproculture were applied and compared for the detection of parasitic helminths as well as protozoan infec- tions. The classical broad-spectrum techniques FEC and Kato smear proved useful for the detection of intestinal helminths: hookworm, T. trichiura and A. lumbricoides. Table 2. Discordance between microscopy and PCR in microscopy-positives. Parasite species Number of microscopy-positive cases being PCR-negative (number of parasites observed per technique) Total number of microscopy-positives Direct smear FEC Kato smear Baermann Coproculture Total S. stercoralis 1 (1) 0 NA 7 a 5 (6–141) a 12 100 Hookworm 0 1 (2) b 3 (1–47) b NA 7 (2–1000) b 9 98 A. lumbricoides 2 (1) 5 (1–4) 0 NA NA 7 161 S. mansoni 0 0 1 (1) c NA NA 1 5 G. intestinalis d 0 1 (1–10) NA NA NA 1 24 copy-positive cases being PCR-negative (number of parasites i ) Total number of microscopy-positives es Number of microscopy-positive cases being PCR-negative (number of parasites observed per technique) Total num FEC, Formal-ether concentration; NA, Not applicable PLOS Neglected Tropical Diseases | DOI:10.1371/journal.pntd.0005310 January 23, 2017 11 / 18 PLOS Neglected Tropical Diseases | DOI:10.1371/journal.pntd.0005310 January 23, 2017 Diagnosis of Intestinal Parasites in Mozambique results from the five microscopic techniques were combined. These findings are in line with earlier studies, despite differences in endemicity and geographic locations [44]. For example, Arndt et al. showed the same helminth PCRs that were used in the present study (in a multi- plex format [36]) to be more sensitive for the detection of S. stercoralis, hookworm and Schisto- soma spp. than the combination of direct smear, FEC, and Kato smear [57]. Also Easton et al. recently reported that real-time PCR outperformed Kato-Katz for the detection of soil-trans- mitted helminths and has the advantage of demonstrating parasite species which are not diag- nosed by Kato-Katz such as G. intestinalis, E. histolytica and S. stercoralis [58]. Earlier findings from our group comparing Kato smear and PCR in Kenyan schoolchildren showed that PCR outperformed the Kato smear for the detection of N. americanus as well as S. mansoni. Even when three consecutive stool samples were analyzed using Kato smear, while PCR was per- formed on only one stool sample, PCR detected more cases [41;59]. When focusing on the diagnosis of Schistosoma only, previous studies found PCR to be more sensitive than microscopy, [41], also when using other Schistosoma-specific targets [60– 64]. It should be noted that S. haematobium was detected in our study populations. This para- site was seen in six out of 95 examined urine samples (6%). PCR results indicated that Schisto- soma DNA was absent in stool samples from people with S. haematobium eggs in urine but without S. mansoni infection according to microscopy. In accordance with the Kenyan study [41], S. haematobium co-endemicity is thus unlikely to explain the relatively low sensitivity of microscopy as compared to PCR for the detection of S. mansoni. Yet, we cannot entirely rule out that “leakage” of S. haematobium DNA may have resulted in occasional false-positive PCRs. To our knowledge, only one study reported a lower diagnostic accuracy of PCR relative to microscopy. Knopp et al. showed real-time PCR for the detection of hookworm and of S. ster- coralis to be less sensitive than microscopy when applied on stool samples collected in a low- endemicity area in Tanzania [65]. PLOS Neglected Tropical Diseases | DOI:10.1371/journal.pntd.0005310 January 23, 2017 The Kato smear may be more informative than FEC as it is a quantitative technique, and in accordance with a recent meta-analysis by Nikolay et al., it tended to detect more STH infections—more hook- worm, T. trichiura, and A. lumbricoides—than FEC [44]. On the other hand, the Kato smear is not suitable for the detection of pathogenic intestinal protozoa. In this respect, the two tech- niques are complementary. In order to also detect S. stercoralis, coproculture and/or the Baer- mann method should be added to the diagnostic work-up. Consistent with a recent meta- analysis by Campo Polanco et al. [56], coproculture tended to have a higher sensitivity for the detection of S. stercoralis than the Baermann method. We demonstrated that coproculture has the additional advantage that it has a higher sensitivity for the detection of hookworm than the other techniques, while the Baermann method is suited for the detection of S. stercoralis only. This was also reported by Knopp et al. [48]. The disadvantage of coproculture however, is that it takes a week to obtain test results while the Baermann method takes several hours, and that it is more difficult to perform under field conditions [48]. In addition, coproculture harbors the occupational hazard of infection by L3 larvae. In concordance with earlier reports [44], the direct smear was inferior to all the other diagnostic tests, for each of the endemic intestinal par- asite species. This confirms that the direct smear is not at all suitable for the diagnosis of intes- tinal parasite infections. In practice however, it is widely used in endemic areas, in both research and clinical settings—including the Beira hospital—resulting in gross underestima- tions of the spread of intestinal parasites. We aimed to compare the diagnostic accuracy of the different microscopic techniques to that of real-time PCR. The detection of parasite-specific DNA had a higher sensitivity than microscopy for S. stercoralis, hookworm, Schistosoma spp., and G. intestinalis, even when PLOS Neglected Tropical Diseases | DOI:10.1371/journal.pntd.0005310 January 23, 2017 12 / 18 Supporting Information S1 Checklist. (DOCX) S1 Database. (XLSX) S1 Checklist. (DOCX) S1 Database. (XLSX) Diagnosis of Intestinal Parasites in Mozambique proposed that residual DNA may persist and be detected by PCR after parasite clearance [67], while others have showed fast DNA clearance after treatment [68;69]. More research is needed to clarify these issues. proposed that residual DNA may persist and be detected by PCR after parasite clearance [67], while others have showed fast DNA clearance after treatment [68;69]. More research is needed to clarify these issues. Acknowledgments We gratefully thank the population of Inhamudima, Beira, Mozambique for participation in this study, Idelson for washing materials, and Sandra for translating. Also, we would like to thank the anonymous reviewer who so generously shared his/her encyclopedic parasitology knowledge. Conclusion We demonstrate that intestinal helminth and protozoan infections and co-infections are wide- spread in Inhamudima, Beira. We showed that classical techniques such as FEC are useful for the detection of some intestinal helminths such as hookworm, T. trichiura and A. lumbricoides. However, they lack the sensitivity to reliably characterize the wide range of intestinal parasites that may coexist in a population or individual. PCR can detect intestinal parasites more accu- rately but in most endemic areas it is not (yet) possible to perform this technique, at least not in the more peripheral laboratories. So, until a more field-friendly approach becomes available, infection levels of intestinal parasites—and polyparasitism—are best approximated by combin- ing multiple and relatively simple microscopic techniques. Although this study used the same PCR procedures as in the present study, this inconsistency could still be explained by minor technical differences in the performance of the DNA extraction or the general PCR set-up, resulting in a reduced test effi- ciency which becomes more obvious when infection levels are already low. Hence, standardi- zation of laboratory procedures and the implementation of external quality assessment schemes are warranted. It is widely recognized that PCR could be particularly useful for the detection of intestinal parasites in low transmission areas and in post-control settings [48;58]. Here, we show that PCR can also have added value in high transmission areas, particularly in polyparasitized pop- ulations. It not only has a higher sensitivity than microscopy for all the intestinal parasites investigated in this study, but it also has the advantage that it can differentiate between mor- phologically identical species where microscopy cannot. This is important because infections caused by species passing morphologically indistinguishable eggs, such as the hookworms, can have different epidemiology. Moreover, it also detects infections such as microsporidia which are extremely difficult to diagnose by conventional light microscopy. Although it was not yet possible to detect T. trichiura by PCR at the time of study, currently available multiplex PCRs do include this helminth. PCR may thus be a useful tool for evaluation of public health inter- ventions, for quality control of microscopy procedures as well as for research on the distribu- tion of intestinal parasitic infections in different endemic settings [46;57]. However, it should be noted that PCR also has disadvantages. PCR consumables are expensive, and it requires high-tech equipment which is often not available in laboratories in affected regions. Transfer of samples to other, better equipped laboratories, may prolong the turnaround time of PCR results. Secondly, the clinical relevance of submicroscopic intestinal parasitic infections is not yet fully understood, and therefore remains a matter of debate [16;57;66]. It has also been PLOS Neglected Tropical Diseases | DOI:10.1371/journal.pntd.0005310 January 23, 2017 13 / 18 Conceptualization: AMP. Data curation: LvL LM. Funding acquisition: AMP LvL MCCL RE DHH. Investigation: EATB DHH MCCL RE MM FM. Methodology: AMP LvL JJV. References 1. 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Pillay P, Taylor M, Zulu SG, Gundersen SG, Verweij JJ, Hoekstra P, et al. Real-Time Polymerase Chain Reaction for Detection of Schistosoma DNA in Small-Volume Urine Samples Reflects Focal Dis- tribution of Urogenital Schistosomiasis in Primary School Girls in KwaZulu Natal, South Africa. Am J Trop Med Hyg 2014 Mar; 90(3):546–52. doi: 10.4269/ajtmh.13-0406 PMID: 24470560 40. Vinkeles Melchers NV, van Dam GJ, Shaproski D, Kahama AI, Brienen EA, Vennervald BJ, et al. Diag- nostic performance of Schistosoma real-time PCR in urine samples from Kenyan children infected with Schistosoma haematobium: day-to-day variation and follow-up after praziquantel treatment. PLoS Negl Trop Dis 2014 Apr; 8(4):e2807. doi: 10.1371/journal.pntd.0002807 PMID: 24743389 41. Meurs L, Brienen E, Mbow M, Ochola EA, Mboup S, Karanja DM, et al. Is PCR the Next Reference Standard for the Diagnosis of Schistosoma in Stool? A Comparison with Microscopy in Senegal and Kenya. PLoS Negl Trop Dis 2015 Jul; 9(7):e0003959. doi: 10.1371/journal.pntd.0003959 PMID: 26217948 42. Bruijnesteijn van Coppenraet LE, Wallinga JA, Ruijs GJ, Bruins MJ, Verweij JJ. Parasitological diagno- sis combining an internally controlled real-time PCR assay for the detection of four protozoa in stool samples with a testing algorithm for microscopy. Clin Microbiol Infect 2009 Sep; 15(9):869–74. doi: 10. 1111/j.1469-0691.2009.02894.x PMID: 19624500 PLOS Neglected Tropical Diseases | DOI:10.1371/journal.pntd.0005310 January 23, 2017 16 / 18 PLOS Neglected Tropical Diseases | DOI:10.1371/journal.pntd.0005310 January 23, 2017 PLOS Neglected Tropical Diseases | DOI:10.1371/journal.pntd.0005310 January 23, 2017 Real-time PCR demonstrates Ancy- lostoma duodenale is a key factor in the etiology of severe anemia and iron deficiency in Malawian pre- school children. PLoS Negl Trop Dis 2012; 6(3):e1555. doi: 10.1371/journal.pntd.0001555 PMID: 22514750 54. Palmer PES, Reeder MM. Ancylostomiasis (hookworm disease). In: The imaging of tropical diseases: With epidemiological, pathological and clinical correlation. 2nd ed. Heidelberg: Springer-Verlag; 2001. p. 51–65. 55. Crompton DW, Savioli L. Intestinal parasitic infections and urbanization. Bull World Health Organ 1993; 71(1):1–7. PMID: 8440028 56. Campo Polanco L, Gutierrez LA, Cardona Arias J. Diagnosis of Strongyloides stercoralis infection: meta-analysis on evaluation of conventional parasitological methods (1980–2013). Rev Esp Salud Publica 2014 Oct; 88(5):581–600. doi: 10.4321/S1135-57272014000500004 PMID: 25327268 57. Arndt MB, John-Stewart G, Richardson BA, Singa B, van Lieshout L, Verweij JJ, et al. Impact of hel- minth diagnostic test performance on estimation of risk factors and outcomes in HIV-positive adults. PLoS One 2013; 8(12):e81915. doi: 10.1371/journal.pone.0081915 PMID: 24324729 58. Easton AV, Oliveira RG, O’Connell EM, Kepha S, Mwandawiro CS, Njenga SM, et al. Multi-parallel qPCR provides increased sensitivity and diagnostic breadth for gastrointestinal parasites of humans: field-based inferences on the impact of mass deworming. Parasit Vectors 2016; 9:38. doi: 10.1186/ s13071-016-1314-y PMID: 26813411 59. van Mens SP, Aryeetey Y, Yazdanbakhsh M, van Lieshout L, Boakye D, Verweij JJ. Comparison of real-time PCR and Kato smear microscopy for the detection of hookworm infections in three consecu- tive faecal samples from schoolchildren in Ghana. Trans R Soc Trop Med Hyg 2013 Apr; 107(4):269– 71. doi: 10.1093/trstmh/trs094 PMID: 23426113 60. Allam AF, Kader O, Zaki A, Shehab AY, Farag HF. Assessing the marginal error in diagnosis and cure of Schistosoma mansoni in areas of low endemicity using Percoll and PCR techniques. Trop Med Int Health 2009 Mar; 14(3):316–21. doi: 10.1111/j.1365-3156.2009.02225.x PMID: 19278527 61. Pontes LA, Dias-Neto E, Rabello A. Detection by polymerase chain reaction of Schistosoma mansoni DNA in human serum and feces. Am J Trop Med Hyg 2002 Feb; 66(2):157–62. PMID: 12135287 17 / 18 PLOS Neglected Tropical Diseases | DOI:10.1371/journal.pntd.0005310 January 23, 2017 Diagnosis of Intestinal Parasites in Mozambique 62. Pontes LA, Oliveira MC, Katz N, Dias-Neto E, Rabello A. Comparison of a polymerase chain reaction and the Kato-Katz technique for diagnosing infection with Schistosoma mansoni. Am J Trop Med Hyg 2003 Jun; 68(6):652–6. PMID: 12887022 63. Gomes LI, Marques LH, Enk MJ, Coelho PM, Rabello A. Further evaluation of an updated PCR assay for the detection of Schistosoma mansoni DNA in human stool samples. Mem Inst Oswaldo Cruz 2009 Dec; 104(8):1194–6. PMID: 20140385 64. Carvalho GC, Marques LH, Gomes LI, Rabello A, Ribeiro LC, Scopel KK, et al. Polymerase chain reac- tion for the evaluation of Schistosoma mansoni infection in two low endemicity areas of Minas Gerais, Brazil. Mem Inst Oswaldo Cruz 2012 Nov; 107(7):899–902. PMID: 23147146 65. Knopp S, Salim N, Schindler T, Karagiannis Voules DA, Rothen J, Lweno O, et al. Diagnostic accuracy of Kato-Katz, FLOTAC, Baermann, and PCR methods for the detection of light-intensity hookworm and Strongyloides stercoralis infections in Tanzania. Am J Trop Med Hyg 2014 Mar; 90(3):535–45. doi: 10. 4269/ajtmh.13-0268 PMID: 24445211 66. Pierce KK, Kirkpatrick BD. Update on human infections caused by intestinal protozoa. Curr Opin Gas- troenterol 2009 Jan; 25(1):12–7. PMID: 19119509 67. Frickmann H, Schwarz NG, Rakotozandrindrainy R, May J, Hagen RM. PCR for enteric pathogens in high-prevalence settings. What does a positive signal tell us? Infect Dis (Lond) 2015 Jul; 47(7):491–8. 68. Mejia R, Vicuna Y, Broncano N, Sandoval C, Vaca M, Chico M, et al. A novel, multi-parallel, real-time polymerase chain reaction approach for eight gastrointestinal parasites provides improved diagnostic capabilities to resource-limited at-risk populations. Am J Trop Med Hyg 2013 Jun; 88(6):1041–7. doi: 10.4269/ajtmh.12-0726 PMID: 23509117 69. van den Bijllaardt W, Overdevest IT, Buiting AG, Verweij JJ. Rapid clearance of Giardia lamblia DNA from the gut after successful treatment. Clin Microbiol Infect 2014 Nov; 20(11):O972–O974. doi: 10. 1111/1469-0691.12626 PMID: 24655130 18 / 18
https://openalex.org/W2121310997
https://www.scielo.br/j/rbh/a/5t8SPLNx6sxhLRNNyXQfSkr/?lang=pt&format=pdf
Portuguese
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As cidades da juventude em Fortaleza
Revista brasileira de história
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cc-by
12,926
ABSTRACT A cidade de Fortaleza é apresentada a partir da experiência juvenil, de suas organizações, de seus deslocamentos e de suas formas específicas de apropria- ção da cidade.Desta observação delineio a idéia de Experiência Musical e de Des- locamentos Geo-Estéticos na cidade. As organizações e experiências juvenis são tomadas como reveladoras de outros possíveis e suas movimentações como manifestação do “novo”urbano contem- porâneo. A cidade de Fortaleza é apresentada a partir da experiência juvenil, de suas organizações, de seus deslocamentos e de suas formas específicas de apropria- ção da cidade.Desta observação delineio a idéia de Experiência Musical e de Des- locamentos Geo-Estéticos na cidade. As organizações e experiências juvenis são tomadas como reveladoras de outros possíveis e suas movimentações como manifestação do “novo”urbano contem- porâneo. The city of Fortaleza is presented through the juvenile experience of its organiza- tions, its displacements and its specific city appropriation ways. It is from this observation that I take the idea of Musi- cal Experience and the Geo-esthetic Displacements of the city. The orga- nizations and juvenile experiences are considered to be revealing of other possi- bilities and their movements, as a man- ifestation of the “new” contemporary urbanity. Palavras-chave:Juventude;cultura;cida- de; música; hip hop; punk. Keywords:Youth,culture,city,music,hip hop, punk. Este texto está dividido em cinco partes, em que a juventude e sua atua- ção na cidade de Fortaleza se esboçam com base na compreensão de sua or- ganização em dois importantes movimentos: o Movimento Hip-Hop e o Mo- vimento Punk. Na mesma medida em que esses jovens se organizam e criam suas organizações, interferem na cidade e nas diversas formas de se organizar e de viver no final do século XX e início do século XXI. As cidades da juventude em Fortaleza Francisco José Gomes Damasceno1 UECE BAILES E CLUBES: A FORTALEZA DAS AFINIDADES ELETIVAS Freqüentemente pensei nas cartas geográficas como uma grande metá- fora do conhecimento, como tensão entre compreensibilidade e desorienta- mento. O que faz o cartógrafo? Transfere para uma folha plana de papel o ‘mundo real’ sob a forma de símbolos e sinais. O que se transcreve é uma ‘di- Revista Brasileira de História. São Paulo, v. 27, nº 53, p. 215-242 - 2007 Francisco José Gomes Damasceno ferença’: a fonte de toda a informação e, portanto, da comunicação. Ainda as- sim, enquanto algumas dessas diferenças são imediatamente decodificáveis por qualquer leitor de mapas, outras têm de expressar códigos totalmente no- vos, de forma tal que até o viajante mais experimentado verá pela primeira vez o lugar representado, mesmo no caso em que tenha estado ali a vida toda. E como a escala 1:1 não adianta, pode servir no máximo a um eu dilatado do narcisismo irrefreável, quanto mais a proporção for reduzida — é micrológi- ca — tanto mais teremos de inventar os códigos a serem transcritos no mapa para deslocar o leitor. Então talvez descobriremos com esses microcódigos que o nosso objeto — a comunicação sincrética — já está incorporando, nas partes terminais de nossos sentidos, enxertos de sensores étnicos, cyber e punk. Mudanças marronizadas. Quilombos patchwork.2 No final dos anos 70 a cidade de Fortaleza fervilhava aos sons de inúme- ras novidades oriundas da produção da chamada indústria cultural e das cul- turas de massa. Alguns vindos literalmente ‘de fora’ e outros considerados ‘lo- cais’e ainda não inseridos no contexto maior de comercialização internacional. Era o caso, por exemplo, do rock, que aportava com força pela musicali- dade de Led Zeppelin, Black Sabbath, Ramones e Sex Pistols, entre outros. Já o predomínio do local se matizava nas inúmeras gravações de forró, bem re- presentados em grupos como Trio Nordestino, Três do Nordeste, Luís Gon- zaga, Jackson do Pandeiro e tantos outros. Estudos sobre o rock em Fortaleza dão conta de uma trajetória iniciada ainda na década de 1950 e um predomínio hegemônico do forró, motivo pe- lo qual se apontaria, até meados dos anos 90, a dificuldade de ‘explosão’ dessa manifestação em Fortaleza, bem como do surgimento e consolidação de ban- das e espaços destinados a essas manifestações. Revista Brasileira de História, vol. 27, nº 53 BAILES E CLUBES: A FORTALEZA DAS AFINIDADES ELETIVAS Entretanto, o rock praticado em Fortaleza era ‘regionalizado’, com ban- das (ou grupos de bailes) como Os Faraós, Os Belgas, Os Diferentes ou, um pouco depois, O Peso, que seguiam linhas melódicas mais leves e pouco liga- das às perspectivas mais ‘transgressivas’: Na década de 50, o rock era tocado por grupos de baile — o mais conhecido era Iranildo e seu conjunto — que animavam as festas de clubes sociais como o Náutico, Líbano e Maguary ... Na década de 60, com a explosão do rock em todo o mundo, esses grupos tocavam os sucessos do momento, além de músicas bra- sileiras, mambo, bolero e rumba. O grande destaque era Os Faraós, banda de Luizinho que ainda hoje toca em festas ... Outro destaque era Os Belgas, cujo guitarrista Júlio Sena era o maior sucesso. Tanto um como o outro tinha em seu 216 Revista Brasileira de História, vol. 27, nº 53 As cidades da juventude em Fortaleza repertório, basicamente sucessos dos Beatles e as versões de Renato e seus Blue Caps. Também vale lembrar d’Os Diferentes, que a exemplo dos demais tocava música dos outros, mas a diferença estava no fato de cantarem músicas próprias e fazerem arranjos diferentes para músicas dos outros ... Nos anos 70, destacou- se Luís Carlos Porto, vocalista da banda O Peso, que talvez seja a única banda cearense de rock a ter tido projeção nacional ... No final da década surgiu a ban- da Posh, fazendo um rock mais para o pop.3 Assim, a manifestação nos anos 70 se encontrava na encruzilhada dessas diferentes formas de apreciar e curtir o rock, parecendo ter havido uma apro- priação dupla da manifestação: 1) a primeira, do ponto de vista estético-político, com o surgimento de bandas ‘mais radicais’, com mensagens politizadas e fazendo parte de um ‘movimento’ que atentava contra os pressupostos anteriores do próprio rock e da cultura de massas — o punk; e 2) a segunda, do ponto de vista dessas manifestações na própria cidade de Fortaleza,e de setores de sua sociedade antes relegados ao ‘silêncio’. 2) a segunda, do ponto de vista dessas manifestações na própria cidade de Fortaleza,e de setores de sua sociedade antes relegados ao ‘silêncio’. junho de 2007 BAILES E CLUBES: A FORTALEZA DAS AFINIDADES ELETIVAS Trata-se de uma complexa articulação de manifestações, intenções, pa- drões estéticos, éticos e práticas de incorporação musical, que dão origem a uma forma inusitada de vivência da cidade e da própria música, que sai dos clubes de elite como o Náutico, Líbano e ou Maguary e passa aos pequenos clubes de periferia, tais como o Apache Clube, o Mênfis Clube do Antônio Bezerra, o Keops Clube, o Detroit, ou ainda o Grêmio recreativo do Conjun- to José Walter, entre tantos outros. É claro que a essa ‘transposição geo-estética’ (ou ‘deslocamento geo- estético’)4 correspondia uma apropriação de outros setores sociais presentes na cidade de Fortaleza, e, além disso, marcava de forma definitiva a emergên- cia de novos atores sócio-históricos: os jovens pobres. Situados nas periferias da então pequena cidade de Fortaleza,5 eles pas- sam a se manifestar de forma muito mais presente e freqüente, e, além disso, essa manifestação se dá em um campo particular, próprio, singular e inova- dor: o campo da arte e do lazer. Assim, os inúmeros bailes que surgem na cidade são mais do que sim- ples diversão e assumem a conotação de manifestação juvenil, que dentro de uma trajetória própria, em pouco tempo, guardando a sua característica his- tórica de transmutação, assumiria uma outra e mais radical forma de se ma- nifestar, como veremos adiante. 217 Francisco José Gomes Damasceno Os inúmeros bailes geram práticas de sociabilidade, de compreensão es- tética, ética e musical que são instauradoras de ‘territórios existenciais’ e pos- suem suas sutilezas e perspectivas próprias. Vejamos como Flor, à época ain- da menina, passa a experimentar o que ocorria: Eu não sei quando é que a coisa virou movimento punk, porque isso vem de um outro movimento. Eu tinha 12 ou 13 anos, conheci essa menina, a Guacira, ela curtia rock e tinha uns festivais de rock que aconteciam no Apache, no Mên- fis Clube do Antônio Bezerra, no Keops Clube, lá no Detroit, várias casas de su- búrbio, shows de rock com playback. Era Led Zeppelin, Black Sabbath, Pink Floyd, Kiss, heavy metal. E aí começou uma reunião aqui na Praça do Ferreira. Eu estu- dava no Anchieta e gazeava aula, ficava no meio dessa galera porque eu gostava de banda e curtia essas coisas. Revista Brasileira de História, vol. 27, nº 53 BAILES E CLUBES: A FORTALEZA DAS AFINIDADES ELETIVAS E aí a gente começou a se reunir pra ouvir esses sons, e tinha concursos de dança...6 Como se pode perceber na lembrança de Flor, iniciava-se na cidade a produção de pequenas festas nos clubes de subúrbio. Nessas festas, uma das estratégias para envolver os grupos eram competições de rock ao som dos con- juntos7 por eles preferidos: Sempre nos finais de semana uma leva de fãs gravitava pelos clubes suburba- nos atrás de diversão. ‘Competições’ eram travadas entre as diversas turmas. A turma do Baby do Bairro do Monte Castelo, do Conjunto José Walter, do Par- que Araxá entre outras, disputava quem agitava mais parecido com seus ídolos (Robert Plant, Ramones), com direitos a guitarras artesanais, feitas de madeira ou papelão e até troféus para as turmas vencedoras.8 Além disso, outro aspecto que se deve considerar é a apropriação da ci- dade de forma itinerante, não fixa, feita de forma ‘errante’, em deslocamentos pela diversidade de cada bairro ou clube, ou ainda das diversas ‘turmas’ en- volvidas: Essa coisa do rock não era fixa não, o cara fazia um evento e divulgava, uma semana num bairro, outra semana em outro totalmente diferente ou distante. Acho que era isso que perpetuava a adrenalina. De repente o cara já tem a adre- nalina lá em cima, aí faz um evento hoje aqui, no outro final de semana não é mais aqui, é lá...9 218 Revista Brasileira de História, vol. 27, nº 53 As cidades da juventude em Fortaleza As cidades da juventude em Fortaleza A novidade dessa experiência juvenil que tomava o lazer em suas mãos, articulava os diversos bairros da cidade, se grupalizava a partir de afinidades, identificava grupos distantes e/ou rivais, estabelecia uma rede de relações, de espaços, constituía sua vivência com base em desejos e prazeres dos quais não se dissociava, e, que em breve se tornariam a referência de suas próprias vidas de forma completa.10 Estavam constituindo seus espaços: Existe espaço sempre que tomam em conta vetores de direção, quantidade de velocidade e a variável tempo. O espaço é um cruzamento de móveis. É de certo modo animado pelo conjunto dos movimentos que aí se desdobram. junho de 2007 BAILES E CLUBES: A FORTALEZA DAS AFINIDADES ELETIVAS O espaço é o efeito produzido pelas operações que o orientam, o circunstanciam, o tem- poralizam e o levam a funcionar em unidade polivalente de programas confli- tuais ou de proximidades contratuais.11 A constituição desses ‘territórios existenciais’ 12 marca de forma peculiar toda a manifestação juvenil no mundo contemporâneo. Alguns autores têm intitulado essa característica de elaboração de grupos a partir das afinidades e diferenças de ‘tribos’,13 numa dupla alusão à historicidade de processos se- melhantes na antropologia e à constituição de redes no universo contempo- râneo dito globalizado. Enquanto para os punks essa articulação inicial se dá no final dos anos 70, para os hip-hopers ocorre processo semelhante no início dos anos 80, com a chegada, primeiro, da dança (break), e depois do canto (rap), e, finalmente, algum tempo depois, do próprio grafite, considerados os três elementos fun- damentais do hip-hop. Os bailes nos quais as diversas sonoridades eram experimentadas em par- tes específicas para cada estilo musical eram uma prática comum na Fortale- za do final dos anos 70 e dos anos 80. Assim, os amantes de cada tipo de mú- sica tinham uma parte específica das festas para se deleitarem,e a elas recorriam nos mais diversos cantos da cidade. Não há ‘oferta’ de bailes específicos, ou seja, de festas com a predominân- cia de uma única manifestação musical, que só aparece, ou se torna forte, pre- dominante, em meados dos anos 90. Talvez isso indique o pequeno número de freqüentadores dos estilos que depois comporiam o underground em For- taleza, juntando-os numa estratégia de produção, venda e consumo que aten- dia naquele momento suas expectativas e possibilidades; ou talvez fosse refle- xo de uma outra compreensão de diversão.O certo é que essa junção contribuía 219 Francisco José Gomes Damasceno na formação dos grupos pelo convívio com a diferença, no nascedouro de muitas dessas manifestações como é o caso do rock, do punk, do próprio hip- hop e até do forró, que durante algum tempo freqüentam os mesmos peque- nos clubes nesses ‘bailes mistos’. Nesse contexto, é interessante lembrar o que afirma Pesavento: a construção do nós identitário pressupõe a existência do outro. O outro é a concretização da diferença, contraposto como alteridade à identidade que se anuncia. BAILES E CLUBES: A FORTALEZA DAS AFINIDADES ELETIVAS A visualização, identificação e avaliação classificatória do outro aconte- ce sob o signo da estrangeiridade, e é pelo distanciamento — contrastivo, anta- gônico ou de semelhança — que se pode construir uma noção de pertencimen- to social. Esta lógica de sentido é, pois, relacional e implica construções que permitem um reconhecimento com sentido. Como diz Ginsburg, todos nós so- mos em certa medida, estrangeiros com relação a algo ou alguém.14 Assim, essa mistura inicial nos pequenos clubes da periferia da cidade criou em Fortaleza uma proximidade entre os diversos grupos que posterior- mente se identificam com este ou aquele estilo musical, e, ao mesmo tempo, nesse relacional se identificavam entre si como iguais–diferentes, essencial- mente compondo um mesmo campo: o de oposição à sociedade excludente, aspecto que posteriormente será incorporado às suas letras, manifestos, mú- sicas e práticas. Nesses bailes, nos pequenos clubes dos muitos (e à época distantes) bair- ros da cidade, integrantes dos diversos grupos se encontravam e aprendiam a se identificar e respeitar: Inter Dance, no Monte Castelo, era um point dos punks, e em 85, 86, 87 o break estava em ascensão, era de certa forma uma novidade. Lá, o pessoal tinha a festa de rock, punk, e tinha o pessoal que dançava o break, se congregava lá. Aí, alguns deles migraram aqui pro Conjunto Ceará...15 Assim, perceba-se: o universo juvenil e o underground eram marcados por um conjunto de práticas que independiam das musicalidades eleitas. Há nesse caso um entrelaçamento entre as perspectivas musicais, as visões de mundo, os interesses mais imediatos e uma aura estética transgressiva que aportava com essas manifestações musicais e que definiu aos poucos o pró- prio campo dessas manifestações (punk e hip-hop). Nessas duas manifestações musicais, corpóreas, estéticas, se fundamenta 220 Revista Brasileira de História, vol. 27, nº 53 As cidades da juventude em Fortaleza um conjunto de vínculos articulados pelo gosto, pela afinidade, pela identifica- ção e identidade comuns e que consubstanciaria nesse período, e nesse proces- so, os movimentos em suas perspectivas de coletivismo, de crítica e de interven- ção social, política e cultural, sendo este último, um ‘campo’ entendido como essencialmente político e se estabelecendo como o lócus de atuação desses jovens. No decorrer dos anos 80, tanto o punk como o hip-hop passam por um in- tenso processo de articulação interna, de articulações políticas, culturais e so- ciais, no sentido de se fundarem. BAILES E CLUBES: A FORTALEZA DAS AFINIDADES ELETIVAS Eles se criam, por assim dizer, em si mesmos. Estruturam-se enquanto organizações de cunho libertário e de ação contracul- tural, numa consistente postura de enfrentamento e negação do mercado de bens culturais. Assim, o que era apenas festa, lazer, tornou-se movimentação, e essa movimentação das culturas — como se chamava nos primórdios do mo- vimento — se institui como movimento sócio-político-cultural, como o en- tendemos com base nos estudos realizados nos últimos anos.16 OS MOVIMENTOS Hoje existem novos atores, novos movimentos sociais e culturais, tornando visíveis redes direcionadas a valores referenciados no binômio liberdade e so- brevivência, em contraposição às condutas de crise da sociedade contemporâ- Foto 1 – Internacional (Inter Dance), no bairro Monte Castelo. Fotografia do autor, 2003. Foto 1 – Internacional (Inter Dance), no bairro Monte Castelo. Fotografia do autor, 2003. 221 junho de 2007 221 junho de 2007 Francisco José Gomes Damasceno nea ... As ações coletivas dos anos 90 abrem o leque do próprio conceito de movi- mento social, permitindo-nos pensar em sua fragmentariedade/descontinuida- de como referente de um tempo em que o espaço democrático se relaciona dire- tamente com a dinâmica entre ação coletiva, cultura política e formação da consciência social das novas gerações.17 Na medida em que essas musicalidades tomavam corpo na cidade, como resultado da experiência direta desses jovens com a música e a própria cida- de, foram tomando feições próprias, articulando-se de forma singular como resultado dessa fusão entre os elementos dispostos e construídos. Em outras palavras: foram se tornando ‘movimentos’. Ao que tudo indica, como observa Melucci, nas sociedades contemporâ- neas, a identidade seria vivenciada como ação e não como situação, sendo construída no complexo de relações sociais estabelecidas no processo de vida e dentro dos meios e lugares sociais ocupados. Assim, haveria uma alteração na noção de identidade que, baseada nesse pressuposto de constituição permanente, auto-reflexiva, e, sobretudo, opera- da por cada pessoa, precisaria de novos termos: “A mesma palavra identidade não é mais apropriada para exprimir essa mudança e será necessário falar de identificação para exprimir o caráter processual, auto-reflexivo e construído da definição de nós mesmos”.18 Dessa forma, o autor quer dar conta da transitoriedade do contemporâneo, da multiplicidade de papéis que cada indivíduo assume cotidianamente. Entre- tanto, no caso dos jovens desses grupos (punk e hip-hop), uma das suas muitas identidades surgidas no momento de identificação pela música, pela dança, por manifestações afins, e de uma definição de papéis, acaba tornando-se duradou- ra; o que ocorre é que a grande maioria acaba assumindo de forma definitiva es- sas identificações (que em princípio são transitórias). O fato de esses jovens assumirem uma dessas identificações de forma per- manente não significa que o caráter de autoconstrução, de auto-reflexão, seja dispensado, mas parece que o processo se dá de forma intensa, dentro do pró- prio estilo que assumem. Revista Brasileira de História, vol. 27, nº 53 OS MOVIMENTOS Assim, pode-se falar em identidades da mesma for- ma ou deslocar o conceito de identidade nesse sentido. Se, como afirma Maffesoli,“a sensibilidade coletiva, originária da forma estética acaba por constituir uma relação ética”,19 então pode-se considerar que esse conjunto de relações instituídas no campo ético/estético, pode se tor- nar o centro do complexo processo de identificação/identidade, como de fato acontece com muito dos entrevistados.20 222 Revista Brasileira de História, vol. 27, nº 53 As cidades da juventude em Fortaleza As cidades da juventude em Fortaleza As cidades da juventude em Fortaleza Dessa forma, constituem uma ‘linguagem’ própria, que articula não só suas falas, mas também os tipos de música, o uso de determinadas roupas e adereços, os símbolos, os locais de freqüência, os usos da cidade, ou as cons- tituições de suas trajetórias da/na cidade, as manifestações do próprio corpo, os cortes de cabelo, as gírias, os aliados, e também, por que não dizer, os pró- prios adversários.21 Essa linguagem articulada dentro do cotidiano é manifesta de forma os- tentatória, com a sutileza de seu uso dirigido apenas aos iniciados, de tal for- ma que mesmo numa multidão, pode-se passar por eles sem percebê-los, num jogo do visível/invisível, em alguns casos. Em outros casos, o visual é assumido para chocar, impactar, como acon- tece com os punks, nessa fase. Entretanto, mais recentemente, essa caracterís- tica é abandonada, em detrimento de uma postura em que esse aspecto se tor- na opcional. Oliveira nos dá conta de um congresso ocorrido em João Pessoa, em que se discutiu e se deliberou o seguinte: “os que ali estavam concluíram que, o mais importante na sua estética não é a forma ‘X’ ou ‘Y’, mas os princí- pios que a guiam...”.22 Assim, pode-se inferir que essa linguagem constituída de símbolos e si- nais, pontos geográficos e espaciais, sentimentos e formas diversas de sentir e participar, são incorporadas e desenvolvidas com base em uma noção maior que acaba assumindo proporções cada vez mais significativas tanto em suas vidas como,em resultado disso,na própria vida social e política: o movimento. junho de 2007 OS MOVIMENTOS É em torno do movimento que tanto punks quanto hip-hopers se insur- gem no espaço urbano contra a exploração, a mesmice, a guerra, o capitalis- mo, a anti-cidadania, a falta de condições de vida dignas e justas, e tantas ou- tras questões que os afligem e incomodam e com o que imaginam estar produzindo um mundo melhor e reinventando sua cidadania. No início dos anos 90 cria-se na cidade de Fortaleza uma entidade orga- nizativa dos hip-hopers (MH2O — “Movimento Hip-Hop Organizado do Cea- rá”) em torno da qual todas as atividades ligadas a essa manifestação se de- senrolaram a partir de então. No final da mesma década, muitas outras entidades surgem como ‘rachas’ da primeira. Hoje, uma outra além dela é for- te e ativa na cidade — O “Movimento Cultura de Rua” ou MCR. Esta entida- de se coloca como um movimento social organizado, ou pretende se produ- zir como tal. Nesse mesmo período a entidade MH2O se institui como movimento e cria um estatuto jurídico, tornando-se uma organização não-governamental (ONG), o mesmo ocorrendo com o MCR pouco tempo depois. O trabalho 223 Francisco José Gomes Damasceno das duas é muito semelhante: música, dança e arte são utilizadas no engaja- mento político, social e profissional dos jovens pertencentes ao movimento. Sutileza que diferencia as entidades das demais, já que o trabalho político vem à frente do musical. Embora o foco inicial do hip-hop em Fortaleza tenha sido o bairro do Conjunto Ceará, onde até hoje o movimento é muito forte e manifesta-se co- tidianamente, com a criação das entidades, com o intenso bombardeio das mídias e a divulgação do estilo, e, principalmente pela atuação do próprio movimento e de suas diversas entidades, toda a cidade, em seus diversos bair- ros, já é possuída pelos jovens. De acordo com a nucleação das entidades há uma maior presença de um ou outro movimento nesse ou naquele bairro. Assim, na Quadra de Santa Ce- cília, onde reside boa parte dos jovens ligados ao MCR, onde se situa sua ‘se- de’ e se desenvolvem suas principais atividades e projetos pilotos, tem-se “uma área do MCR”. O Conjunto Ceará “se constitui em uma área do MH2O”, gra- ças aos vínculos históricos com esse bairro. Revista Brasileira de História, vol. 27, nº 53 MOVIMENTAÇÕES JUVENIS E TRANSPOSIÇÕES GEO-ESTÉTICAS O fato de os punks não possuírem um território fixo na cidade também me conduziu a pensar no território como corpo, ainda mais ao perceber que é nas roupas que o punk estampa seus pensamentos, colocando-se na rua como uma espécie de bandeira do movimento ... E os critérios dessa seleção não foram os que eu esperava: apego ao lugar, sen- timento de pertença, de segurança etc., mas sim critérios de praticidade, de pro- ximidade e de possibilidade. Essas considerações foram tornando a dimensão territorial do punk em Londrina muito mais confusa, pois era diferente do que eu havia tomado conhecimento em teoria ... Nesse caso, o território não seria propriamente o bar, mas sim a calçada, e os limites do território seriam os corpos. As pessoas que transitam pela calçada des- viam do grupo e lançam olhares desconfiados.24 O que de fato se pode ler nessa movimentação25 é uma apropriação da cidade de forma diferenciada da que ocorria até então. Primeiro, é interes- sante lembrar que essa nova forma é feita pelo viés da arte, da cultura e do la- zer, o que, por si só, já se constitui em uma novidade para essa cidade e sob a influência direta dela, em outras palavras, em resposta aos desafios colocados pela própria vida na cidade. Segundo, pelo inusitado do papel que a música assume nesse processo.26 Há um duplo giratório de apropriação da juventu- de. Por um lado, do ponto de vista social: trata-se de uma juventude de ‘peri- feria’ que se manifesta de forma política, social e cultural e insere-se nos mais variados contextos pela sua arte, pela sua música, fazendo disso sua forma de intervenção. Do ponto de vista estético, dão uma conotação radical ao rock e ao hip- hop pela incorporação das condições de vida por eles enfrentadas nesse pro- cesso de re-invenção de si próprios,como também pela re-invenção da própria arte por eles praticada.27 A apropriação ‘geo-estética’ da cidade dá-se na medida em que os jovens utilizando-se da arte de viver — como passo a entender sua arte, produzida enquanto ética e estética de vida — produzem outras ‘Fortalezas’ para vive- rem, onde os espaços de convivência se tornam referência de bem viver, rega- dos a músicas, festas, prazer e trabalho coletivo de construção dessas utopias juvenis. OS MOVIMENTOS No entanto, suas atuações não se limitam a esses locais e se desenrolam, como já disse, em toda a cidade, mui- tas vezes concomitantemente, em relações complexas, nem sempre harmo- niosas. Com os punks o processo é semelhante, mas a orientação anarquista im- prime um outro viés: a criação de coletivos de convivência alternativa. O pri- meiro é criado ainda nos anos 80 (o Núcleo Coletivo de Consciência Libertá- ria — NCCL), e durou até o final dessa mesma década. Depois disso muitos outros vieram, e nos anos 90 o mais presente foi o ‘Coletivo Ruptura’. Até re- centemente a principal articulação se dava em torno da ‘Comuna Libertária’, uma entidade sediada no bairro de Parangaba e que encaminha a luta de punks e anarquistas sob essa ótica. As muitas formas de manifestação de seus desejos e necessidades têm apontado para uma construção alternativa nesses moldes. Além disso, uma articulação local e global23 se fortalece com vínculos de solidariedade e traba- lho com jovens e entidades de outros estados, como suas movimentações mais freqüentes grafitam, cantam e dançam por aí... A música iniciada nos ‘bailinhos’ quebrou as paredes dos clubes de clas- se média, espalhou-se como um rastro de pólvora, e botou fogo em tudo ao seu redor. Ao lazer puro e simples, juntou-se um outro com prazer misturado à política, à arte, à própria vida, como resposta direta aos desafios cotidianos. O canto falado e o grito primal nunca foram tão ouvidos e fortes. 224 Revista Brasileira de História, vol. 27, nº 53 Revista Brasileira de História, vol. 27, nº 53 As cidades da juventude em Fortaleza junho de 2007 MOVIMENTAÇÕES JUVENIS E TRANSPOSIÇÕES GEO-ESTÉTICAS A cidade, constituída por essas outras trajetórias, torna-se ao mesmo tempo múltipla, e, além de palco, sujeito, com os jovens em questão, de ou- 225 Francisco José Gomes Damasceno tras histórias. O rock e, pouco tempo depois, o hip-hop, apropriados pelos jo- vens, servem de elementos de ressignificação da cidade e de suas próprias ex- periências, e a cidade existente, junto com a cidade criada por eles, servem (ambas) de baliza para sua própria recriação. A reinvenção se dá nesse múlti- plo onde tudo e todos se insinuam uns sobre os outros. O rock que chegou a Fortaleza, em meados dos anos 50, nos salões dos clubes de elite ou das camadas médias urbanas e dentro de certos padrões de socialidade, sendo associado a jovens das famílias mais abastadas, como já o mostramos, aos poucos se altera, é tomado por outros sujeitos (jovens) e os toma, re-inventando a si mesmo, como resultado desse processo de tomar e ser tomado, de inventar e ser inventado, mas também, e, sobretudo, como re- sultado direto de experiências que se dão com as especificidades do contexto em questão e do meio (cidade) no qual se dão. Se, no início, o principal clube da cidade e das elites de Fortaleza (Náuti- co) abrigava o rock, bem como clubes elitizados, embora menos tradicionais (Maguary e Líbano), clubes estes situados à beira-mar (Náutico) ou em re- giões centrais e ocupadas por essas elites da cidade, e os grupos de baile ‘arra- savam’em ritmos ‘frenéticos’com um ‘balanço quente, mas respeitoso’(o mais conhecido foi “Iranildo e seu conjunto”), logo em seguida o que se operaria seria muito diferente. Ocorreria uma mudança, dessas regiões das elites para as periferias, sobretudo das camadas pobres da população, e também desses clubes para outros, criados no processo de expansão da cidade rumo às di- versas saídas para o interior.28 Silva esclarece esse modelo de urbanização, que é duramente criticado pelos jovens punks e hip-hopers em suas letras, em sua arte e intervenção sócio- histórica: Parte do monstro urbano que conhecemos foi gerado por medidas autoritá- rias tomadas em gabinetes oficiais, com forte influência de políticos e empresá- rios. Revista Brasileira de História, vol. 27, nº 53 MOVIMENTAÇÕES JUVENIS E TRANSPOSIÇÕES GEO-ESTÉTICAS A formação de extensas periferias e bolsões de miséria, constituídos quase que unicamente por conjuntos habitacionais distantes e mal servidos, a opção pelo transporte individual em detrimento do transporte coletivo, a facilidade de se implantar novas infra-estruturas que ocasionam a degradação de setores ou bairros tradicionais, expressam essa forma equivocada de produzir a cidade.29 Nos anos 60 o rock explode mundo afora e a repercussão dessa onda no Ceará foi inevitável, desta feita misturada a outros ritmos, mas há uma ver- dadeira proliferação de bandas, como as já apontadas anteriormente — Os 226 Revista Brasileira de História, vol. 27, nº 53 As cidades da juventude em Fortaleza As cidades da juventude em Fortaleza Faraós; Os Belgas tocando basicamente músicas de outros conjuntos como Beatles e Renato e Seus Blue Caps; e ainda Os Diferentes que tanto tocavam músicas de outros conjuntos com arranjos próprios, como as próprias com- posições. Faraós; Os Belgas tocando basicamente músicas de outros conjuntos como Beatles e Renato e Seus Blue Caps; e ainda Os Diferentes que tanto tocavam músicas de outros conjuntos com arranjos próprios, como as próprias com- posições. Nos anos 70 as bandas continuam a sua atuação em bailes e clubes de eli- te, como os já citados, mas há certo desgaste na fórmula, tanto pela sua exaus- tão, por certa repetição, como por outros dois fatores: 1) O rock em todo o mundo sofre mais uma de suas ‘revoluções internas’, desta vez o punk opera sua obra; e, 2) se inicia um processo, que nos anos 90 se tornaria hegemôni- co, de construção do forró como ritmo preponderante na cidade, colocado como “autêntico reflexo de nossa regionalidade”. Mesmo assim, bandas como a Peso ou a Posh se tornaram bastante conhecidas no cenário local e tiveram suas contribuições nesse contexto. junho de 2007 MOVIMENTAÇÕES JUVENIS E TRANSPOSIÇÕES GEO-ESTÉTICAS Assim, nos encontramos com os nossos sujeitos-jovens, jovens-sujeitos, no final dos anos 70, em um cotidiano delicado, refletindo um acentuado cres- cimento da então pequenina cidade de Fortaleza, graças ao processo de in- chaço sofrido pela chegada constante de migrantes oriundos do interior do Estado, que se reflete em espaços compactados, com o surgimento de bairros e loteamentos, e mesmo de favelas (apesar dos altos investimentos nos con- juntos habitacionais planejados e construídos a partir de então) sem os ne- cessários investimentos: lazer, emprego e opções de vida para uma população marcadamente jovem.30 A semelhança com os aspectos que dão origem às manifestações de jo- vens punks (no final dos anos 70) e de jovens hip-hopers (no início dos anos 80) não é coincidência. E a chegada de elementos culturais que traduziam es- sas realidades em outras partes do mundo parece ter sido amalgamada por esses jovens, incorporada e ter servido de instrumento em seus processos de apropriação do que se constituía para eles: a vida urbana. Não havia, até então, uma distinção musical, que só viria com o ‘amadu- recimento’ das manifestações. Apenas algum tempo depois é que alguma di- ferenciação foi percebida e estabelecida, e fomentou, assim, a formação de grupos por afinidades musicais e estéticas: “assim que a gente gostava, que tu- do era rock, mas a diferenciação que a gente não tinha conhecimento; que a gente foi conhecendo até por um acaso, rolou de conhecer... aí conheci Sex Pistols, The Clash, The Jam, The Breathers...”.31 Em outras palavras, o rock e o hip-hop (sofrendo a influência do próprio rock) aportam enquanto musicalidades, agrupam pelo gosto, pela afinidade, pela eleição do ritmo, pela incorporação estética, até mesmo de elementos co- 227 Francisco José Gomes Damasceno mo a dança e outros de dimensão plástica (como o grafite, ou mesmo o da constituição dos visuais), e deste ponto servem de elemento de aglutinação dos jovens identificados com essas manifestações, que passam então a re- inventá-las a partir de suas próprias experiências, expectativas e desejos. MOVIMENTAÇÕES JUVENIS E TRANSPOSIÇÕES GEO-ESTÉTICAS Esse ponto de partida — a eletividade32 e a comunhão estética e ética — tem sustentação em um cotidiano de experiências comuns, de perspectivas de vida semelhantes, e constitui o mais caro no que denominamos de ‘expe- riência musical’, algo que está na musicalidade, na música propriamente dita, mas que se constitui diferentemente, apenas a partir do momento em que o que está nas entrelinhas da pauta musical é absorvido, construído por quem ouve e faz música. Revista Brasileira de História, vol. 27, nº 53 OS SONS E A DANÇA – UMA OUTRA EXPERIÊNCIA Seu caráter mais importante reside na afirmação da existência de um mun- do universalmente obrigatório ... um mundo essencialmente diferente do mundo factual da luta cotidiana pela existência, mas que cada indivíduo pode realizar ... por si mesmo, ‘do interior’, sem transformação alguma do real ... Na verdade, mesmo quando a arte protesta contra a miséria social e a soli- dão, esse protesto é reapropriado esteticamente e solto de maneira gratificante, na experiência imaginária individual, como parte integrante do prazer que a obra lhe proporciona, e não como crítica real e desejo de mudar o mundo. As- sim, Marcuse conclui que mesmo ‘a idéia rebelde se torna um acessório para a justificação’.33 É desta forma que se constitui aos poucos o que denominamos de expe- riência musical, entre encontros furtivos no centro da cidade, nas praças dos diversos bairros periféricos da cidade de Fortaleza, em encontros nas casas para a audição do que ‘vem de fora’, em bailes e festas para a prática pública da escuta e da dança, no exercício, enfim, da manifestação da forma específi- ca como se resolve constituí-la. Disto resultam os deslocamentos diferenciados pela cidade em busca de diversão e arte, em busca dos iguais a si, dos diferentes de si, dos espaços para as práticas recém-instauradas de escuta, dança e vivência da música, então novidade na pequena cidade de Fortaleza. Com ela surge toda uma angústia de ser jovem naquele momento, associada a certa rebeldia presente nas musi- calidades em questão, veiculadas, sobretudo, em suas letras. 228 Revista Brasileira de História, vol. 27, nº 53 As cidades da juventude em Fortaleza As cidades da juventude em Fortaleza As cidades da juventude em Fortaleza O que ocorre é que ‘música’ deixa de ser apenas uma construção meló- dica e passa a dar sustentação a suas vidas de forma mais ampla. Passa a cons- tituir eticamente, se não todas, pelo menos grande parte de suas ações. Orien- ta para a vida ou pela vida afora, de tal modo que nos casos dos universos pesquisados nos últimos dez anos (hip-hop e punk rock), todos os jovens en- volvidos com essas musicalidades e ou movimentos continuam se pautando por essas perspectivas. A noção de música se amplia, e tem-se uma outra, a de ‘experiência mu- sical’, que se entende dentro desse complexo quadro de fusão entre experiên- cias de vida, eletividades afetivas, estéticas e práticas sócio-musicais. junho de 2007 OS SONS E A DANÇA – UMA OUTRA EXPERIÊNCIA E tam- bém a dimensão de uma arte enquanto prática, saber-fazer específico, e como tal com uma categoria de agentes ou sujeitos que detêm esse conhecimento.34 Essa arte transformada em experiência musical passa a ser construída de forma social por seus sujeitos, que atribuem significados a ela, extrapolando o campo de uma arte fechada em si mesma e se tornando o ponto de articu- lação entre suas concepções de mundo e suas experiências de vida, dadas no universo do sensível. Nesse sentido, a dança é outro aspecto da ‘experiência musical’ que deve ser observado como parte indissociável da musicalidade. O rock trouxe à ce- na no mundo contemporâneo essa dimensão de forma explícita e irrevogá- vel, e essa apropriação que, em certa medida, rompia com uma postura de contemplação estática, dos modelos anteriores, associados à música de câma- ra, é, talvez, sua marca mais registrada.35 Há uma espécie de dissolução dos sentidos como os conhecemos, de for- ma que cada um possua uma função específica, empregada quando necessá- rio. É como se todos os sentidos se tornassem um só e fossem empregados na mesma tarefa, ao mesmo tempo. A força da música, das letras, do ambiente, da bebida, da catarse promovida pelos efeitos de tudo isso junto, cria uma forma de sentir diferente, feita com o corpo como um todo homogêneo e in- divisível: “Ouvir com o corpo e empregar no ato da escuta não apenas os ou- vidos, mas a pele toda, que vibra ao contato com o dado sonoro: é sentir em estado bruto. É misturar o pulsar do som com as batidas do coração, é um quase não pensar”.36 Da mesma forma que essa dissolução se opera na musicalidade e nas suas danças, opera-se também uma fusão ética/estética, de tal modo que o ideal de beleza das manifestações musicais se transfere para a vida, a vida bela é ‘dançada’ aos ritmos de suas músicas, e mesmo compreendida diferentemen- te do que encontram no desigual quadro social no qual estão inseridos. 229 Francisco José Gomes Damasceno Francisco José Gomes Damasceno Torna-se, então, uma forma política de entendimento e de manifestação aos poucos burilada em uma interação política e social com outros atores so- ciais, tais como partidos, sindicatos, coletivos, universidades e grupos. OS SONS E A DANÇA – UMA OUTRA EXPERIÊNCIA A par- tir disso criam-se os movimentos, caudalosos, com adeptos onde quer que ocorram, em uma ‘nação imaginada’, ligada pelos sentimentos despertados pela música e construídos com suas próprias especificidades e sutilezas. As singularidades dos movimentos em Fortaleza estão justamente na cria- ção de suas entidades em torno de uma manifestação inicialmente musical, ou, em outras palavras, na constituição de um movimento sócio-político- cultural em torno do hip-hop e do punk. Portanto, o que antes era música, aos poucos fundamenta-se e se torna movimento.37 Revista Brasileira de História, vol. 27, nº 53 A INVENÇÃO PELA MÚSICA – DESLOCAMENTOS DA/NA CIDADE Nós vemos, toda Cidade é uma espécie de comunidade, e que toda comuni- dade é constituída em função de um certo bem (porque é em vista de obter aqui- lo que lhes aparece como um bem realizar sempre seus atos): daí resulta clara- mente que se todas as comunidades visam um bem determinado, aquela que está mais alta de todas e que engloba todas as outras, visa também, mais que as ou- tras, um bem que é o mais alto de todos. Esta comunidade é aquela que é cha- mada Cidade, é a comunidade política.38 Assim, a experiência musical desses jovens é um aspecto que deve ser le- vado em consideração no deslocamento dos clubes de elite para os clubes de periferia. Trata-se de uma juventude pobre e de periferia se manifestando e tendo a atenção de setores sociais diversos. Saímos do Maguary,do Líbano e do Náutico,de grupos de baile como “Ira- nildo e seu conjunto”, e, de uma musicalidade comportada e aceita, para clubes de periferia como o Apache, o Mênfis Clube do Antônio Bezerra, o Keops Clu- be, o Detroit, entre tantos outros, produzidos enquanto espaços por jovens po- bres, muitas vezes radicais, e de periferia, com bandas como Estado Indigente, Zueira, Desnutrição, e rappers como Titio e Cachorrão, W Man, Poeta Urbano e ZMC, entre tantos outros. Esse deslocamento que é ao mesmo tempo geográfico, posto que se faz dos espaços das elites para os espaços populares, tem ao mesmo tempo um caráter de classe e etário, já que é operado por jovens trabalhadores, ou mes- mo jovens sem profissão, desempregados e pobres, ou ainda estudantes, por- 230 Revista Brasileira de História, vol. 27, nº 53 As cidades da juventude em Fortaleza tanto, em formação para o mundo do trabalho, habitantes dos bairros mais pobres de minha cidade. Também tem um caráter estético, já que dentro das duas manifestações o que ocorre de forma invariável é uma sucessão de transgressões estéticas de cada uma dessas musicalidades, além de ser uma transgressão também para o tipo de música (mesmo de rock e de hip-hop) feito em Fortaleza no perío- do. Fortaleza jamais havia visto algo tão ‘radical’. Além disso, essa dimensão transgressiva de alguma forma constituiu os seus repertórios de compreen- são e de articulação estética e posteriormente também política. A INVENÇÃO PELA MÚSICA – DESLOCAMENTOS DA/NA CIDADE Amalgamando todos esses aspectos, consubstanciados nesse deslocamen- to, estão suas experiências de vida, suas experiências musicais, que dão a esse movimento um caráter de duplo deslocamento, por um lado estético e por outro geográfico, ou mesmo geopolítico, daí por que falamos em deslocamen- tos ‘geo-estéticos’ em Fortaleza. Utilizo a noção de ‘deslocamento geo-estético’ 39 como uma fusão do es- paço geográfico e da manifestação estética e que dá sentido a esses espaços, configurando assim, com base na idéia de que o espaço é o lugar praticado,40 uma outra dimensão: a dimensão da arte de viver, de viver com as referências de beleza por eles instituídas, e, assim, elegendo as trajetórias a serem estabe- lecidas dentro do universo urbano, que redimensiona o espaço a partir da ar- te (música, dança) e instaura a cidade na qual vivem, dentro das muitas cida- des existentes na cidade de Fortaleza. junho de 2007 EXPERIÊNCIAS MUSICAIS, DESLOCAMENTOS GEO-ESTÉTICOS: AS CIDADES E OS JOVENS As leituras construídas pelos jovens em suas letras e manifestos guardam características peculiares. Vejamos especificamente e de forma rápida o caso de algumas noções dos jovens punks e hip-hopers. Em primeiro lugar, como a sociedade que emerge de suas letras nos é re-apresentada; em seguida, a no- ção que têm de si próprios. Emerge de suas letras uma cidade identificada com a divisão social, ou uma ‘sociedade’ constituída a partir de suas experiências na/da cidade. Assim se caracteriza uma cidade/sociedade marcadamente repressora, desigual, di- vidida, exploradora e injusta. É entendida como a máxima manifestação or- ganizacional humana e, portanto, o que se representa é uma humanidade di- vidida e à beira de um tipo qualquer de colapso. 231 Francisco José Gomes Damasceno Francisco José Gomes Damasceno Divididos em classes, os raps e punk rocks são construídos com a inten- ção de serem a fala das minorias sem voz, dos injustiçados, marginais, prosti- tutas, mendigos, meninos de rua, trabalhadores, e, sobretudo, das periferias, do povo pobre, operários e desvalidos.41 pois nós somos a voz da periferia / o grito do gueto / o argumento da pobreza / pra lutar-brigar...42 Ossos quebrados / Mente vazia / Olhos arrancados / Intestino apodrecido / Re- trato operário / Pobre indigente / Largado jogado / Vítimas inocentes / Largado jogado / Estado esquecido / De putrefação / De imperialismo / Diferença social / Grito de dor / Invasão militar / Só carnificina / Não há sobrevivência / Minha vida esquecida / Não há sobrevivência / Sua vida apodrecida.43 A sociedade para eles é a ‘encarnação’ dessas diferenças, e, portanto, a sua manutenção é necessária para quem delas se beneficia. Identificados como os inimigos, os ricos são o principal alvo: a sociedade fecha as portas na cara de quem nada tem / pois as portas são fecha- das / grades seladas / pois é isso que convém antes de você / cometer o delito / delito ou ato de sobreviver...44 Em outro rap se mostra concretamente como essa sociedade de ricos se fecha: Em outro rap se mostra concretamente como essa sociedade de ricos se fecha: De que vale o Beach Park se eu não posso entrar? / de que me serve o Iguatemi se eu não tenho ‘grana’ pra gastar? / carro importado pra quê se eu não posso comprar? EXPERIÊNCIAS MUSICAIS, DESLOCAMENTOS GEO-ESTÉTICOS: AS CIDADES E OS JOVENS 45 A inclinação desses jovens à esquerda (não única) a partir da criação de seus movimentos e de articulações com partidos políticos, sindicatos, ONGs, coletivos e outras instituições revela que a cidadania não é uma construção meramente voltada e construída pelo políti- co, embora possa vir a ser desta forma enunciada. A questão social aparece co- mo um problema posto pelo desenvolvimento das cidades, e uma das formas do seu enfrentamento é o deflagrar da construção da cidadania.46 A aparição no universo citadino é uma intervenção de conquista dos di- reitos elementares negados, e as suas letras, os seus manifestos podem ser en- 232 Revista Brasileira de História, vol. 27, nº 53 As cidades da juventude em Fortaleza tendidos como a fala de sujeitos sociais em ebulição na dividida cidade de ri- cos e pobres, na Fortaleza de muitos caminhos possíveis e instituição de seus desejos de cidades outras. Eles se apresentam aos poucos, em indícios sutis do lugar social de suas falas. Isso pode ser observado em um dos primeiros raps do movimento a fa- zer sucesso, em toda a cidade, o “Rap da Dora” da dupla Titio e Cachorrão. Nele o trabalho é identificado ao sofrimento — leia-se o trabalho que não re- compensa — criando a necessidade de espaços onde o sofrimento deve ser substituído pela alegria e pela vida. EXPERIÊNCIAS MUSICAIS, DESLOCAMENTOS GEO-ESTÉTICOS: AS CIDADES E OS JOVENS Expressa de forma ambígua a Dora:“boto a mão na cabeça é tão bom co- mo me sinto / pois nesses dias não tem que trabalhar fico em casa a se ajeitar / pois nesses dias quer também se divertir...” — observe-se que o sujeito ‘eu’ elíptico e depois indefinido pelo uso da partícula ‘se’, provocando uma auto- mática identificação/confusão autores/Dora — como se por sua boca eles fa- lassem, mostrando como se sentem estes jovens que, imersos no mundo do trabalho, na tensão nele estabelecida, encontram nos bailes, nos momentos de diversão e lazer, uma das poucas alegrias que têm.47 Também nos punk rocks a condição operária é sentida em manifestos, documentos, músicas: A vida de operário é um tormento / A vida de operário é um sacrifício / Pra vi- ver (2x) / Sem razão (2x) / Escravo moderno nos campos / Escravo moderno nas fábricas / Pra viver (2x) / Sem razão (2x) / Viver pelas leis dos patrões / Conde- nados à pena de morte / Pra viver (2x) / Sem razão (2x) 48 Eis que se revelam suas faces: jovens trabalhadores da cidade. Pequenos operários, desempregados, mão-de-obra pouco qualificada e barata, que na cidade através de sua arte se produz enquanto grupo e aponta os sérios pro- blemas que os cercam e demandam solução imediata. EXPERIÊNCIAS MUSICAIS, DESLOCAMENTOS GEO-ESTÉTICOS: AS CIDADES E OS JOVENS A cidade apartada aparece em vários momentos, como neste trecho: quem vê Fortaleza como no resto do Brasil / vê estupro, assassinato, corrupção desenfreada / temos brigas de gangues / assaltos a mão armada, traficantes de maconha e polícia bem safada / os garotos roubando lá na praça da estação / e no passeio público tem até prostituição / sem esquecer dos mendigos lá do cen- tro da cidade / e dos apatoladores que existem aos milhares / temos mortes no trânsito e muitos viciados / motoristas imprudentes e hospitais superlotados / temos roubo de carro e cheques sem fundos / pode crer na capital nós temos de 233 junho de 2007 junho de 2007 Francisco José Gomes Damasceno tudo / o nosso Ceará com todos seus problemas / é o rap do Conscientes do Sis- tema (3x) / aqui também tem desemprego aumentando a violência / e escolas do governo numa tremenda decadência / temos rádio e TV / revista e jornal / essa merda que ilude e nos fazem passar mal / distorcendo as notícias isso eles fazem muito bem / transformando em marginais muitas pessoas de bem / essa é a dura realidade quem tem grana mata e rouba e ainda fica em liberdade / ao contrário do pobre que não tem nada e é fodido / só por causa da aparência leva nome de bandido / temos o Iguatemi e o Shopping Aldeota / a rota dos burgue- ses que nos vêem como idiotas / temos fome e miséria e até racionamento / gen- te inocente passando o maior tormento / “eu sou a nata do lixo eu sou o luxo da aldeia eu sou do Ceará” (sampler) / o nosso Ceará com todos seus problemas / é o rap do Conscientes do Sistema (3x) 49 Outra letra bastante significativa de suas idéias sobre si e sobre a cidade é “Nativo Urbano”, da banda Estado Indigente: Mesmo que eu morasse / No mundo sozinho / Mesmo que eu morasse / Em mundo perfeito / Tocava hardcore pra vida / Com prazer (2x) / E nada me im- pediria / Consciência, capacidade e prazer / Virtudes de poucas viventes / No ha- bitar sonolento da vida / Almeja sonho impotente / Desmotiva pela inconsciên- cia / De outros / Paralelo, direto, nostálgico. Revista Brasileira de História, vol. 27, nº 53 EXPERIÊNCIAS MUSICAIS, DESLOCAMENTOS GEO-ESTÉTICOS: AS CIDADES E OS JOVENS A consciência de si e dos outros, a possibilidade de intervenção direta, operada pelo sujeito consciente, apesar da identificação da vida aos bailes, re- vela certa tristeza, oriunda decerto de suas origens, ou dos meios nos quais se manifestam: as ruas.50 O espaço da rua, longe de ser o espaço de ‘deambulação’ como é signifi- cado para os rockers,51 constitui-se em espaço de vivências, de violência, de amores, de lutas, de revolta, torna-se o palco de suas vidas, o local onde vi- vendo, buscam inspiração para suas letras, ou ainda onde vivem as histórias que suas letras contam. Locais perigosos e instáveis, onde se corre o risco de viver. Seus habitan- tes, longe da calma das casas, da segurança das famílias, da limpeza e higiene dos lares normais, são múltiplos e dividem a agitação, o vai-e-vem de tran- seuntes, a insegurança da luta pela sobrevivência, a sujeira causada pelas aglo- merações etc. É o espaço da violência, da morte, da dor, da fome, da miséria, dos des- validos, dos marginalizados, dos menores abandonados, delinqüentes, prosti- 234 Revista Brasileira de História, vol. 27, nº 53 As cidades da juventude em Fortaleza tutas, pobres e desocupados. Tipos como o ‘vagabundo’, que, como nos mos- tram B.Girl e Rainha,“vive pelas ruas do centro da cidade”: tutas, pobres e desocupados. Tipos como o ‘vagabundo’, que, como nos mos- tram B.Girl e Rainha,“vive pelas ruas do centro da cidade”: aonde a cada esquina um assalto acontece ... marginais andam a solta, tromba- dinhas cheiram cola / se não preferem o roubo muitos deles pedem esmola / são jogadores nas ruas pra ganhar algum dinheiro / mas as ruas lhes ensinam o ver- dadeiro desacerto...52 se os meus filhos estão nas ruas / suas vidas aventuram lances, mortes, extermínio ... Rap e Hip-Hop nas calçadas da cidade / o Break violento e forte... (grifo meu)53 se os meus filhos estão nas ruas / suas vidas aventuram lances, mortes, extermínio ... Rap e Hip-Hop nas calçadas da cidade / o Break violento e forte... (grifo meu)53 As ruas tomam conta desses seres / menores carentes ninguém quer saber deles ... aqui trombadinhas aumentam a cada dia / menores nas ruas, crianças perdi- das / filhos das ruas, quem vai tirá-los da rua? / quem os fará viver? / ... e você...? EXPERIÊNCIAS MUSICAIS, DESLOCAMENTOS GEO-ESTÉTICOS: AS CIDADES E OS JOVENS / mas vocês são todos iguais / em suas casas tão em paz / longe das ruas, longe da sujeira, longe da vida imunda / eu aqui e você na sua / e os menores carentes estão todos nas ruas / filhos da rua / filhos da rua / sem-tetos dormindo nas pra- ças / ... matar ou morrer faz parte da vida / da vida na verdade não são inocen- tes são vítimas conseqüentes / crianças delinqüentes / ... prostituir o próprio corpo em troca de quase nada ... / flanelas na mão pedindo um trocado / sem esperanças, sem família, nas ruas jogados / não existe para eles sonho algum / vivem o presente sem futuro nenhum...54 No percurso pelas trilhas abertas na cidade, na instauração das muitas cidades, se estabelecem marcações, se estabelecem territórios no andamento da vida. Eles instituem os seus marcos, ou pequenos portos no mar da cidade. Depois de algum tempo, as turmas punks passaram a se encontrar na re- gião do centro da cidade, na Praça do Ferreira, depois na Praça José de Alen- car; os rappers em locais específicos nos bairros, como o Pólo de Lazer do Conjunto Ceará, que se constituíram em points, onde eram ‘trocadas as idéias’ e traçadas as trajetórias de cada final de semana: Eu estudava ainda, vinha à Praça José de Alencar, que de certa forma sempre foi um point, aqui em frente ao teatro todas as sextas tinha um grupo tocando MPB, música daqui mesmo. Aí, os mesmos roqueiros que pintavam lá no Grê- mio do José Walter se reuniam todas as sextas aqui nessa praça ou então na an- 235 junho de 2007 junho de 2007 Francisco José Gomes Damasceno tiga Praça do Ferreira, e aí a gente foi se entrosando. Quando não queria ir pra casa, ia pro Monte Castelo, a gente virava a noite, ficava ouvindo som...55 Nos points se decidia a programação do final de semana, marcavam-se os encontros, escolhiam-se os melhores programas, além, é claro, da tradicio- nal ‘troca de idéias’: Nessa época eu não tinha muito tempo, porque eu trabalhava durante o dia e estudava à noite, mas foi uma das épocas que eu vacilei mais com o estudo. Eu deixava de ir pro colégio pra pintar nos points às quartas e sextas, ali na Praça do Ferreira. EXPERIÊNCIAS MUSICAIS, DESLOCAMENTOS GEO-ESTÉTICOS: AS CIDADES E OS JOVENS E tinha um dos primeiros clubes com sons de bandas, só Ramortes que tocava na época, o Davis Clube, lá na pista da Barra. Que lá o cara aloprava todas as quartas, sextas, sábados e domingos, quando a banda não tocava era som de fita...56 O point nunca era o fim de uma trajetória e ou de uma jornada, mas sem- pre o seu ponto inicial, o local de encontro, o ponto de referência em torno do qual todos aqueles que queriam podiam se encontrar. O fim da jornada, quando não havia um baile em jogo, era a experiência comum de ouvir mú- sica, de trocar idéias e depois fazer as próprias músicas, aprender como tecer sua arte: a gente se reunia ali, e ia pra casa dum, pra casa do outro, do João Wilsom, aí fi- cava lá, ouvindo o som, trocando idéia, fazendo música... Era isso quase todo dia. Nos finais de semana, sempre tinha os locais pra gente ir, uma banda que ia tocar... Descia pro Davis Clube, que quase todo dia tinha som lá...57 O point, no entanto, era mais do que um local. Em inúmeros momentos andei, divaguei, pontuei, trilhei as cidades dos desejos dos jovens. Nosso des- locamento em bando causava medo e reações diversas nas pessoas pois a caminhada vai adquirindo uma autonomia de point, nesse durante con- versamos, um espera o outro ou se adianta para falar com alguém que está na frente. Poderíamos talvez não chegar. Big me diz rindo: ‘acho que a gente está andando por andar’.58 Ruas e praças, clubes, centros comunitários, casas e universidades, sedes de sindicatos, movimentação na cidade e pela cidade que suas experiências di- retas instituem, criam como espaço de vida. As apropriações feitas revelam ou- tras possibilidades, outras formas de perceber o mundo e também de criá-lo. 236 Revista Brasileira de História, vol. 27, nº 53 As cidades da juventude em Fortaleza As cidades da juventude em Fortaleza Talvez caminhem esses jovens por caminhos que não vemos, mesmo quan- do os seus deslocamentos se fazem nas ruas que vemos, nas praças nas quais sentamos, conversamos... talvez cantem esses jovens realidades que não conse- guimos sequer imaginar, compreender.... 1 Pesquisador ligado ao Grupo de Práticas Urbanas do Mestrado Acadêmico em História — Mahis, e do Labvida — Laboratório de Direitos Humanos, Cidadania e Ética do Mes- trado em Políticas Públicas também da UECE. 2 CANEVACCI, Massimo. Conclusão em forma-de-mapa. In.: Sincretismos: uma explora- ção das hibridações culturais. São Paulo: Studio Nobel; Instituto Cultural Ítalo Brasilei- ro–Instituto Italiano de Cultura, 1996. p.91. 3 VIEIRA, Roberto César. Pedras que não rolam criam limo: rock cearense — consumo e mercado. Monografia apresentada ao curso de Comunicação Social, Universidade Federal do Ceará. Fortaleza, 1994. p.57-8. EXPERIÊNCIAS MUSICAIS, DESLOCAMENTOS GEO-ESTÉTICOS: AS CIDADES E OS JOVENS talvez essas outras realidades, cidades, estejam no limiar de suas experiências e reflitam essa ambigüidade, pois: Enquanto o adulto vive ainda sob o impacto de um modelo de sociedade que se decompõe, o jovem já vive em um mundo radicalmente novo, cujas catego- rias de inteligibilidade ele ajuda a construir. Interrogar essas categorias permite não somente uma melhor compreensão do universo de referências de um grupo etário particular, mas também da nova sociedade transformada pela mutação.59 Foto 2 – Davis Clube (extinto). Fotografia do autor, 2003. Foto 2 – Davis Clube (extinto). Fotografia do autor, 2003. NOTAS Muitas outras no- tícias dão a dimensão das carências da cidade, como por exemplo: “Comunidades carentes reclamam das péssimas condições de vida”, Tribuna do Ceará, 21 jan. 1980, p.7; ou ainda: “Passagem cara faz o povo andar a pé”, Diário do Nordeste, 24. dez. 1981, capa, onde se anunciava o presente de Natal dos fortalezenses, um aumento das passagens de ônibus. Algum tempo depois, em reação explícita a essa situação o movimento punk decreta uma campanha pela gratuidade do transporte coletivo em Fortaleza, com o ‘desça por trás’. Cf. “Fora aumentos, chega de exploração, coletivo grátis já!”, nota/panfleto, Fortaleza, s.d. Di- ziam eles em determinado momento: “Chega! Chega!... Tá na hora de dizer um basta a tu- do isso. Só vão aprender quando a população se revoltar. Por isto estamos propondo que os trabalhadores reajam diante dessa situação. Vamos subir por trás, pular a catraca, con- versar entre nós e vamos lutar pelo transporte coletivo gratuito. Já estamos pagando caro demais em impostos e ainda sermos obrigados a enriquecer mais e mais os bolsos desses safados, enquanto o povo morre de fome? Não! Chega! NCCL — Núcleo Coletivo de Cons- ciência Libertária e MAP — Movimento Anarco-Punk”. 6 Entrevista realizada por este pesquisador, com a ex-integrante do movimento punk, à época chamada Flor Punk, na Praça dos Leões, centro da cidade de Fortaleza, em 6 mar. 2003. p.2-3. 6 Entrevista realizada por este pesquisador, com a ex-integrante do movimento punk, à época chamada Flor Punk, na Praça dos Leões, centro da cidade de Fortaleza, em 6 mar. 2003. p.2-3. 7 Carmo nos informa que, nesse período, o termo ‘bandas’não era empregado. Cf. CARMO, Paulo Sérgio do. Culturas da rebeldia: a juventude em questão. São Paulo: Ed. Senac, 2001. 279p. 8 XIMENES, Amaudson. A música underground em Fortaleza: resistência ou crise de iden- tidade? Monografia apresentada ao curso de Ciências Sociais, Universidade Estadual do Ceará. Fortaleza, 1998. p.34. É interessante frisar que Ximenes é guitarrista da banda Obs- kure, e fundador da Associação Cearense do Rock (ACR). Roqueiro ‘de primeira hora’, vi- venciou esse período e alguns dos acontecimentos que narra, tendo em dado momento desenvolvido alguns trabalhos com os punks em Fortaleza. Foi entrevistado no sentido de delinear a ação dos grupos punks, e de identificar as relações estabelecidas nesse momento de surgimento do punk na cidade. 9 Entrevista realizada por este pesquisador com ‘Magoo’, em 28 jan. NOTAS NOTAS 1 Pesquisador ligado ao Grupo de Práticas Urbanas do Mestrado Acadêmico em História — Mahis, e do Labvida — Laboratório de Direitos Humanos, Cidadania e Ética do Mes- trado em Políticas Públicas também da UECE. 2 CANEVACCI, Massimo. Conclusão em forma-de-mapa. In.: Sincretismos: uma explora- ção das hibridações culturais. São Paulo: Studio Nobel; Instituto Cultural Ítalo Brasilei- ro–Instituto Italiano de Cultura, 1996. p.91. 3 VIEIRA, Roberto César. Pedras que não rolam criam limo: rock cearense — consumo e mercado. Monografia apresentada ao curso de Comunicação Social, Universidade Federal do Ceará. Fortaleza, 1994. p.57-8. 3 VIEIRA, Roberto César. Pedras que não rolam criam limo: rock cearense — consumo e mercado. Monografia apresentada ao curso de Comunicação Social, Universidade Federal do Ceará. Fortaleza, 1994. p.57-8. 237 junho de 2007 Francisco José Gomes Damasceno 4 Mais à frente comentarei a noção de deslocamento geo-estético. 5 A matéria do Diário do Nordeste, 16 jan. 1982, p.7, dava conta das dimensões da cidade: “Com um milhão e trezentas mil pessoas, Fortaleza só tem sete creches”. Muitas outras no- tícias dão a dimensão das carências da cidade, como por exemplo: “Comunidades carentes reclamam das péssimas condições de vida”, Tribuna do Ceará, 21 jan. 1980, p.7; ou ainda: “Passagem cara faz o povo andar a pé”, Diário do Nordeste, 24. dez. 1981, capa, onde se anunciava o presente de Natal dos fortalezenses, um aumento das passagens de ônibus. Algum tempo depois, em reação explícita a essa situação o movimento punk decreta uma campanha pela gratuidade do transporte coletivo em Fortaleza, com o ‘desça por trás’. Cf. “Fora aumentos, chega de exploração, coletivo grátis já!”, nota/panfleto, Fortaleza, s.d. Di- ziam eles em determinado momento: “Chega! Chega!... Tá na hora de dizer um basta a tu- do isso. Só vão aprender quando a população se revoltar. Por isto estamos propondo que os trabalhadores reajam diante dessa situação. Vamos subir por trás, pular a catraca, con- versar entre nós e vamos lutar pelo transporte coletivo gratuito. Já estamos pagando caro demais em impostos e ainda sermos obrigados a enriquecer mais e mais os bolsos desses safados, enquanto o povo morre de fome? Não! Chega! NCCL — Núcleo Coletivo de Cons- ciência Libertária e MAP — Movimento Anarco-Punk”. 5 A matéria do Diário do Nordeste, 16 jan. 1982, p.7, dava conta das dimensões da cidade: “Com um milhão e trezentas mil pessoas, Fortaleza só tem sete creches”. Revista Brasileira de História, vol. 27, nº 53 NOTAS 18 Melucci, citado em CARRANO, P. C. R. Juventude — as identidades são múltiplas. Re- vista Movimento, São Paulo: DP&A, n.1, maio 2000. O mesmo Melucci (1997) propõe em outro artigo que se deveria falar em redes conflituosas, em vez de em movimentos, dado o caráter de produção de formas culturais. Apesar de interessante, no caso dos movimentos aqui estudados não se aplica, posto que os significados construídos por esses jovens pas- sam pela noção de movimento. 18 Melucci, citado em CARRANO, P. C. R. Juventude — as identidades são múltiplas. Re- vista Movimento, São Paulo: DP&A, n.1, maio 2000. O mesmo Melucci (1997) propõe em outro artigo que se deveria falar em redes conflituosas, em vez de em movimentos, dado o caráter de produção de formas culturais. Apesar de interessante, no caso dos movimentos aqui estudados não se aplica, posto que os significados construídos por esses jovens pas- sam pela noção de movimento. NOTAS 2003, em sua residên- cia na cidade de Caucaia, região metropolitana de Fortaleza. p.1. Magoo foi um dos prin- cipais expoentes do punk durante os anos 80 e 90, distanciou-se do movimento organiza- do, mas continua com a banda Estado Indigente, que formou ainda no final dos anos 80. 10 A priorização total do estilo ocorre com a maioria dos entrevistados da pesquisa realiza- da com punks e hip-hopers na cidade de Fortaleza. 11 CERTEAU, Michel de. A invenção do cotidiano. Artes de Fazer. Petrópolis (RJ): Vozes, 1999. p.202. 12 Cf. JANOTTI JR., Jéder S. Heavy metal: o universo tribal e o espaço dos sonhos. Disser- tação (Mestrado) — Depto. de Comunicação Social, programa de mestrado em Multi- 238 Revista Brasileira de História, vol. 27, nº 53 As cidades da juventude em Fortaleza meios, Unicamp. Campinas, 1994. Segundo ele através “do vestuário, dos shows e dos lo- cais de encontro do grupo, os jovens procuram construir ‘territórios existenciais’ que pos- sibilitem exercitar sua subjetividade situada além dos espaços normatizados (tais como fa- mília, escola e trabalho)”. meios, Unicamp. Campinas, 1994. Segundo ele através “do vestuário, dos shows e dos lo- cais de encontro do grupo, os jovens procuram construir ‘territórios existenciais’ que pos- sibilitem exercitar sua subjetividade situada além dos espaços normatizados (tais como fa- mília, escola e trabalho)”. 13 MAFFESOLI, Michel. O tempo das tribos: o declínio do individualismo nas sociedades de massa. Rio de Janeiro: Forense-Universitária, 1987. 14 PESAVENTO, Sandra Jatahy. Uma outra cidade: o mundo dos excluídos no final do sé- culo XIX. São Paulo: Cia. Ed. Nacional, 2001. p.11-2. 15 Entrevista realizada por este pesquisador com o rapper Sátiro Silvestre, no dia 10 ago. 2003, no bairro do Conjunto Ceará, em Fortaleza, p.1. Atual integrante da coordenação do MH2O e um dos primeiros dançarinos de break da cidade, foi um dos fundadores da Striking gangue de break nos anos 80. 16 Cf. DAMASCENO, Francisco José Gomes. O movimento hip-hop organizado do Ceará / MH2O-Ce (1990-1995) Dissertação (Mestrado) — Pontifícia Universidade Católica de São Paulo. São Paulo, 1997. 333p.; e DAMASCENO, Francisco José Gomes. Sutil diferença: o movimento punk e o movimento hip-hop em Fortaleza — grupos mistos no universo ci- tadino contemporâneo. Tese (Doutorado) — PUC-SP, São Paulo, 2004. 511p. 17 SOUZA, Janice Tirelli Ponte de. Reinvenções da utopia: A militância política de jovens nos anos 90. São Paulo: Hacker, 1999. p.93-4. junho de 2007 19 MAFFESOLI, Michel. O tempo das tribos..., cit., p.27. 19 MAFFESOLI, Michel. O tempo das tribos..., cit., p.27. 20 É interessante observar que todos os entrevistados desta pesquisa têm o estilo no centro de suas atividades, sejam elas profissionais, sociais, políticas etc. Há a priorização total do estilo, que assume a dimensão de ordenador das relações. Neste caso, o termo ‘identidade’ se aplica perfeitamente. 21 De um modo geral, cada entrevistado tem sempre uma história de como ocorreu consi- go, ou mesmo com outros do grupo. 22 OLIVEIRA, Vantiê Clínio de Carvalho. O movimento anarco-punk (A identidade e a au- tonomia nas produções e nas vivências de uma tribo urbana juvenil). Dissertação (Mes- trado) — Programa de Pós-Graduação em Ciências Sociais, UFRN. Natal, 2003. p.47. Ape- sar de o estudo versar sobre a cidade de Natal,a referência é feita com relação a um congresso dos anarco-punks ocorrido na cidade de João Pessoa. 23 “No interior desses remoinhos flutuantes e plurais de panoramas glocal, emerge com for- ça a produção, a difusão e o consumo de sincretismos culturais. Essa palavra nova, fruto de recíprocas contaminações entre global e local, foi forjada justamente na tentativa de 239 Francisco José Gomes Damasceno captar a complexidade dos processos atuais. Nela foi incorporado o sentido irrequieto do sincretismo. O sincretismo é glocal” (grifos meus). CANEVACCI, op. cit., p.25. captar a complexidade dos processos atuais. Nela foi incorporado o sentido irrequieto do sincretismo. O sincretismo é glocal” (grifos meus). CANEVACCI, op. cit., p.25. 24 TURRA NETO, Nécio. Enterrado vivo: identidade punk e território em Londrina. São Paulo: Ed. Unesp, 2004. p.100, 104, 117. 25 ‘Movimentação’ aqui se entende por ‘tornar-se movimento’ e, ao mesmo tempo, como ação dinâmica dos jovens em fazer, criar, inventar-se enquanto processo complexo de rela- ções sociais, políticas, culturais, artísticas etc. 26 É interessante frisar que música, aqui, se configura basicamente pelo rock e demais ma- nifestações underground, que tendo o rock como referência se posicionam como elemento de transgressão de determinados setores, sobretudo da juventude contemporânea, assu- mindo o caráter de instrumento de crítica social e política. 27 Refiro-me aqui a arte como prática, como defendido por SHUSTERMAN, Richard. Vi- vendo a arte: o pensamento pragmatista e a estética popular. São Paulo: Ed. 34, 1998. Revista Brasileira de História, vol. 27, nº 53 19 MAFFESOLI, Michel. O tempo das tribos..., cit., p.27. Vivendo a arte…, cit., p.68-9. 34 A idéia da arte enquanto prática sócio-histórica, desenvolvida por Shusterman, foi uma das que mais me auxiliaram na compreensão dos fenômenos da arte e da cultura juvenil. 35 Alguns autores sugerem essa prática como uma metáfora da vida cotidiana urbana con- temporânea. Aqui nos interessa apenas anotar a dança como parte indissociável da expe- riência musical. 35 Alguns autores sugerem essa prática como uma metáfora da vida cotidiana urbana con- temporânea. Aqui nos interessa apenas anotar a dança como parte indissociável da expe- riência musical. 36 MORAES, Jota de. O que é música. São Paulo: Nova Cultural; Brasiliense, 1986. 37 Cf. DAMASCENO, cit., 2004. A constituição dos movimentos punk e hip-hop como mo- vimentos juvenis sociais contemporâneos é desenvolvida nesse trabalho. 37 Cf. DAMASCENO, cit., 2004. A constituição dos movimentos punk e hip-hop como mo- vimentos juvenis sociais contemporâneos é desenvolvida nesse trabalho. 38 Aristóteles, cit. em PESAVENTO, Sandra Jatahy. A vitória de Antígona sob o signo de Ba- bel, a cidade brasileira dessacralizada. In: PESAVENTO, Sandra Jatahy (Org.) Escrita, lin- guagem, objetos: leituras de história cultural. Bauru (SP): Edusc, 2004. p.168. 38 Aristóteles, cit. em PESAVENTO, Sandra Jatahy. A vitória de Antígona sob o signo de Ba- bel, a cidade brasileira dessacralizada. In: PESAVENTO, Sandra Jatahy (Org.) Escrita, lin- guagem, objetos: leituras de história cultural. Bauru (SP): Edusc, 2004. p.168. 39 Faço assim uma apropriação livre de conceitos como o de ‘geopolítica’, por exemplo, e, dilato seu sentido, reinvento o seu uso e o avalio sob a ótica de que a manifestação juvenil contemporânea é essencialmente cultural e nisso reside a articulação entre cultura e polí- tica para esses sujeitos: uma intervenção cultural, social e artística na cidade, que é marca- damente política pela crítica e pelas práticas que estabelecem. 40 Cf. CERTEAU, Michel de. A invenção do cotidiano..., cit. 40 Cf. CERTEAU, Michel de. A invenção do cotidiano..., cit. 41 Essa é uma pretensão assumida por eles próprios, e colocada e reexposta constantemen- te. Sente-se isso nas entrevistas realizadas com eles no Ceará, e essa também é uma carac- terística mais geral do rap. Mano Brown, líder dos Racionais MCs declara (Folha de S. Pau- lo, Ilustrada, 7 maio 1994, “A vez do Rap”.): “Não sou porta-voz do movimento hip-hop, mas da periferia talvez!”. Cf. 19 MAFFESOLI, Michel. O tempo das tribos..., cit., p.27. 28 “Nossa cidade teve seu processo de metropolização e expansão da malha urbana direcio- nado pelas suas principais vias rádio-concêntricas, herança de seus antigos caminhos co- mo o do Soure, antigo nome de Caucaia, que provocou o adensamento urbano na porção oeste da cidade”. SILVA, José Borzacchiello da. Nas trilhas da cidade. Fortaleza: Museu do Ceará; Secretaria de Cultura e Desporto do Ceará, 2001. (Coleção Outras Histórias). p.70. 29 Ibidem, p.103. 30 Cf. SILVA, José Borzacchiello da. Movimentos sociais populares em Fortaleza: uma abor- dagem geográfica. Tese (Doutorado) — Departamento de Geografia, Universidade de São Paulo. São Paulo, 1986. Os inúmeros migrantes expulsos do interior e sem a certeza de qualidade de vida em terras distantes fazem de Fortaleza um pólo atrativo, não só pela sua proximidade. Este dado evidencia a enorme influência do ‘sertão’ ou do ‘campo’ na cidade de Fortaleza e em suas manifestações culturais. 31 Magoo, cit., p.1. 31 Magoo, cit., p.1. 32 São muitas as narrativas que citam esse aspecto, de tal modo que se pode avaliar o papel da eletividade como fundamental: Sátiro Silvestre, um rapper, ex-breaker, e desde o princí- pio ligado ao hip-hop, em tom de brincadeira, alude ao tempo que já tem de ‘movimento’: “Era uma vez... (risos) cara meu contato com o hip-hop foi... desde os 12 anos, faz tem- po... mas foi aqui na comunidade mesmo, nas festas que tinha, que eu via a galera dançan- do break, a rapaziada que antecedeu a gente, que era o Lula, não tinha nada organizado, mas o pessoal que dançava lá no Inter ... Inter dance, no Monte Castelo, era um point on- de os punks, e no tempo... 85, 86, 87 o break tava em ascensão, era de certa forma uma no- vidade, aí lá o pessoal tinha a festa de rock, punk, e tinha o pessoal que dançava o break se congregava lá, aí eles migraram alguns aqui pro Conjunto Ceará, aí foi quando eu vi o Lu- la fazendo o Moinho de Vento eu endoidei, ‘meu irmão, como é que esse cara faz isso?’. A partir daí, fui chegando na roda e me engajei, a a partir daí foi o 1o contato mesmo com o hip-hop... (Entrevista realizada com Sátiro em 10 ago. 2003, no bairro do Conjunto Ceará em Fortaleza). 240 Revista Brasileira de História, vol. 27, nº 53 As cidades da juventude em Fortaleza 33 SHUSTERMAN, Richard. 19 MAFFESOLI, Michel. O tempo das tribos..., cit., p.27. ainda as matérias “Tiroteio na Periferia”, em O Globo, 30 jun.1994; “Rap é a língua oficial da periferia” Estado de S. Paulo, 27 nov. 1992, e ainda “Rap’eriferia” no jornal O Povo, caderno Tribos, 15 maio 1994. 42 “Mais do que nunca a Fortaleza é DEF” — O grupo Ataque Frontal era composto pelos rappers ZMC e Ligado. Ambos participam do movimento, aproximadamente desde 1993. ZMC é um dos membros mais engajados com setores diversos das ‘esquerdas’ como o Mo- vimento Estudantil e Popular. Depois de trabalhar como lavador de carros à noite no esta- cionamento de casas noturnas da cidade, tem trabalhado como prestador de serviços na Câmara de Vereadores de Fortaleza. Ligado, além de seu vizinho na favela da Quadra, no coração da Aldeota, sem profissão definida, trabalha como vendedor de pequenas lojas de grifes locais e como servente de pedreiro, quando não faz pequenos biscates. 44 “Sem Título” — Realidade em Cadeia. A pergunta insistentemente repetida nesse rap é “Quem são os mandantes, quem são?” e “Olhe pra cima e veja então, quem é que mata sem arma na mão / me diga agora aonde devo ir? Me diga agora mano? Me diga agora!” 45 “Mais do que nunca a Fortaleza é DEF” — Ataque Frontal (ZMC e Ligado). 46 PESAVENTO Sandra cit p 15 241 junho de 2007 junho de 2007 Francisco José Gomes Damasceno 47 É interessante a reflexão feita por STAM, Robert. Bakhtin: da teoria literária à cultura de massa. São Paulo: Ática, 1992. p.75-6: “Porém os mesmos mecanismos dialógicos básicos operam também dentro da chamada ‘cultura popular’. Veja-se, por exemplo, o fenômeno do hip-hop nos Estados Unidos, que inter-relaciona os universos culturais do rap, do gra- fite e da dança break. O rap, forma de música popular que utiliza os efeitos pirotécnicos executados em toca-discos combinados com um discurso verbal agressivo e ritmado — a própria palavra rap significa conversar, dialogar —, pode ser considerado uma esperta vi- são ‘de rua’ das teorias bakhtinianas sobre o dialogismo. Criado por adolescentes negros e hispânicos da classe operária ou lúmpen,o rap é intensamente,exuberantemente dialógico”. 48 “Tortura” Estado Indigente 48 “Tortura” — Estado Indigente. Revista Brasileira de História, vol. 27, nº 53 48 “Tortura” — Estado Indigente. 49 “Ceará e seus problemas” — Conscientes do Sistema. Este rap dá título ao primeiro dis- co inteiramente de rap do Ceará. O grupo era ligado ao MH2O, de onde saiu para fazer carreira solo. Recentemente se desmembrou em dois, ficando uma parte ainda com o no- me de Conscientes do Sistema, e o outro passando a chamar-se de Consciência Armada, nome de uma das composições desse CD. 50 Ambas as manifestações se consideram “culturas de rua”. 51 “O cotidiano que se apreende no rock é o da deambulação. Trata-se da negação de um cotidiano tradicional e da afirmação de um cotidiano de rua. O lugar do dia-a-dia deixa de ser a casa para ser a cidade. Uma cidade com suas ruas e esquinas, com pistas de carro em alta velocidade, dos gigantescos anúncios luminosos, da multiplicidade de informa- ções, da multidão nas calçadas, da intensa movimentação nos bares, dos encontros furti- vos e, sobretudo, do anonimato. Seus personagens não têm nomes...”. GUERREIRO, Al- merinda Sales. Retratos de uma tribo urbana: rock brasileiro. Dissertação (Mestrado) — Faculdade de Filosofia, Letras e Ciências Humanas, Depto. de Antropologia, USP. São Pau- lo, 1991. p.83. 52 “O Poder da Palavra” — Rap Força. 52 “O Poder da Palavra” — Rap Força. 53 “Sangue Negro” — Conexão Racial. 54 “Filhos da Rua” — Flip Rap. 54 “Filhos da Rua” — Flip Rap. 55 Entrevista realizada por este pesquisador com Eliane ‘Zueira’ em 12 fev. 2003 na Praça José de Alencar, Fortaleza. p.2. 55 Entrevista realizada por este pesquisador com Eliane ‘Zueira’ em 12 fev. 2003 na Praça José de Alencar, Fortaleza. p.2. 56 Flor Punk, cit., p.3. 57 Entrevista realizada por este pesquisador com o punk Lezado, em 20 fev. 2003, em sua residência no bairro do Conjunto Ceará, Fortaleza. p.1. 57 Entrevista realizada por este pesquisador com o punk Lezado, em 20 fev. 2003, em sua residência no bairro do Conjunto Ceará, Fortaleza. p.1. 58 CAIAFA, Janice. Movimento Punk na Cidade: a invasão dos bandos sub. Rio de Janeiro: Jorge Zahar, 1985. p.49. 59 PERALVA, Angeline. O jovem como modelo cultural. Juventude/Contemporaneidade, Re- vista Brasileira de Educação, São Paulo: Anped, n.5-6, 1997. p.23. 242 Revista Brasileira de História, vol. 27, nº 53 Revista Brasileira de História, vol. 27, nº 53
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https://www.frontiersin.org/articles/10.3389/fnins.2014.00186/pdf
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Pairmate-dependent pup retrieval as parental behavior in male mice
Frontiers in neuroscience
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ORIGINAL RESEARCH ARTICLE published: 11 July 2014 doi: 10.3389/fnins.2014.00186 Pairmate-dependent pup retrieval as parental behavior in male mice Mingkun Liang 1,2 , Jing Zhong 1,2 , Hong-Xiang Liu 1 , Olga Lopatina 1 , Ryusuke Nakada 2 , Agnes-Mikiko Yamauchi 2 and Haruhiro Higashida 1* 1 2 Department of Basic Research on Social Recognition and Memory, Research Center for Child Mental Development, Kanazawa University, Kanazawa, Japan Department of Biophysical Genetics, Graduate School of Medical Sciences, Kanazawa University, Kanazawa, Japan Edited by: Sonoko Ogawa, University of Tsukuba, Japan Reviewed by: Aldo Lucion, Universidade Federal do Rio Grande do Sul, Brazil Tomoko Soga, Monash University, Malaysia *Correspondence: Haruhiro Higashida, Department of Basic Research on Social Recognition and Memory, Research Center for Child Mental Development, Kanazawa University, 13-1 Takara-machi, Kanazawa 920-8640, Japan e-mail: haruhiro@ med.kanazawa-u.ac.jp Appropriate parental care by fathers can greatly facilitate healthy human family life. However, much less is known about paternal behavior in animals compared to those regarding maternal behavior. Previously, we reported that male ICR strain laboratory mice, although not spontaneously parental, can be induced to display maternal-like parental care (pup retrieval) when separated from their pups by signals from the pairmate dam (Liu et al., 2013). This parental behavior by the ICR sires, which are not genetically biparental, is novel and has been designated as pairmate-dependent paternal behavior. However, the factors critical for this paternal behavior are unclear. Here, we report that the pairmate-dependent paternal retrieval behavior is observed especially in the ICR strain and not in C57BL/6 or BALB/c mice. An ICR sire displays retrieval behavior only toward his biological pups. A sire co-housed with an unrelated non-pairing dam in a new environment, under which 38-kHz ultrasonic vocalizations are not detected, does not show parenting behavior. It is important for sires to establish their own home territory (cage) by continuous housing and testing to display retrieval behavior. These results indicated that the ICR sires display distinct paternity, including father-child social interaction, and shed light on parental behavior, although further analyses of paternal care at the neuroendocrinological and neurocircuitry levels are required. Keywords: parental behavior, paternal care, pup retrieval behavior, paternity, mouse INTRODUCTION According to Schor and others, “a stable, well-functioning family that consists of two parents and children is potentially the most secure, supportive, and nurturing environment in which children may be raised” (Schor and American Academy of Pediatrics Task Force on the Family, 2003; Fortunato and Archetti, 2010; Benbassat and Priel, 2012). Thus, the role of a father in the home is highly significant, and currently, the physical absence of the father in the home is seen as a major problem facing families worldwide (Feinberg, 2002; Fleming et al., 2002; Amato, 2005; Benbassat and Priel, 2012; Morrongiello et al., 2013; Bornovalova et al., 2014). This raises questions regarding which factors determine paternal care and how they are maintained. This may be addressed by behavioral studies and neuroendocrinological analysis of oxytocin, stress hormones, sex hormones, or epigenetic mechanisms (Ogawa et al., 1998; Pfaff et al., 1999; Nunes et al., 2001; Gammie, 2005; Jin et al., 2007; Bridges, 2008; Nishimori et al., 2008; Lee et al., 2009; Neumann, 2009; Chourbaji et al., 2011; Douglas, 2011; Morgan and Bale, 2011; Hashimoto et al., 2012; Higashida et al., 2012a,b; Parhar et al., 2012; Soga et al., 2012; Bambico et al., 2013; Salmina et al., 2013; Morrison et al., 2014). Although a number of animal models have been used in experimental studies of parental care (Reburn and Wynne-Edwards, 1999; Carter et al., 2009; de Jong et al., 2009; McGraw and Young, 2010; Ozawa et al., 2010; Kuroda et al., 2011; Mogi et al., 2011; www.frontiersin.org Saltzman and Maestripieri, 2011; Lambert et al., 2013; Tachikawa et al., 2013; Yoshida et al., 2013), given its value for genetic studies, a mouse model of paternal behavior may be especially useful (Hager and Johnstone, 2003; Jin et al., 2007; Liu et al., 2013). While some strains of the laboratory mouse Mus musculus become biparental (Wright and Brown, 2000; Chourbaji et al., 2011), a phenomenon called sensitization (Rosenblatt, 1967; Rosenblatt et al., 1996), little information is available regarding the factors that specifically induce male parental behavior (Gubernick and Alberts, 1987, 1989; Lonstein and De Vries, 2000; Kentner et al., 2010; Leuner et al., 2010). Previously, we reported that the outbred ICR strain is uniparental and is a good model for studies of parental behavior (Jin et al., 2007; Liu et al., 2008, 2013; Higashida et al., 2012a), because these mice actively reproduce offspring and exhibit easily monitored pup retrieval after separation (Fujimoto et al., 2013; Liu et al., 2013), which is a reliable indicator of parental behavior (Gammie, 2005; Wynne-Edwards and Timonin, 2007; Yoshida et al., 2013). We demonstrated that male ICR mice display robust parental care, which is induced by signaling from the pairmate dam, after separation from the pups (Liu et al., 2013). We demonstrated that this signaling is mediated through as yet unidentified olfactory pheromonal cues and auditory 38-kHz ultrasonic vocalization (USV) cues (Liu et al., 2013), that the male response can be modified hormonally via oxytocin (Akther et al., 2013), that CD38 in the nucleus accumbens is critical (Akther et al., 2013), July 2014 | Volume 8 | Article 186 | 1 Liang et al. and that the central cholinergic system is involved (Fujimoto et al., 2013). However, the factors influencing singly isolated sires in which there is no direct communicative interaction between dams and sires remain unclear. In the present study, to simplify fatherhood evaluation, we used an all-or-nothing type of pup retrieval behavior by calculating the percentage of sires that displayed retrieval behavior (Liu et al., 2013). We investigated paternal behavior in terms of the types of conditions that can induce or maintain paternal retrieval behavior by sires when the males are isolated before the offspring are delivered by pregnant mates, and the males are held separately to prevent them being sires by physically separating them from other family members for 3 days. Then, family ties are formed with or without mate information. In other experiments, we examined isolation from pups under different housing conditions in which either pairmate dam and pup olfactory information is present or excluded. MATERIALS AND METHODS ANIMALS Male and female Slc:ICR, C57BL/6, and BALB/c mice were obtained from Japan SLC, Inc. (Hamamatsu, Japan) via a local distributor (Sankyo Laboratory Service Corporation, Toyama, Japan). The ICR mice were originally obtained from Charles River Laboratories in 1965 and since then bred in Japan with the alternative name Swiss CD1. The offspring of these mice were born in our laboratory colony, weaned at 21–28 days of age, and housed in same-sex groups of 3–5 animals until pairing (Liu et al., 2013). The animals were paired and kept in our laboratory under standard conditions (24◦ C; 12-h light/dark cycle, lights on at 08:00) with food and water ad libitum. The mice were housed together continuously in standard mouse maternity cages. The experiments were performed in accordance with the Guidelines for the Care and Use of Laboratory Animals of Kanazawa University. BEHAVIORAL TESTING Virgin males and females were paired at 45–55 d. A single male and a single female were continuously housed together in a standard mouse maternity cage from the mating period until the delivery of pups. In some experiments, the males were separated in new cages 1 day before parturition to prevent formation of family relationships and kept in the new cages for 3 days. Then, the males were allowed to meet their pups with or without pairmates from day 3 to day 5. All family units composed of a new sire (first-time father), dam, and their first litter were experimentally naïve. One male parent was placed for 10 min in the original cage or new cage alone or with his pairmate (separation environment). Five pups were randomly selected from the litter and placed individually at a site remote from the nest in the original cage. The sires were returned to the original home cage or a new cage in the presence of their five biological or foster pups to assess parental behavior. Parental retrieval behavior (percentage of sires exhibiting retrieval) was examined for 10 min following reunion. The behavioral tests were performed in a randomly mixed sequence of experimental groups. Experiments were usually performed at Frontiers in Neuroscience | Neuroendocrine Science Mate-dependent paternal behavior 10:00–15:00. We defined retrieval as positive if the sires carried all 5 pups to the original nesting place or within two thirds of the distance between the nest and the place at which the pups had been placed (Liu et al., 2013). We also observed other parental behaviors (grooming, crouching, and huddling) as defined by Gubernick and Alberts (1987, 1989). The animals in this and subsequent experiments were tested only once. MEASUREMENT OF USVs Experiments were carried out in a soundproof chamber measuring 600 × 500 × 500 mm (model MC-050/VA; Muromachi Kikai, Tokyo, Japan). USVs were detected with a condenser microphone (Type 7016; Aco, Tokyo, Japan) and a preamplifier (type 4116; Aco) designed for sound pressure level (SPL) measurements between 20 Hz and 90 kHz. A 4-kHz band-pass filter was used to minimize background noise during recordings; however, most WAV files still contained a considerable amount of “non-USV” signal. Extraneous noise was identified and removed from the sonograms as far as possible. When a rater found an ultrasound signal that was difficult to interpret, the call was evaluated by a minimum of one additional trained observer and identification required a consensus by all raters. Each sonogram was then evaluated with a series of automated parameters. The microphone was placed 50 cm above the cage in a soundproof chamber and connected to an amplifier (model UMA-2; Muromachi Kikai). Acoustic signals were transmitted to a vocalization analyzer system (model MK-1500; Muromachi Kikai) with functions such as an analog-to-digital converter (192 kHz), frequency filters, a digital fast-Fourier-transform analyzer, and signal input—output terminals. Input signals were visualized on SpectraLAB (Sound Technology Inc., State College, PA) in the analyzer system on a personal computer. USVs were recorded as WAVE files and analyzed; the number of calls, frequency, and wave width (>40 ms) were measured using a USV monitor (Muromachi Kikai). STATISTICAL ANALYSIS The data were calculated as the means or the means ± s.e.m. Two-tailed Fisher’s exact probability test was used for single comparisons of retrieval behaviors. The remaining data were analyzed by two-tailed Student t-test. RESULTS It has been reported that parental behavior in mice is dependent on the strain (Wright and Brown, 2000). Therefore, we first examined and compared parent–pup family units in three strains, i.e., ICR, C57BL/6, and BALB/c mice, under various experimental settings. The data are summarized in Table 1. Maternal nurturing behavior was observed in dams of all three strains, in a strainnonspecific fashion, except for the low rate of retrieval by the BALB/c dams. In contrast, paternal behavior was variable between the strains. No retrieval behavior was observed by BALB/c sires (n = 15). C57BL/6 sires displayed retrieval during reunion after single-separation in new cages (approximately 40%, n = 15). However, isolation together with the partner in new cages did not potentiate but rather decreased this rate to 13.3% (n = 15). This parental behavior suggests that C57BL/6 males display mateindependent paternal behavior. Interestingly, 38-kHz USVs were July 2014 | Volume 8 | Article 186 | 2 Liang et al. Mate-dependent paternal behavior Table 1 | Parental behaviors in three strains of mice. Behavior Dam Sire Mouse strain ICR (n = 15) C57BL/6 (n = 15) BALB/c (n = 15) Retrieval 100% fast, rhythmic 100% fast, rhythmic 60% slow, interrupted Crouching Over all pups Over not all pups Over all pups Grooming Rare Rare Rare Nest building Sometimes Sometimes Sometimes Retrieval by separation 10% 40% 0% After co-housing pairmates 60% 10% 0% Fast (<4 min) Very slow – Smooth Intermittent – Over not all pups Not often – Crouching Grooming Rare Rare – Nest building Not often Rare – Number of pups per litter ∼15 ∼5 ∼7 Survival ratio ∼100% 60–70% ∼100% USVs >70 calls/2 min <20 calls/2 min >80 calls/2 min Communication from dams to sires with 38 kHz USVs Detected Not detected Not detected Pattern of paternal care Mate-dependent Mate-independent None Pup not recorded from any dam–sire pairs of C57BL/6 and BALB/c strains separated in new cages for 10 min. These results indicated that pairmate-dependent care is specific to the ICR strain. Therefore, in the following experiments, we examined various critical conditions under which ICR strain males did or did not show paternal behavior. RETRIEVAL BEHAVIOR BY SIRES SEPARATED ALONE IN HOME CAGES The experimental paradigms for each experiment are shown schematically in each figure. In Figure 1, we first reproduced our previous results (Liu et al., 2013). Male and female ICR strain mice were paired and housed together continuously in a standard mouse maternity cage (Figure 1A). The mice were left undisturbed during the first 3 days after the birth of their pups (Figure 1B), during which they displayed distinct paternal and maternal behaviors as described previously (Liu et al., 2013). The sire and dam nursed the pups. This involved nest-building, pup retrieval, licking, and huddling over the pups and lactating. However, as described in the Methods section, we mainly analyzed the male’s retrieval behavior, as a parental role, in the following experiments. The sire in the first family was left alone in the vacated cage during the period of separation (Figure 1C), whereas the pups and dam were removed and placed in a new cage (Figure 1D) separated from the family cage. After 10 min, the five selected pups of the sire (biological offspring) were returned to the nursing cage in a remote area away from the nest, where the sire was present (Figure 1E). The sire retrieved the offspring over 10 min (86% of the sires, n = 15; Figures 1F,S). www.frontiersin.org If the non-biological (foster) pups (Figure 1N) of the third family (Figures 1M,N) were introduced into the vacated home cage with the second sire (Figures 1I,K) in the second family (Figures 1G,H), instead of the biological pups (Figures 1H,J), 33% of the 15 sires displayed pup retrieval (Figures 1L,S; twotailed Fisher’s exact probability test between sires toward biological (F) and non-biological (L) pups, P < 0.01). When a sire from the third family (Figure 1N) was placed and isolated for 10 min in the home cage of the second family (Figure 1O), the third sire did not retrieve any of the foster (second family’s) pups (Figures 1R,S; n = 15, two-tailed Fisher’s exact probability test between unrelated sires (R) and sires with non-biological (L) or biological (F) pups, P < 0.05 and P < 0.0001, respectively). These results suggested that paternal pup retrieval behavior in the home cage is maintained by biological family cues of their mate dams and remaining pups. RETRIEVAL BEHAVIOR BY SIRES AFTER SEPARATION IN NEW CAGES Male parental care in Figure 1 may have been induced by the fact that the males were left in the nursing environment during parent–pup separation. To select out pup information during isolation, we used the co-housing paradigm presented in Figure 2. We examined whether sires developed paternal behavior following time spent with the family. Pup retrieval increased on a daily basis after parturition, while dams displayed a higher retrieval ratio from the first day of parturition than the sires (Table 2). The sires alone (Figure 2D) or together with the mate dams (Figure 2H) were placed in a new cage for 10 min, whereas the pups alone (Figure 2G) or together with dams (Figure 2C) July 2014 | Volume 8 | Article 186 | 3 Liang et al. FIGURE 1 | Parental retrieval test in ICR mice for biological and non-biological pups. Schematic representations of the parental care test in three mated pairs (A,G,M). After cohabiting with their pups as a family for 3 days from postnatal day 1 (P1) until postnatal day 3 (P3) (B,H,N), the sires were separated in the home cage (C,I,O) from the pups and pairmates (D,J,P) for 10 min. The sires were then reunited with five biological (E) or non-biological (K) pups. Subsequent pup retrieval behavior over a 10-min period was then observed (F,L). The third sire (M,N) was placed in the home cage (O) of were left in the home cage. Then, the sires were returned to the home cages in which five pups remained (Figures 2E,I). The male’s retrieval behavior was undiminished when housed with the pairmate (66%, n = 30; Figures 2J,U) but was strongly reduced when housed alone (24%, n = 41; Figures 2F,U). As expected, a high level of sire care was displayed after isolation in the new environment together with mate dams and pups (as the whole family (Figures 2K,L) (66%, n = 15; Figure 2U): twotailed Fisher’s exact probability test between sires separated alone (F) and together (J) or as a whole family (N), P < 0.001, equally. The latter was specifically associated with co-habitation with the pairmate dam during the separation period (Figure 2H), because negligible retrieval behavior was apparent if the sire was housed with the dam of another brood (Figures 2O–T; 20%, n = 15); two-tailed Fisher’s exact probability test shows no significant difference between sires separated together with unrelated dams (T) and alone (F); and separated together (J), P < 0.01; and separated as a whole family (N), P < 0.05, Figure 2U). Thus, it appears that the mate dam provides some signal(s) during the separation period to induce parental behavior in the sire, in agreement with the results reported previously (Liu et al., 2013). Whereas parental care by the dam is independent of the presence of the male or the housing environment, that by the male is strongly dependent on cues from the pairmate dam and/or home cage. We recorded USVs (with >40 ms in wave width) to determine their role as one form of critical interactive information in this Frontiers in Neuroscience | Neuroendocrine Science Mate-dependent paternal behavior another family (H), and retrieval was tested for non-biological (another family’s) pups in an unrelated cage (Q,R). The numbers of positive mice/number of mice tested are shown in parentheses. The number of sires displaying retrieval behavior out of sires tested was expressed as a percentage (S). N = 15 for each experiment. Two-tailed Fisher’s exact probability test: between sires toward biological (F) and non-biological (L) pups or unrelated sires (R), ∗∗ P < 0.01 and ∗∗∗∗ P < 0.0001, respectively; and between sires tested toward non-biological pups (L) and unrelated sires (R), ∗ P < 0.05. paradigm. We detected 38-kHz USVs identical to those reported previously (Liu et al., 2013) under isolation conditions in new cages for 10 min between sires and mate dams at a frequency of 25.9 ± 4.8 calls/10 min (n = 8, Table 3; P < 0.01 from other values, two-tailed Student t-test). No identical 38-kHz USVs were recorded between sires and unrelated dams. Instead, 30–80-kHz USVs were recorded infrequently at 40.7 ± 26.7 calls/10 min (n = 11) between unfamiliar couples. These 30–80-kHz USVs were emitted when a sire was co-housed with a virgin female at 313.6 ± 64.9 calls/10 min (n = 11, P < 0.001 from two other values, twotailed Student t-test). These data clearly support the suggestion that paternal retrieval is essentially triggered by the pairmate’s 38-kHz USVs. RETRIEVAL BY ISOLATED BEFORE PAIRMATE PARTURITION The retrieval behavior displayed by males may have been induced by family formation in the nursing cage environment. To assess this possibility, data were obtained from parting males (Figures 3, 4) that remained with the paired pregnant females 1 day before parturition of their first litter and were then separated into a new cage (Figures 3A–C). The males were then isolated alone for 3 days (Figure 3E). When sire paternal retrieval was examined immediately on day 3 in the sire home cage (Figure 3K), 21.4% of sires with no prior contact with their biological pups and pairmate dam, i.e., the paternity unformed state (Figure 3E), displayed retrieval behavior (n = 42; Figure 3N). Next, when the isolated males were relocated in the home cage July 2014 | Volume 8 | Article 186 | 4 Liang et al. Mate-dependent paternal behavior FIGURE 2 | Paternal retrieval test in ICR mice isolated in new cages. Paired couples were kept in rearing cages from mating to postnatal day 3 (P3) (A,B,O,P). In (C,D), the pups and the mating dam were left in their home cages (C), and the sire alone was placed in a new holding cage (D). In (G,H), the pups were kept in the original cage, and the parents were placed in a new cage (H). In (K,L), the whole family was moved to a new cage (L). In (Q,R), the sire was kept during the separation period (Q) with a non-mating dam of another family (O,P). After isolation for 10 min in each cage, pup retrieval behavior over a 10-min period was observed in each case (E,I,M,S). The number of sires displaying retrieval behavior was scored (F,J,N,T). The numbers of positive mice/number of mice tested are shown in parentheses and expressed as percentages (U). Two-tailed Fisher’s exact probability test: between sires separated alone (F) and together (J) or as a whole family (N), ∗∗∗ P < 0.001 equally; between sires separated together (J) and as a whole family (N), ∗∗ P < 0.01; between sires separated alone (F) and co-housed with unrelated dams (T), not significant (n.s.); sires separated as a whole family (N) and co-housed with unrelated dams (T), ∗ P < 0.05. Table 2 | Percentages of sire’s or dam’s exhibiting retrieval behavior Table 3 | Number of USVs recorded from cages of sires co-housed during the postpartum period. with different types of females for 10 min. Postnatal day Percentage of exhibiting of pups Type* Number of USVs retrieval behavior By sires (n) (calls/10 min) By dams 38-kHz 30–80-kHz 1 14 (15) 55 (20) With pairmate dam 25.9 ± 4.8** 0 (8) 2 40 (20) 90* (20) With unrelated dam 0 40.7 ± 26.7 (11) With virgin female 0 313.6 ± 64.9*** 3 65** (20) 90* (20) 4 70** (17) 85 (20) 5 65** (20) 75 (16) USVs (with >40 ms in duration) were recorded in n pairs. *Judging from the previous results (Liu et al., 2013), 38-kHz USVs appear to be Number of mice tested are shown in parentheses. *, **Significantly different from day 1, *P < 0.05 and **P < 0.01, respectively, two-tailed Fisher’s exact emitted from pairmate dams and 30–80-kHz USVs from sires. **P < 0.01 or ***P = 0.001, from pairmate dams, unrelated dams or virgin probability test. females, respectively, two-tailed Student’s t-test. and stayed with the family (pups and pairmate dam) for 3 days (Figure 3F), the rate of retrieval in their home cage was only 4% (n = 25; Figures 3G–J). Although the sire lived together with the family for 3 days, such treatment made no contribution to the formation of paternity (two values in Figure 3O were equally very low; no significance, two-tailed Fisher’s exact probability test). To further analyze the relevance of family interaction during the stay as a whole family on postnatal days 3–5 (P3–P5) (Figure 3), we used the short-term pup exposure method (twice for 3 h for a total 6 h a day; Figure 4) to acquire or learn the process of paternity for the family. Males were isolated in new cages prior to parturition (Figures 4B,C) and kept in the cages for 2 days (Figures 4D,E). Then, pairmate dams and pups were relocated to the male’s cage, and the whole family was kept there for 3 days (Figure 4F). Retrieval behavior was displayed by 8 (62%) of 13 sires (Figures 4G–J). The high level of retrieval appears to www.frontiersin.org (11) July 2014 | Volume 8 | Article 186 | 5 Liang et al. FIGURE 3 | Paternal retrieval test in ICR mice isolated prior parturition from the mating pair and then united as a whole family. A paired couple was kept in a rearing cage from mating (A) to 1 day before parturition, and the female and male were then kept in a home cage (B) or in a new cage (C). The next day, the female delivered her pups (D) and remained until postnatal day 2 (P2). The male was kept continuously in the new cage until P2 (E). From P3 to P5, the sire was introduced to the have been caused by continuously living in new cages that had been established as the male’s territory. In this suitable condition, we examined whether the presence of the dam was necessary for parental behavior by the isolated males. From P3 to P5, the pups and dam were kept together in their original home cages (Figure 4K), but the pups were temporarily transferred to the sire’s cage twice for 3 h (a total of 6 h) per day (Figure 4L), and the males were otherwise alone for the rest of the day (18 h; Figure 4M). These sires showed retrieval behavior at a very high rate (17 (85%) of 20 sires tested; Figures 4O–Q). In both cases, the sires displayed a very high frequency of retrieval after living as the whole family or only with pups shortly in new cages that had, nevertheless, been established as the territory and established nest of the male, although no significant differences were observed between two types of sire (J and Q in Figure 4R; not significant, two-tailed Fisher’s exact probability test). Furthermore, these results indicated that direct interaction with the mate dam is not necessary if the home territory is established by the sires. Finally, we further examined the impact of territorial information on male retrieval behavior. Family cues were learned by individual sires in a manner identical to that shown in Figure 4 (Figures 5A–F) during P3–P5, but in this case, via Frontiers in Neuroscience | Neuroendocrine Science Mate-dependent paternal behavior family cage with the dam and pups (F). In another experiment, pup retrieval behavior over a 10-min period was examined for sires at P2 (K–N) or at P5 (G–J). The number of sires displaying retrieval behavior was scored (J,N). The numbers of positive mice/number of mice tested are shown in parentheses and expressed as percentages (O). Note that two values in O are equally very low: no significance (n.s.) between (J) and (N), two-tailed Fisher’s exact probability test. short exposure by transferring of their biological pups with their dams in new cages to the nursing cage with the sires. Then, retrieval behavior was examined under two housing conditions: in the sire’s home cage in which the sire had stayed continuously (Figures 5F,G,I–L), or in a new cage (to the sires) in which the mate dams and pups had been staying (Figures 5G,H,M–P). In the home cages, 10 (50%) of 20 sires showed retrieval (Figure 5L), whereas only 3 (15%) of 20 sires in new cages displayed retrieval behavior (P < 0.05 between testing in old (L) and new (P) cages shown in Figure 5Q, two-tailed Fisher’s exact probability test). In both cages, nests were established by the sire and dam. However, the new cages established by the sires’ mate dams were quite new to the sires, even if the cages were fully filled with the mate dam’s olfactory information. DISCUSSION The studies described here were performed to test several hypotheses that had not been explored previously (Akther et al., 2013; Fujimoto et al., 2013; Liu et al., 2013), pertaining to the various conditions responsible for parental behaviors other than the communicative interaction between sires and dams. Four findings are of particular interest: (1) among the mouse strains tested, the mate-dependent paternal retrieval behavior was observed only in July 2014 | Volume 8 | Article 186 | 6 Liang et al. FIGURE 4 | Paternal retrieval test in mice isolated prior parturition from the mating pair and then united as a whole family or with pups only. A paired couple was kept in a rearing cage from mating (A) to 1 day before parturition. The female was kept in a home cage (B) and delivered her pups (D) and remained until postnatal day 2 (P2) (D). The male was kept in a new cage before meeting the pups (C) and kept until P2 (E). The dam and pups were introduced in the sire’s own (new) cage and stayed as a whole family until P5 (F). Instead of the whole family, in another experiment, only pups in home cages with their dams (K) were transferred the ICR strain (Table 1), and acquisition of such paternal behavior increased slowly following parturition of the dam (Table 2); (2) the ICR sires displayed parental retrieval behavior only for their own biological pups (Figure 1); (3) interaction between the sires and unrelated non-mating dams is not effective (Figure 2) and does not involve 38-kHz USVs (Table 3); (4) it is important for the sire to establish its home cage (territory) by continuous housing to display parental retrieval behavior (Figures 3–5). After separation from pups in the home or new cages with the sires alone or together with the pairmate dam, the sires displayed retrieval behavior, as shown in Figures 2J,N, in agreement with previous reports (Liu et al., 2013). We designated this behavior of the sire as mate-dependent parental behavior. In the present study, this particular behavior was specific to the ICR strain and was not observed in two other laboratory strains, i.e., C57BL/6 and BALB/c mice. Therefore, the ICR strain’s mate-dependent retrieval is not a general behavior observed equally in all mice but is strain-specific. However, this does not reduce the value of our findings because the observed paternal behavior is unique. Furthermore, when considering human society, human males are not completely and genetically predisposed to display parental behavior. In this context, the behavior of the ICR strain may be www.frontiersin.org Mate-dependent paternal behavior twice for 3 h (total 6 h) a day to the sire’s cage (L). During the rest of the time from P3 to P5, the sire stayed alone (M), and pups were located with the dam (K). Pup retrieval behavior over a period of 10 min was examined (G–J and N–Q, respectively). The number of sires displaying retrieval behavior was scored (J,Q). The numbers of positive mice/number of mice tested are shown in parentheses, and the numbers of sires displaying retrieval were expressed as percentages (R). Note that the retrieval rate in two cases (J,Q) was high enough to have no significance (n.s.), two-tailed Fisher’s exact probability test. a more suitable and novel model for investigating paternal behavior, comparing the genetically determined paternity, observed in animals such as voles or California mice (de Jong et al., 2009; Ahern et al., 2011). The ICR sires displayed parental retrieval behavior only for their biological pups, indicating that they can discriminate between their biological and non-biological offspring. This discrimination likely depends on odor or USV (Kuroda et al., 2007, 2011). The characteristic 38-kHz USVs were not recorded during co-housing of ICR sires with non-mate dams, suggesting that the sires can distinguish the mate from non-mate dams or that the dams can distinguish the mate from non-mate sires. These results strongly support our suggestion that 38-kHz USVs are critical and have context for sires to induce retrieval behavior. In these experiments, we examined the olfactory information of pups and cages (homes) for the sires prior to separation from the mate dam and their offspring. In habituation as a family, the presence of the mate was not completely essential. Interestingly, we estimated that the territory information is much more important to sires than the pheromones in the cages once they had established their home cage. Surprisingly, when the sires were continuously housed in their newly established home July 2014 | Volume 8 | Article 186 | 7 Liang et al. FIGURE 5 | Paternal retrieval test in ICR mice isolated prior parturition from the mating pair and then united with pups only. A paired couple was kept in a rearing cage from mating to 1 day before parturition (A). The male was kept in the old cage (B) before meeting the pups and kept until P2 (D). The female was kept in a new cage (C), delivered her pups, and remained until postnatal day 2 (E). The pups were transferred twice for 3 h (total 6 h) a day (G) from the dam’s (new) cage (H) during P3 to P5. During cages, they displayed paternal retrieval. In sharp contrast, if the cage was new to the sire, even though the dam’s and sire’s olfactory information was there, the sire failed to display retrieval behavior. These observations suggested that territory establishment is critical to maintaining paternity (Wright and Brown, 2000). Pup retrieval as a parental behavior is rare among laboratory mice that are not genetically monogamous (Wright and Brown, 2000; Kalueff et al., 2007). We found conditions in which the ICR sires retrieved their pups related to their family structure. This unique ability of the ICR sires will contribute to the increased survival rate after reproduction and to the high level of social attachment and interaction. We have recently reported that central cholinergic cellular signaling (Fujimoto et al., 2013) and CD38 and oxytocin signaling in the nucleus accumbens (NAcc) (Akther et al., 2013) are critical for the expression of paternal care of the ICR mice. We also demonstrated the modulatory roles of the mPOA and VP on parental behavior in rodents (Akther et al., 2014). These published findings suggest that the neural circuitry mediating paternal behavior includes the mPOA, VTA, NAcc, and VP, and may be similar to those that mediate maternal behavior as proposed by Numan and others (Numan et al., 2005; Lee and Brown, 2007; Wynne-Edwards and Timonin, 2007; Numan and Stolzenberg, 2009). In addition, it is particularly interesting to test if mPOA galanin neurons regulate mate-dependent parental behavior in the ICR strain (Wu et al., 2014). Further neuroendocrinological and neurocircuitry analyses in ICR mice will be useful for understanding disorders with social impairment, such as autism spectrum disorders and schizophrenia (Insel, 2010; Munesue et al., 2010; Riebold et al., 2011; Feldman et al., 2012; Salmina et al., 2013). Frontiers in Neuroscience | Neuroendocrine Science Mate-dependent paternal behavior the rest of the time, the sires stayed alone in the home cages (F) and the dams were with the pups (H). Pup retrieval behavior over a period of 10 min was examined at P5 (I–L and M–P, respectively). The number of sires displaying retrieval behavior was scored (L,P), and the numbers of positive mice/number of mice tested are shown in parentheses. Pup retrieval was expressed as percentages (Q; ∗ P < 0.05 between old (L) and new (P) cages, two-tailed Fisher’s exact probability test). AUTHOR CONTRIBUTIONS Haruhiro Higashida designed experiments. Mingkun Liang, Jing Zhong, Hong-Xiang Liu, Olga Lopatina, Ryusuke Nakada, Agnes-Mikiko Yamauchi, and Haruhiro Higashida performed animal experiments. Haruhiro Higashida and Mingkun Liang wrote the manuscript. ACKNOWLEDGMENTS This work was supported in part by the Core Research for Evolutional Science and Technology (CREST) from the Japan Science and Technology Agency and by the Strategic Research Program for Brain Sciences from Ministry of Education, Culture, Sports, Science, and Technology, Japan. REFERENCES Ahern, T. H., Hammock, E. A., and Young, L. J. (2011). Parental division of labor, coordination, and the effects of family structure on parenting in monogamous prairie voles (Microtus ochrogaster). Dev. Psychobiol. 53, 118–131. doi: 10.1002/dev.20498 Akther, S., Fakhrul, A. 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Transport Response is a filial-specific behavioral response to maternal carrying in C57BL/6 mice. Front. Zool. 10:50. doi: 10.1186/1742-9994-10-50 Conflict of Interest Statement: The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest. Received: 24 February 2014; accepted: 11 June 2014; published online: 11 July 2014. Citation: Liang M, Zhong J, Liu H-X, Lopatina O, Nakada R, Yamauchi A-M and Higashida H (2014) Pairmate-dependent pup retrieval as parental behavior in male mice. Front. Neurosci. 8:186. doi: 10.3389/fnins.2014.00186 This article was submitted to Neuroendocrine Science, a section of the journal Frontiers in Neuroscience. Copyright © 2014 Liang, Zhong, Liu, Lopatina, Nakada, Yamauchi and Higashida. 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. July 2014 | Volume 8 | Article 186 | 10
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Immigration Discourse and the Prospects of Russian Language in Lithuania
Slavistica Vilnensis
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Received: 24/11/2019. Accepted: 15/12/2019 Copyright © 2019 Алла Борисовна Лихачева. Published by Vilnius University Press. This is an Open Access article distributed under the terms of the Creative Commons Attribution Licence, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited. Immigration Discourse and the Prospects of Russian Language in Lithuania Summary. In contrast to the ongoing emigration, immigration to Lithuania cannot be called intensive, and this topic as a whole remains on the periphery of public and academic attention. This article offers a description of the situation of immigration from the perspective of sociolinguistics. In particular, an analysis of the discourse of today’s Russian-speaking immigration to Lithuania reveals the linguistic attitudes of immigrants with a native Russian language or their own, their adaptation strategies, family language planning, and, ultimately, makes it possible to formulate general conclusions about the pros- pects of preserving / not preserving Russian-speaking community in the host country. The paper summarizes interview materials with immigrants from Russia and some post-Soviet coun- tries posted on the Lithuanian Internet, as well as recorded during this study. Key words: Russian-speaking immigrants; immigration discourse; language attitudes; interviews; the language group vitality Contents lists available at Vilnius University Press Slavistica Vilnensis ISSN 2351-6895 eISSN 2424-6115 2019, vol. 64(2), pp. 128–142 DOI: https://doi.org/10.15388/SlavViln.2019.64(2).26 Contents lists available at Vilnius University Press Slavistica Vilnensis ISSN 2351-6895 eISSN 2424-6115 2019, vol. 64(2), pp. 128–142 DOI: https://doi.org/10.15388/SlavViln.2019.64(2).26 Contents lists available at Vilnius University Press Slavistica Vilnensis 2019, vol. 64(2), pp. 128–142 ISSN 2351-6895 eISSN 2424-6115 DOI: https://doi.org/10.15388/SlavViln.2019.64(2).26 ISSN 2351-6895 eISSN 2424-6115 DOI: https://doi.org/10.15388/SlavViln.2019.64(2).26 Алла Борисовна Лихачева Алла Борисовна Лихачева Вильнюсский университет (Литва) E-mail: ala.lichaciova@flf.vu.lt Вильнюсский университет (Литва) E-mail: ala.lichaciova@flf.vu.lt Вильнюсский университет (Литва) E-mail: ala.lichaciova@flf.vu.lt Аннотация. В отличие от непрекращающейся эмиграции, иммиграцию в Литву нельзя назвать интенсивной, и эта тема в целом остается на периферии общественного и академического вни- мания. В данной статье предлагается описание ситуации иммиграции с позиций социолингви- стики. В частности, анализ дискурса сегодняшней русскоязычной иммиграции в Литву позволя- ет выявить языковые установки иммигрантов с родным русским языком или владеющих им, их адаптационные стратегии, семейное языковое планирование, а в конечном итоге — сформули- ровать общие выводы о перспективах сохранения/несохранения русскоязычия в принимающей стране. В работе обобщаются материалы интервью с иммигрантами из России и некоторых постсовет- ских стран, выложенных в литовском Интернете, а также материалы интервью, записанных в ходе данного исследования. Ключевые слова: русскоязычные иммигранты; дискурс иммиграции; языковые установки; ин- тервью; витальность языковой группы Received: 24/11/2019. Accepted: 15/12/2019 Imigracijos diskursas ir rusakalbiškumo perspektyvos Lietuvoje Santrauka. Kitaip nei neslūgstama emigracija, imigracija į Lietuvą nėra intensyvi, tad ši tema išlieka visuomenės ir akademinės bendruomenės dėmesio periferijoje. Šiame straipsnyje yra siūlomas imigra- cijos situacijos aprašymas iš sociolingvistikos pozicijų. Konkrečiai – šiuolaikinės rusakalbės imigraci- 128 Статьи. Алла Борисовна Лихачева. jos diskursas leidžia išsiaiškinti imigrantų su gimtąja ar gerai mokama rusų kalba kalbines nuostatas, jų adaptacijos strategijas, šeimos kalbinį planavimą, o galiausiai – suformuluoti bendras išvadas apie rusų kalbos gyvybingumo perspektyvas priimančioje šalyje. Straipsnyje apibendrinama medžiaga iš įdėtų internete, taip pat šiam darbui specialiai užrašytų interviu su imigrantais iš Rusijos ir kai kurių kitų posovietinių šalių. Reikšminiai žodžiai: rusakalbiai imigrantai; imigracijos diskursas; kalbinės nuostatos; interviu; kal­ binės grupės gyvybingumas Проблемы, обусловленные активными миграционными процессами, все больше привлекают внимание ученых во всем мире. Одна из основ­ ных тем, исследуемых представителями социальных и гуманитарных наук, работающими в области критического анализа дискурса, связана с имиджем иммигрантов в принимающих странах. Выявляются типич­ ные приемы описания иммигрантов в политическом, научном, медий­ ном, правоохранительном дискурсах. Как показывают исследования, вне зависимости от страны, о которой идет речь, в разных типах дискурса процессы иммиграции и их участники в основном получают негативные характеристики (см. об этом [Матицына 2018; Бардин 2016; Richardson, Wodak 2013; Tereškinas 2009; Sipavičienė, Gaidys, Jeršovas 2010; Collins 2007; Van der Valk 2000] и др.). Миграционная ситуация “глазами” самих переселенцев, их самоощущение и способы адаптации в новой стране становятся объектом анализа значительно реже; как правило, это работы социологического, правового, экономического характера (напр.: [Зимова 2017; Bartkevičienė 2015; Kuznecovienė 2009; Li Xue 2007]). В Литве центральной темой медийных и научных публикаций по­ следних лет является интенсивная эмиграция литовцев. В частности, появилась целая серия работ социолингвистической направленности, из­ учающих языковую жизнь и идентичность представителей эмигрантских сообществ в Великобритании, США, Ирландии, Норвегии и других стра­ нах традиционно активного “исхода” литовцев (таковы, например, кол­ лективные монографии о языках и идентичности эмигрантов [Ramonienė 2015; Ramonienė 2019]). В Литве центральной темой медийных и научных публикаций по­ следних лет является интенсивная эмиграция литовцев. В частности, появилась целая серия работ социолингвистической направленности, из­ учающих языковую жизнь и идентичность представителей эмигрантских сообществ в Великобритании, США, Ирландии, Норвегии и других стра­ нах традиционно активного “исхода” литовцев (таковы, например, кол­ лективные монографии о языках и идентичности эмигрантов [Ramonienė 2015; Ramonienė 2019]). Imigracijos diskursas ir rusakalbiškumo perspektyvos Lietuvoje Сегодняшняя иммиграция в Литву освещается в основном средствами массовой информации: на телевидении и в Интернете периодически по­ являются репортажи и интервью с иностранцами, приезжающими в Лит­ ву на учебу, желающими получить временное разрешение на работу, по­ литэмигрантами и др. (см. материалы delfi.lt, 15min.lt, Žinių radijas, LRT и др.). Академические исследования, связанные с “новыми” иммигран­ тами в Литву, немногочисленны, в частности, можно назвать работы со­ циологов [Petrušauskaitė, Batuchina 2015; Žibas 2014, 2012; Beresnevičiūtė, Leončikas, Žibas 2009]. В этих работах затрагивается, но не является цен­ 129 ISSN 2351-6895 eISSN 2424-6115 Slavistica Vilnensis тральной, и основная для настоящей статьи тема языкового существова­ ния иммигрантов в принимающем обществе. Вначале некоторые официальные данные. По определению литовско­ го подразделения Европейской сети миграции (EMN) [Migracija skaičiais], иммиграцией называется прибытие в страну с целью проживания в ней дольше одного года; понятие иммиграции применимо не только к ино­ странцам, планирующим проживание в Литве более года, но и к возвра­ щающимся из эмиграции литовцам. Иностранцами считаются лица, не имеющие гражданства Литвы. С 2004 по 2016 г. в среднем 80% потока иммигрантов составляли возвращающиеся литовцы. В 2017 г. эта тенден­ ция изменилась: литовцы и иностранцы составляют по 50% въезжающих в Литву на проживание. В 2017 г. большую часть иностранцев, живущих в Литве, составляли граждане России (26 %), Украины (24 %), Белару­ си (19 %). В течение первых месяцев 2018 г. большинство иностранцев, получивших разрешение на временное проживание в Литве, состави­ ли украинцы (56,2 %), белорусы (23 %) и россияне (5,4 %) [Migracija skaičiais]. Таким образом, картина иммиграции остается неизменной: по ко­ личеству новых иностранцев в Литве лидируют славянские соседи, да­ лее следуют жители некоторых бывших советских републик. В связи с устойчивостью этой тенденции представляется актуальным выявление установок иммигрирующих в Литву граждан относительно использова­ ния русского языка как родного или привычного для них как жителей постсоветского пространства лингва франка, а также относительно ис­ пользования в их языковом существовании международного английского и готовности к овладению литовским как государственным языком стра­ ны иммиграции. По мнению ученых, “отбор данных для анализа дискурса может осу­ ществляться несколькими способами в зависимости от функций текстов. В случае если сами тексты являются объектами изучения, исследователь идентифицирует все тексты соответствующего типа и тематики и анали­ зирует всю совокупность или определенную выборку текстов. Однако, если тексты изучаются в качестве более широкой репрезентации соци­ альных явлений или процессов, <…> предлагается начинать с индиви­ дов, с поиска текста или текстов, артикулируемых в социальных группах и возникающих в определенной ситуации. Imigracijos diskursas ir rusakalbiškumo perspektyvos Lietuvoje Точно так же, как в первом случае, исследователь может анализировать всю совокупность текстов или конктретную выборку” (S. Titscher (2002) – цит. по: Tereškinas 2009, 303). Для данной статьи применялся второй тип отбора текстов, посколь­ ку всех участвовавших или упоминавшихся в исследовании людей без­ условно можно отнести к социальной группе иммигрантов, к субъектам 130 Статьи. Алла Борисовна Лихачева. Статьи. Алла Борисовна Лихачева. Дискурс иммиграции и перспективы русскоязычия в Литве р Дискурс иммиграции и перспективы русскоязычия в Литве ситуации иммиграции, а следовательно — к непосредственным и посто­ янным участникам дискурса иммиграции (о таких понятиях анализа дис­ курса как ситуации и их участники см. [van Dijk 1987, 161–163, 191]). Причем следует отметить, что все они становятся участниками несколь­ ких ситуативных разновидностей, или типов дискурса (о типах дискурса см. [Карасик 2004, 231-240]): институционального — в общении с пред­ ставителями государственных учреждений, частных фирм; академиче­ ского — актуального для студентов университетов; медицинского — в поликлиниках и больницах; делового — в рабочих коллективах; быто­ вого — в повседневном общении с соседями, продавцами, прохожими, новыми друзьями и знакомыми, и др. Общее представление о жизни недавно прибывших в Литву имми­ грантов дают материалы социальной группы “Жизнь в Литве” [facebook. com/groups/porusski.lt/]. Как пишут ее администраторы, все желающие приглашаются обмениваться информацией о событиях, происходящих в Литве, описывать свой опыт проживания в Литве, задавать вопросы и отвечать на них, а также просто общаться на разные темы по-русски, т.к. группа объединяет людей, для которых русский является родным или хо­ рошо усвоенным языком. Там же приводятся ссылки на короткие видео­ сюжеты, снятые самими иммигрантами, об их условиях жизни и работы, экскурсиях по Вильнюсу и Литве, их рассказы о временных коммуника­ тивных трудностях и дальнейших жизненных планах. Два из таких видеосюжетов, снятых в 2017 [youtube...Vu1dQ] и 2018 г. [youtube…TKkU] в виде интервью с иммигрантами из России, анализи­ руются в настоящей работе. Также используется видеозапись обсужде­ ния пребывания в Литве и дальнейших планов с иммигрантами из пост­ советских стран, состоявшегося в мае 2019 г. в студии русской редакции DELFI [delfi.lt/video]. Анализируются и материалы пяти интервью с иммигрантами из Азербайджана, записанных и проанализированных в дипломной работе, защищенной в Вильнюсском университете в 2016 г.1 Для информации о сегодняшней языковой жизни бывших иммигрантов мы обратились также к сделанным автором дипломной работы записям двух интервью с азербайджанцами позднесоветской волны иммиграции, получившими литовское гражданство. Кроме того, анализируются 4 ин­ тервью, записанные в 2019 г. 1 Дипломная работа студентки магистерской программы “Русистика” Гульнар Агаевой “Культурно-языковая адаптация иммигрантов, проживающих в Литве (случай иммигрантов из Азербайджана)” была написана под моим руководством и защищена в Вильнюсском универси­ тете в 2016 г. Imigracijos diskursas ir rusakalbiškumo perspektyvos Lietuvoje специально для данного исследования: одно из них — с сотрудницей миграционной службы, занимающейся приемом и адаптацией иммигрантов, и по одному — с иммигрантами из Беларуси, 131 ISSN 2351-6895 eISSN 2424-6115 Slavistica Vilnensis Украины и России, которые в настоящее время живут в Вильнюсе. Общая продолжительность звучащего текста проанализированных интервью — около 5 часов. Для всех респондентов русский язык является родным или хорошо усвоенным языком. Следует отметить, что за каждым рассказом можно увидеть не только обстоятельства переезда конкретных людей или конкретной семьи, но и их опыт вербализации возникающих проблем и принимаемых решений в общении и дискуссиях с другими представителями местного иммигрант­ ского сообщества. Поэтому собранный материал фактически можно рас­ сматривать как отражение общей ситуации с постсоветскими иммигран­ тами в Литве. С респондентами обсуждались причины выезда из страны проживания, соответствия/несоответствия ожиданий иммигрантов и реальной жизни в Литве, опыта общения с государственными и други­ ми официальными институциями, языковой специфики их сегодняшней жизни, ощущения Литвы как толерантной/интолерантной страны и под. Вопросы, касающиеся языкового существования иммигрантов, в ходе интервью не выделялись как основные для данной работы. В формулировке социологов, “социальные процессы, происходящие в этнических группах, являются не только частью личного опыта от­ дельных индивидов, но и результатом развития общества” [Kasatkina, Leončikas 2003, 9]. Это утверждение актуально как при анализе причин эмиграции жителей конкретной страны (в частности, литовцев), так и при изучении стремлений и адаптационных интенций приезжающих в страну на проживание, в нашем случае — “новых” иммигрантов в Литву. Обобщить результаты анализа текстов интервью можно следующим образом: 1. В первую очередь очевидно, что тема языка актуальна не только для филолога-социолингвиста, изучающего дискурс иммиграции как “сово­ купность тематически соотнесенных текстов” [Чернявская 2001, 16] и анализирующего языковые аспекты данного социального процесса. Это центральная тема в судьбах самих иммигрантов, в рассказах об обстоя­ тельствах их переезда и адаптации в новой стране. В рассуждениях о по­ иске работы, о дальнейших планах для себя и своих детей, информанты постоянно возвращались к роли языка/языков на новом жизненном этапе. В беседе с работницей миграционной службы обсуждался вопрос об иммигрантах из постсоветских стран. По ее наблюдениям, среди них есть русские, но есть и представители других национальностей: бело­ русы, украинцы, чеченцы, таджики, азербайджанцы, армяне, грузины, евреи. Их всех можно назвать русскоязычной или относительно рус­ скоязычной иммиграцией, и для них существование русского языка в бывшей советской республике чрезвычайно важно — они оценивают 132 Статьи. Алла Борисовна Лихачева. Imigracijos diskursas ir rusakalbiškumo perspektyvos Lietuvoje р Дискурс иммиграции и перспективы русскоязычия в Литве русский язык как значительную помощь и опору на начальном этапе адаптации в чужой стране. Вот иллюстрации этого мнения из интервью. Женщина-программист, в 2016 г. переехавшая из Москвы вместе с 10-летней тогда дочерью, рассказывает: До решения перебраться в Литву я бывала здесь у знакомой, тоже бывшей мо­ Д р р р у у , сквички, и поняла, что здесь (в Вильнюсе) достаточно много русских и пото­ му ассимиляция будет достаточно легкой, для ребенка в первую очередь (РЖ44, 2019)2. сквички, и поняла, что здесь (в Вильнюсе) достаточно много русских и пото­ му ассимиляция будет достаточно легкой, для ребенка в первую очередь (РЖ44, 2019)2. Из рассказа женщины из Азербайджана, в 2010 г. вышедшей замуж за гражданина Литвы: Я приехала в страну, которую вообще не знала. Пугало сначала то, что не знаю языка, но здесь многие говорят на русском, поэтому мне очень повезло (AЖ29, 2016). Я приехала в страну, которую вообще не знала. Пугало сначала то, что не знаю языка, но здесь многие говорят на русском, поэтому мне очень повезло (AЖ29, 2016). 2. Многие иммигранты владеют русским и английским языками, причем первый язык они воспринимают как традиционный язык меж­ национального общения, все еще используемый на бывшем советском пространстве, а второй — как актуальный, но пока не всеми освоенный глобальный язык-посредник. Показателен фрагмент из интервью с 48-летним иммигрантом из Минска, в детстве не раз бывавшим в Вильнюсе вместе с родителями, а 4 года назад открывшим здесь собственный автосервис: Здесь, как было в советское время, так и осталось: все говорят по-литовски и по-русски. Поэтому Вильнюс для меня — типичный постсоветский город в язы­ ковом отношении. Фактически мне литовский здесь не очень-то и нужен, можно без него обойтись. Если уж кто-то не ответит по-русски, могу обратиться по-английски (БМ48, 2019). Своим опытом адаптации делятся студентки из Азербайджана, живу­ щие в Каунасе: Не было очень сложно установить связь с литовцами, они доброжелательны, во многом мне помогают и отлично знают английский язык. С другими иностран­ цами на самом деле и не общаюсь, только с литовцами и с людьми своей нацио­ нальности. С близкими и родными общаюсь на своем родном языке, а также и на русском (АЖ20, 2016); Чаще всего пользуюсь английским и русским. На английском языке ведётся об­ учение, русский же язык помогает мне “выжить” (АЖ27, 2016). 2 В скобках после фрагментов интервью заглавные буквы указывают на страну эмиграции и пол информанта, сообщается его возраст, если он известен, и год записи интервью. 2 В скобках после фрагментов интервью заглавные буквы указывают на страну эмиграции и пол информанта, сообщается его возраст, если он известен, и год записи интервью. Из интервью с бывшей москвичкой, хорошо владеющей английским и изучавшей литовский язык на начальных курсах для иммигрантов: Из интервью с бывшей москвичкой, хорошо владеющей английским и изучавшей литовский язык на начальных курсах для иммигрантов: Все (в государственных учреждениях, банках, поликлиниках) сразу предлагают говорить по-русски, всюду без проблем, как только слышат мой литовский. Но я, говорить по-русски, всюду без проблем, как только слышат мой литовский. Но я, правда, начинаю с того, что прошу прощения за свой плохой литовский. Говорю им по-литовски: “Gal jūs suprantat rusiškai arba angliškai?” (‘Может, вы понима­ ете по-русски или по-английски?’), и они сразу предлагают общаться по-русски (РЖ44, 2019). правда, начинаю с того, что прошу прощения за свой плохой литовский. Говорю им по-литовски: “Gal jūs suprantat rusiškai arba angliškai?” (‘Может, вы понима­ ете по-русски или по-английски?’), и они сразу предлагают общаться по-русски (РЖ44, 2019). 3. О намерении учить литовский язык говорят все русскоязычные иммигранты, отмечая не столько насущную необходимость этого в се­ годняшнем Вильнюсе и даже в традиционно литовскоязычном Каунасе (особенно при владении английским), сколько свое желание проявить уважение и симпатию к принявшей их стране. Из интервью со студентом из Азербайджана, живущим в Каунасе око­ ло пяти лет к моменту записи интервью и владеющим, кроме азербайд­ жанского, турецким, английским и русским языками: Как только приехал в Литву, поначалу думал, что литовским языком не буду ин­ тересоваться. Со временем узнал, что в университете есть курсы литовского языка, поэтому решил познакомиться с Литвой поближе, узнав её язык. Я счи­ таю, что я неплохо владею литовским. Изучать начал его три года назад, так что разговариваю и понимаю (АМ23, 2016). Еще одно мнение — украинца, в 2015 г. приехавшего учиться в маги­ стратуре по приглашению Вильнюсского университета и оставшегося в Вильнюсе работать экспертом по вопросам дискриминации: Когда я ехал в Литву, совершенно не собирался учить литовский язык. Тем более что мне вообще трудно даются языки. В университете можно было обойтись английским и даже русским, потому что программа была для иностранцев. Но параллельно предлагался двухнедельный вводный курс литовского языка. Ну и, если уж начал здесь жить, подумал, что нужно хоть как-то знать язык. В пер­ вую очередь — это уважение к стране, ведь язык — это культурный код, а во- вторых — элементарное бытовое удобство, если идешь по улице и понимаешь по витринам, что здесь чинят компьютеры, а здесь продается мед… Я и сейчас хотел бы учить литовский, если бы это было бесплатно. Конечно, по характеру работы, я мог бы здесь жить и общаться только по-украински и по-русски. Но ведь язык дает тебе больше возможностей (УМ30, 2019). 4. Imigracijos diskursas ir rusakalbiškumo perspektyvos Lietuvoje 133 ISSN 2351-6895 eISSN 2424-6115 Slavistica Vilnensis Из интервью с бывшей москвичкой, хорошо владеющей английским и изучавшей литовский язык на начальных курсах для иммигрантов: Согласно данным интервью, нередко толчком к изучению государ­ ственного языка для иммигрантов становится ощущение родственности литовской культуры их национальной культуре. Сотрудница службы ми­ грации также обращает внимание на то, что многие иммигранты, особен­ 134 Статьи. Алла Борисовна Лихачева. р Дискурс иммиграции и перспективы русскоязычия в Литве но граждане России, Беларуси, Украины отмечают, что приезжают жить в Литву, поскольку литовцы им эмоционально, ментально близки. Например, в одном из включенных в наш анализ видеоинтервью мо­ лодая пара из Екатеринбурга, вместе с маленьким ребенком приехавшая в Литву по рабочей визе и живущая здесь полтора года, рассказывает, что изучает литовский язык не только на курсах и с помощью аудиосамоу­ чителей, но и постоянно слушая песни в исполнении популярных литов­ ских исполнителей: Литовский очень классный язык… Для нас звучание литовского языка — оно очень крутое… Мы песни слушаем на литовском, мы вообще балдеем от раз­ ных песен… Песни на литовском — это прямо отдельная наша любовь… Нам нравится, что у этой страны (Литвы) своя богатая история и очень интерес­ ная культура, нравится то, что эта культура переплетается с традиционной русской культурой и историей. У нас действительно очень много общего (РМЖ, 2018). Аналогичное мнение высказывают и иммигранты более старшего возраста: Аналогичное мнение высказывают и иммигранты более старшего возраста: Я приехал по делам бизнеса. У меня работа всегда была связана с заграницей. До этого 17 лет работал в Голландии, пробовал начать работу в Польше — там не понравилось. Три с половиной года жил в Москве. Но здесь, в Вильнюсе, мне удоб­ нее, спокойнее. Для белорусов литовский менталитет гораздо ближе, чем рус­ ский… Я еще до переезда сюда начал самостоятельно изучать литовский язык по аудиосамоучителю. Теперь уже по работе все понимаю по-литовски. Сказать не все могу. Но в магазинах говорю по-литовски, я не комплексую по поводу своего уровня (БМ48, 2019). Я приехал по делам бизнеса. У меня работа всегда была связана с заграницей. До этого 17 лет работал в Голландии, пробовал начать работу в Польше — там не понравилось. Три с половиной года жил в Москве. Но здесь, в Вильнюсе, мне удоб­ нее, спокойнее. Для белорусов литовский менталитет гораздо ближе, чем рус­ ский… Я еще до переезда сюда начал самостоятельно изучать литовский язык по аудиосамоучителю. Теперь уже по работе все понимаю по-литовски. Сказать не все могу. Но в магазинах говорю по-литовски, я не комплексую по поводу своего уровня (БМ48, 2019). Сотрудница службы миграции также рассказала о политэмигрантах из Таджикистана, утверждающих, что, выбирая между Литвой и Герма­ нией, останавливаются на Литве из-за более традиционного и близкого им отношения литовцев к воспитанию детей. 5. Можно заметить, что литовский язык изучается иммигрантами не столько из-за его потенциальной полезности в жизни вообще, сколько из благодарности за проявления толерантности и усилия государства и его жителей облегчить адаптацию иммигрантов. Из интервью с бывшей москвичкой: Из интервью с бывшей москвичкой: Знание языка — это знак уважения к стране, которая нас приняла… Мне не хотелось бы, чтобы сюда приезжали люди (из России), которые не хотят учить язык. Мне важно, чтобы к нам, иммигрантам, не возникало отрицательного от­ ношения местных жителей. Это то, чего я не встречала за три года ни разу со стороны литовцев (РЖ44, 2019). 135 ISSN 2351-6895 eISSN 2424-6115 Slavistica Vilnensis На доброжелательность жителей Литвы к русскоязычным иммигран­ там обращают внимание и две молодые пары, выложившие свои видео­ интервью в Интернете. Супруги из Екатеринбурга отмечают: За полтора года ни разу не было какого-то там презрительного отношения или агрессии из-за того, что мы русские или мы не понимаем по-литовски, не гово­ рим по-литовски или плохо говорим. Никогда такого нет, и все литовцы, наобо­ рот, с очень большим интересом… когда узнают, что мы не русские Литвы, а приехавшие. Аналогичное мнение высказывают и иммигранты более старшего возраста: Если у нас всё наладится, мы вер­ немся на родину. Но, может быть, ребята наши (о детях и внуках) тут останут­ ся. Моя внучка уже ходит в первый класс, и она уже пошла в литовскую школу. Мы хотим научить ее культуре литовского государства, литовских людей (ТМ, 2019). Мы не говорим, что навсегда останемся тут. Если у нас всё наладится, мы вер­ немся на родину. Но, может быть, ребята наши (о детях и внуках) тут останут­ ся. Моя внучка уже ходит в первый класс, и она уже пошла в литовскую школу. Мы хотим научить ее культуре литовского государства, литовских людей (ТМ, 2019). Еще одно характерное замечание относительно школы для дочери прозвучало в интервью с российской иммигранткой, покинувшей страну, когда, по ее словам, однозначно стало ясно, что из России надо уезжать: Я выбрала для дочери русскую школу, потому что хотела, чтобы она не поте­ ряла язык. Но я жалею об этом решении. О том, что именно русская школа, я жалею. Я думаю, что в литовской она сразу приобрела бы язык. Да, русский она бы потеряла, и это была бы большая проблема (РЖ44, 2019). 7. В социолингвистическом плане иммигрантский дискурс предостав­ ляет возможность увидеть перспективы сохранения русскоязычия в Лит­ ве. С одной стороны, иммигранты воспринимают как норму использо­ вание государственного языка в официальных сферах общения, и ста­ раются следовать этой норме даже при наличии собственной минималь­ ной языковой компетенции. Распространение русского языка в столице и крупных городах они считают уходящим явлением: — Как вы думаете, долго ли еще хотя бы в Вильнюсе можно будет жить, не зная литовского языка? — Ну, поменяются поколения, молодежь уже не понимает по- русски. Вторым языком станет английский, а русский язык постепенно уйдет. — А приезжие? Или их тут капля в море? — Я думаю, не то чтобы капля в море, а что не они заказывают музыку (РЖ44, 2019). — Как вы думаете, долго ли еще хотя бы в Вильнюсе можно будет жить, не зная литовского языка? — Ну, поменяются поколения, молодежь уже не понимает по- русски. Вторым языком станет английский, а русский язык постепенно уйдет. — А приезжие? Или их тут капля в море? — Я думаю, не то чтобы капля в море, а что не они заказывают музыку (РЖ44, 2019). Аналогичное мнение высказывают и иммигранты более старшего возраста: Их наоборот это сильно интересует, и они задают много вопросов. Но их уровень вопросов реально адекватный, в отличие от того, что спрашива­ ют у нас в России про каких-то там литовских фашистов… Никакой русофобии, даже уже немного скучно развенчивать этот миф… (РМЖ, 2018). Схожее мнение высказывают иммигранты, три месяца прожившие в Литве, а до этого молодой человек успел пожить в Украине, Израиле, Канаде и России, а девушка — в Украине и России: Вид на жительство мы получили и пойдем обязательно изучать литовский язык. Мы считаем, что, если находишься в какой-то стране, у тебя есть какие-то обязанности перед ней, ты не можешь как-то халатно относиться… Если они (иммигранты) приезжают из России, нужно просто забыть, что им там гово­ рили в телевизоре (про русофобию). Просто всё классно, люди открытые, очень спокойно, безопасно… А то такие истории, что вас там выгонят, не дадут вам ничего, нахамят… Они (литовцы) очень классные! У них все предусмотрено, они тебе звонят на дом, если чего-то не хватает из документов: приходи, пожалуй­ ста, в какое время удобно… В кабинетах (о службе миграции) мы видели игруш­ ки… ну нормальный такой подход… (РМЖ, 2017). 6. Как сообщает сотрудница миграционной службы, несмотря на име­ ющийся у иммигрантов начальный энтузиазм в овладении государствен­ ным языком, приоритетом для них неизбежно становится работа, и люди, имеющие высшее образование и не планирующие получить его в Литве, устраиваются на работу туда, где хорошее знание государственного язы­ ка не требуется, например, если документация ведется на английском, а в общении преобладает русский язык. Кроме того, по словам сотрудницы службы миграции, большинство иммигрантов с родным или хорошо усвоенным русским языком выбира­ ют для детей детские сады и школы с русским языком обучения. Из рассказа старшеклассницы-азербайджанки, пять лет назад при­ ехавшей с родителями в Шяуляй и заканчивающей русскую школу: Чаще всего я пользуюсь азербайджанским и русским. Изучать (литовский язык) я начала, как только приехала в Литву, потому что знала, что надо будет сда­ вать экзамен, чтобы получить аттестат. В школе брала дополнительные уроки литовского языка (АЖ18, 2016). 136 Статьи. Алла Борисовна Лихачева. р Дискурс иммиграции и перспективы русскоязычия в Литве Школы с государственным языком обучения, по данным службы ми­ грации, чаще выбирают переселившиеся в Литву украинцы литовского происхождения, некоторые иммигранты из России, называющие в каче­ стве причины переезда политическую ситуацию в их стране, политэми­ гранты из Таджикистана. Например, участник видеобеседы в студии DELFI из Таджикистана, с 2015 г. живущий в Вильнюсе, рассказывает: Мы не говорим, что навсегда останемся тут. Аналогичное мнение высказывают и иммигранты более старшего возраста: С другой стороны, даже те иммигранты, которые приехали в Литву относительно давно — в позднее советское время, и для которых русский язык не является родным, продолжают активно его использовать: Как только приехал сюда, не так тяжело все было, как казалось, проблема была в том, что не знаю литовский язык. Со временем понял, что для начала хватит и русского языка, до сих пор чаще всего пользуюсь русским языком. <…> В семье общаемся только на азербайджанском языке, а в общественных местах, с дру­ зьями и соседями — на русском (АМ51, 2016). 137 ISSN 2351-6895 eISSN 2424-6115 Slavistica Vilnensis Источники В студии DELFI: cтанут ли иммигранты из Украины, Беларуси, России и дру- гих стран национальными меньшинствами Литвы? URL: https://www.delfi.lt/video/ laidos/zinios-rusu-kalba/v-studii-delfi-ctanut-li-immigranty-iz-ukrainy-belarusi-rossii-i- drugih-stran-nacionalnymi-menshinstvami-litvy.d?id=81255171 (21 11 2019). Форум Жизнь в Литве. URL: https://www.facebook.com/groups/porusski.lt/ (21 10 2019). Личный опыт: Иммиграция в Литву из России по Blue Card. URL: https://www. youtube.com/watch?v=OSaw44qTKkU (10 11 2019). О Литве, переезде, националистах и еде. URL: https://www.youtube.com/ watch?v=zOwJx0Vu1dQ (10 11 2019). О Литве, переезде, националистах и еде. URL: https://www.youtube.com/ watch?v=zOwJx0Vu1dQ (10 11 2019). Migracija skaičiais – European Migration Network. URL: http://123.emn.lt/ (10 10 2019). Для позднесоветской волны иммигрантов русский язык был и оста­ ется существенным компонентом их языковой жизни и жизни их семей: Дискурс иммиграции и перспективы русскоязычия в Литве Статьи. Алла Борисовна Лихачева. р Дискурс иммиграции и перспективы русскоязычия в Литве принятыми как выход для Литвы — такой же, как и для многих других стран. Иммигранты, говорящие по-русски, ощущают, что их присутствие в Литве не воспринимается как угроза в политическом или в языковом отношении. Они видят себя субъектами, чей вклад в литовскую экономи- ку, демографию и даже в количественные показатели носителей литов- ского языка должен быть поддержан литовским обществом. Для позднесоветской волны иммигрантов русский язык был и оста­ ется существенным компонентом их языковой жизни и жизни их семей: Для позднесоветской волны иммигрантов русский язык был и оста­ ется существенным компонентом их языковой жизни и жизни их семей: Когда приехала в Литву, говорить начала на русском, поэтому детей отправила в русский садик. Это было удобно для меня, потому что на тот момент я не знала литовского языка. Затем дети пошли в русскую школу. Между собой дети общались в детстве на русском и азербайджанском языке, когда выросли, они выучили литовский язык. Сейчас дети уже большие, с нами, родителями, гово­ рят на азербайджанском, между собой — чаще всего на русском (АЖ50, 2016); Дети у нас пока маленькие. Дочь учится во втором классе в русской школе, сын ходит в русский садик. Выбрали именно русскую школу и садик, потому что нам самим легче общаться на русском, чем на литовском языке. У детей много друзей во дворе, и они общаются на русском (АМ51, 2016). Согласно критериям [Bourhis, Landry 2008], витальность языковой группы (the language group vitality) напрямую зависит от языкового по­ ведения ее членов, частоты использования ими этнического или хорошо усвоенного, общего для членов группы языка, поддержания этого язы­ ка, в частности, в семейном и близком общении. “Использование языка меньшинства не прекратится до тех пор, пока не изменятся групповые нормы использования языка” [Fase, Jaspaert & Kroon 1992, 7]. С учетом этих факторов можно утверждать, что русскоязычие на определенных территориях Литвы в ближайшие годы сохранится и может даже не­ сколько усилить свои позиции за счет новой волны иммигрантов раз­ ных национальностей (о тесной зависимости количественных показате­ лей языковой группы и шансов сохранения ее общего языка см. [Clyne 1992]), для которых русский язык является основным языком общения во многих жизненных ситуациях, в том числе, как показывают материалы интервью, в институциональном и других видах внесемейного дискурса. Важным показателем устойчивого положения языковой группы яв­ ляется также положительное восприятие и оценка языка его носителя­ ми, живущими в иноязычных условиях [Allard, Landry 1992]. В дискур­ се постсоветской русскоязычной иммиграции переезд в Литву, с одной стороны, рисуется как переселение в Европу, а с другой — как переезд в более близкое и понятное, чем западноевропейское, культурное, сим­ волическое (в том числе и за счет присутствия здесь русского языка) пространство. При всем уважении к принимающей стране и ее языку иммигранты не предполагают выбора между литовским и русским, но планируют обязательное дополнительное овладение литовским языком при условии дальнейшей жизни в Литве. Продолжающаяся эмиграция и относительно небольшое количество возвращающихся литовцев имплицирует ожидания иммигрантов быть 138 Статьи. Алла Борисовна Лихачева. Литература Jaspaert and S. Kroon (Eds.). Maintenance and Loss of Minority Languages. Amsterdam: John Benjamins Publishing Company, 17–36. Minority Languages. Amsterdam: John Benjamins Publishing Company, 17–36. COLLINS, J., 2007. Immigrants as Victims of Crime and Criminal Justice Discourse in COLLINS, J., 2007. Immigrants as Victims of Crime and Criminal Justice Discourse in Australia. International Review of Victimol­ogy, Vol. 14, 1, 51–79. COLLINS, J., 2007. Immigrants as Victims of Crime and Criminal Justice Discourse in Australia. International Review of Victimol­ogy, Vol. 14, 1, 51–79. FASE, W., JASPAER, K. & KROON, S. 1992. Maintenance and Loss of Minority Lan- guages: Introductory Remarks. In: W. Fase, K. Jaspaert and S. Kroon (Eds.). Maintenance FASE, W., JASPAER, K. & KROON, S. 1992. Maintenance and Loss of Minority Lan- guages: Introductory Remarks. In: W. Fase, K. Jaspaert and S. Kroon (Eds.). Maintenance and Loss of Minority Languages. Amsterdam: John Benjamins Publishing Company, 3–13. guages: Introductory Remarks. In: W. Fase, K. Jaspaert and S. Kroon (Eds.). Maintenance and Loss of Minority Languages. Amsterdam: John Benjamins Publishing Company, 3–13. and Loss of Minority Languages. Amsterdam: John Benjamins Publishing Company, 3–13. KASATKINA, N., LEONČIKAS, T., 2003. Lietuvos etninių grupių adaptacija: kontekstas ir eiga. Vilnius: Socialinių tyrimų centras. KUZNECOVIENĖ, J., 2009. Lietuvių imigrantų Norvegijoje, Anglijoje ir Ispanijoje įsitraukimo strategijos: nuo konformizmo iki navigacijos. Filosofija. Sociologija, t. 20, nr. 2, 96–103. URL: http://mokslozurnalai.lmaleidykla.lt/publ/0235-7186/2009/2/96-103. pdf (03 11 2019). LANDRY, R., ALLARD, R., 1994. Diglossia, ethnolinguistic vitality, and language behaviour. International Journal of the Sociology of Language, 108, 15–42. LI XUE, 2007. Portrait of an Integration Process. Difficulties encountered and resources relied on for newcomers in their first 4 years in Canada. Evidence from three waves of the Longitudinal Survey of Immigrants to Canada (LSIC). URL: http://www.cic. gc.ca/english/pdf/research-stats/portrait-integr-process-e.pdf (03.11. 2019). PETRUŠAUSKAITĖ, V., BATUCHINA, A., 2015. Migrant workers from non-EU states in Lithuania: the structure of migration and challenges in survey research. Etniškumo studijos, 1, 28–47. RAMONIENĖ, M. (red.), 2015. Emigrantai: kalba ir tapatybė. Vilnius: VU leidykla. RAMONIENĖ, M. (red.), 2015. Emigrantai: kalba ir tapatybė. Vilnius: VU leidykla. RAMONIENĖ, M. (red.), 2019. Emigrantai: kalba ir tapatybė II. Vilnius: VU leidykla. RAMONIENĖ, M. (red.), 2019. Emigrantai: kalba ir tapatybė II. Vilnius: VU leidykla. RICHARDSON, J. E., WODAK, R., 2013. The Impact of Visual Racism: Visual Arguments in Political Leaflets of Austrian and British Far-Right Parties. In: R. Wodak (Ed.). Critical Discourse Analysis. Vol. IV. Applications, Interdisciplinary Perspectives and New Trends. Литература БАРДИН, А., 2016. Миграционная проблема в германском научном дискурсе. По- лис. Политические исследования, № 6, 183–188. БАРДИН, А., 2016. Миграционная проблема в германском научном дискурсе. По- П д №6 183 188 БАРДИН, А., 2016. Миграционная проблема в германском научном дискурсе. По- лис. Политические исследования, № 6, 183–188. ЗИМОВА, Н., 2017. Социальная адаптация трудовых мигрантов из стран Цен- тральной Азии в России. Миграция и социально-экономическое развитие. Т. 2, № 1, 19–28. ЗИМОВА, Н., 2017. Социальная адаптация трудовых мигрантов из стран Цен- тральной Азии в России. Миграция и социально-экономическое развитие. Т. 2, № 1, 19–28. КАРАСИК, В., 2004. Языковой круг: личность, концепты, дискурс. Москва: Гно- зис. МАТИЦЫНА, М., 2018. Деконструирование дискурса иммиграции. Иммигранты: жертвы, герои или угроза? Вестник Омского государственного педагогического уни- верситета. Гуманитарные исследования, № 3 (20), 84–88. ЧЕРНЯВСКАЯ, В., 2001. Дискурс как объект лингвистических исследований. In: Текст и дискурс. Проблемы экономического дискурса. Санкт-Петербург: Изд-во СпбГУЭФ, 11–22. ALLARD, R., LANDRY, R., 1992. Ethnolinguistic Vitality Beliefs and Language Maintenance and Loss. In: W. Fase, K. Jaspaert and S. Kroon (eds.). Maintenance and Loss of Minority Languages. Amsterdam: John Benjamins Publishing Company, 171–196. BARTKEVIČIENĖ, A., 2015. Imigrantų integracijos problemos ir jų sprendimo būdai: trumpalaikės ir ilgalaikės integracijos priemonės. URL: http://sei.lt/wp-content/ uploads/2017/10/IMIGRANTU_INTEGRACIJOS_PRIEMONES.pdf. (20 11 2019). BERESNEVIČIŪTĖ, V., LEONČIKAS, T., ŽIBAS, K., 2009. Migrantų gyvenimas Lietuvoje: visuomenės nuostatos ir migrantų patirtys. Etniškumo studijos, 2, 77–102. 139 ISSN 2351-6895 eISSN 2424-6115 Slavistica Vilnensis BOURHIS, R.Y., LANDRY, R., 2008. Group Vitality, Cultural Autonomy and the Well- BOURHIS, R.Y., LANDRY, R., 2008. Group Vitality, Cultural Autonomy and the Well- ness of Language Minorities. In: R.Y. Bourhis (Ed.). The Vitality of the English-Speaking ness of Language Minorities. In: R.Y. Bourhis (Ed.). The Vitality of the English-Speaking Communities of Quebec: From Community Decline to Revival. Montreal: Quebec, 185–212. CLYNE, M., 1992. Linguistic and Sociolinguistic Aspects of Language Contact, Main- tenance and Loss. In: W. Fase, K. Jaspaert and S. Kroon (Eds.). Maintenance and Loss of Minority Languages Amsterdam: John Benjamins Publishing Company 17 36 CLYNE, M., 1992. Linguistic and Sociolinguistic Aspects of Language Contact, Main- CLYNE, M., 1992. Linguistic and Sociolinguistic Aspects of Language Contact, Main- tenance and Loss In: W Fase K Jaspaert and S Kroon (Eds ) Maintenance and Loss of g g p g g tenance and Loss. In: W. Fase, K. Jaspaert and S. Kroon (Eds.). Maintenance and Loss of Minority Languages. Amsterdam: John Benjamins Publishing Company, 17–36. tenance and Loss. In: W. Fase, K. Литература Los Angeles – London – New Delhi – Singapore – Washington DC, 245–274 SIPAVIČIENĖ, A., GAIDYS, V., JERŠOVAS, M., 2010. Lietuvos gyventojų požiūris į imigraciją ir darbo imigrantus: tyrimo ataskaita. Vilnius. URL: http://www.iom.lt/images/ publikacijos/failai/1427788343_8TMOLietuvos%20gyventoju%20poziuris%20i%20 imigracija%20ir%20darbo%20imigrantus.pdf (03 11 2019). TEREŠKINAS, A., 2009. Imigracijos diskursai JAV ir Airijos spaudoje. Filosofija. Sociologija, t. 20, nr. 4, 302–309. VAN DER VALK, I., 2000. Parliamentary discourse on immigration and nationality in France. In: R. Wodak & T.A. van Dijk (Eds.). Racism at the top: parliamentary discourses on ethnic issues in six Euro­pean states. Klagenfurt: Drava Verlag, 221–260. VAN DIJK, TEUN A., 1987. Episodic Models in Discourse Processing. In: R. Horowitz & S.J. Samuels, (Eds.) Comprehending Oral and Written Language. New York: Academic Press, 161–196. 140 Статьи. Алла Борисовна Лихачева. искурс иммиграции и перспективы русскоязычия в Литв ŽIBAS, K., 2012. Baltarusiai, rusai ir ukrainiečiai Lietuvoje: nuo imigrantų iki ateities piliečių. Etniškumo studijos, nr. 1/2, 145–177. ŽIBAS, K., 2014. Turkų ir kinų imigrantai Lietuvoje. Vilnius: Lietuvos socialinių tyrimų centras. Bibliography (Transliteration) ALLARD, R., LANDRY, R., 1992. Ethnolinguistic Vitality Beliefs and Language Maintenance and Loss. In: W. Fase, K. Jaspaert and S. Kroon (eds.). Maintenance and Loss of Minority Languages. Amsterdam: John Benjamins Publishing Company, 171–196. BARDIN, A., 2016. Migracionnaja problema v germanskom nauchnom diskurse. In: Polis. Politicheskie issledovanija, № 6, 183–188. BARTKEVIČIENĖ, A., 2015. Imigrantų integracijos problemos ir jų sprendimo būdai: trumpalaikės ir ilgalaikės integracijos priemonės. URL: http://sei.lt/wp-content/ uploads/2017/10/IMIGRANTU_INTEGRACIJOS_PRIEMONES.pdf. (20 11 2019). BERESNEVIČIŪTĖ, V., LEONČIKAS, T., ŽIBAS, K., 2009. Migrantų gyvenimas Lietuvoje: visuomenės nuostatos ir migrantų patirtys. Etniškumo studijos, Nr. 2, 77–102. BOURHIS, R.Y., LANDRY, R., 2008. Group Vitality, Cultural Autonomy and the Well- BOURHIS, R.Y., LANDRY, R., 2008. Group Vitality, Cultural Autonomy and the Well- ness of Language Minorities. In: R.Y. Bourhis (Ed.). The Vitality of the English-Speaking , , , , p y, y ness of Language Minorities. In: R.Y. Bourhis (Ed.). The Vitality of the English-Speaking Communities of Quebec: From Community Decline to Revival. Montreal: Quebec, 185–212. CHERNJAVSKAJA, V., 2001. Diskurs kak ob’ekt lingvisticheskih issledovanij. In: Tekst i diskurs. Problemy jekonomicheskogo diskursa. Sankt-Peterburg: Izd-vo SpbGUJeF, 11–22. CLYNE, M., 1992. Linguistic and Sociolinguistic Aspects of Language Contact, CLYNE, M., 1992. Linguistic and Sociolinguistic Aspects of Language Contact, Maintenance and Loss. In: W. Fase, K. Jaspaert and S. Kroon (Eds.). Maintenance and Loss of Minority Languages Amsterdam: John Benjamins Publishing Company 17 36 CLYNE, M., 1992. Linguistic and Sociolinguistic Aspects of Language Contact, Maintenance and Loss. In: W. Fase, K. Jaspaert and S. Kroon (Eds.). Maintenance and CLYNE, M., 1992. Linguistic and Sociolinguistic Aspects of Language Contact, Maintenance and Loss. In: W. Fase, K. Jaspaert and S. Kroon (Eds.). Maintenance and Loss of Minority Languages. Amsterdam: John Benjamins Publishing Company, 17–36. COLLINS J 2007 I i t Vi ti f C i d C i i l J ti Di i CLYNE, M., 1992. Linguistic and Sociolinguistic Aspects of Language Contact, Maintenance and Loss. In: W. Fase, K. Jaspaert and S. Kroon (Eds.). Maintenance and Loss of Minority Languages. Amsterdam: John Benjamins Publishing Company, 17–36. COLLINS, J., 2007. Immigrants as Victims of Crime and Criminal Justice Discourse in COLLINS, J., 2007. Immigrants as Victims of Crime and Criminal Justice Discourse in Australia. International Review of Victimol­ogy, Vol. 14, 1, 51–79. Australia. International Review of Victimol­ogy, Vol. 14, 1, 51–79. Australia. International Review of Victimol­ogy, Vol. 14, 1, 51 79. FASE, W., JASPAER, K. & KROON, S. 1992. Bibliography (Transliteration) Maintenance and Loss of Minority Languages: Introductory Remarks. In: W. Fase, K. Jaspaert and S. Kroon (Eds.). M i t d L f Mi it L A t d J h B j i P bli hi FASE, W., JASPAER, K. & KROON, S. 1992. Maintenance and Loss of Minority Languages: Introductory Remarks. In: W. Fase, K. Jaspaert and S. Kroon (Eds.). Maintenance and Loss of Minority Languages. Amsterdam: John Benjamins Publishing Company 3 13 p y, KARASIK, V., 2004. Jazykovoj krug: lichnost’, koncepty, diskurs. Moskva: Gnozis. KASATKINA, N., LEONČIKAS, T., 2003. Lietuvos etninių grupių adaptacija: kontekstas ir eiga. Vilnius: Socialinių tyrimų institutas. KARASIK, V., 2004. Jazykovoj krug: lichnost’, koncepty, diskurs. Moskva: Gnozis. Č y j g p y KASATKINA, N., LEONČIKAS, T., 2003. Lietuvos etninių grupių adaptacija: kontekstas ir eiga. Vilnius: Socialinių tyrimų institutas. KUZNECOVIENĖ, J., 2009. Lietuvių imigrantų Norvegijoje, Anglijoje ir Ispanijoje įsitraukimo strategijos: nuo konformizmo iki navigacijos, Filosofija. Sociologija, t. 20, nr. 2, 96–103. URL: http://mokslozurnalai.lmaleidykla.lt/publ/0235-7186/2009/2/96-103. pdf (03 11 2019). LANDRY, R., ALLARD, R., 1994. Diglossia, ethnolinguistic vitality, and language behaviour. International Journal of the Sociology of Language, 108, 15–42.i LI XUE, 2007. Portrait of an Integration Process. Difficulties encountered and resources relied on for newcomers in their first 4 years in Canada. Evidence from three waves of the Longitudinal Survey of Immigrants to Canada (LSIC). URL: http://www.cic. gc.ca/english/pdf/research-stats/portrait-integr-process-e.pdf (03.11. 2019). 141 ISSN 2351-6895 eISSN 2424-6115 Slavistica Vilnensis MATICYNA, M., 2018. Dekonstruirovanie diskursa immigracii. Immigranty: zhertvy, MATICYNA, M., 2018. Dekonstruirovanie diskursa immigracii. Immigranty: zhertvy, geroi ili ugroza? Vestnik Omskogo gosudarstvennogo pedagogicheskogo universiteta. Gumanitarnye issledovanija, № 3 (20), 84–88. geroi ili ugroza? Vestnik Omskogo gosudarstvennogo pedagogicheskogo universiteta. Gumanitarnye issledovanija, № 3 (20), 84–88. PETRUŠAUSKAITĖ, V., BATUCHINA, A., 2015. Migrant workers from non-EU states in Lithuania: the structure of migration and challenges in survey research. Etniškumo studijos, 1, 28–47. RAMONIENĖ, M. (red.), 2015. Emigrantai: kalba ir tapatybė. Vilnius: VU leidykla. RAMONIENĖ, M. (red.), 2019. Emigrantai: kalba ir tapatybė II. Vilnius: VU leidykla RICHARDSON, J. E., WODAK, R., 2013. The Impact of Visual Racism: Visual Arguments in Political Leaflets of Austrian and British Far-Right Parties. In: R. Wodak gl g (Ed.). Critical Discourse Analysis. Vol. IV. Applications, Interdisciplinary Perspectives a New Trends. Los Angeles – London – New Delhi – Singapore – Washington DC, 245–274. SIPAVIČIENĖ, A., GAIDYS, V., JERŠOVAS, M., 2010. Lietuvos gyventojų požiūris į imigraciją ir darbo imigrantus: tyrimo ataskaita. Vilnius. Bibliography (Transliteration) URL: http://www.iom.lt/images/ publikacijos/failai/1427788343_8TMOLietuvos%20gyventoju%20poziuris%20i%20 imigracija%20ir%20darbo%20imigrantus.pdf (03 11 2019).i TEREŠKINAS, A., 2009. Imigracijos diskursai JAV ir Airijos spaudoje. Filosofija. Sociologija, t. 20, nr. 4, 302–309. VAN DER VALK, I., 2000. Parliamentary discourse on immigration and nationality in France. In: R. Wodak & T.A. van Dijk (Eds.). Racism at the top: parliamentary discourses on ethnic issues in six Euro­pean states. Klagenfurt: Drava Verlag, 221–260. VAN DIJK, TEUN A., 1987. Episodic Models in Discourse Processing. In: R. Horowitz & S.J. Samuels, (Eds.) Comprehending Oral and Written Language. New York: Academic Press, 161–196. ZIMOVA, N., 2017. Social’naja adaptacija trudovyh migrantov iz stran Central’noj ZIMOVA, N., 2017. Social’naja adaptacija trudovyh migrantov iz stran Central’noj Azii v Rossii. Migracija i social’no-ekonomicheskoe razvitie. T. 2, №1, 19–28. Azii v Rossii. Migracija i social’no-ekonomicheskoe razvitie. T. 2, №1, 19–28. ŽIBAS, K., 2012. Baltarusiai, rusai ir ukrainiečiai Lietuvoje: nuo imigrantų iki ateities piliečių. Etniškumo studijos, nr. 1/2, 145–177. ŽIBAS, K., 2014. Turkų ir kinų imigrantai Lietuvoje. Vilnius: Lietuvos socialinių tyrimų centras. Алла Лихачева, доктор гуманитарных наук, профессор кафедры русской филологии Вильнюсского университета Alla Likhachiova, PhD (Humanities), Professor of the Russian Department, Vilnius University Ala Lichačiova, humanitarinių mokslų daktarė, Vilniaus universiteto Rusų filologijos katedros profesorė Alla Likhachiova, PhD (Humanities), Professor of the Russian Department, Vilnius University Ala Lichačiova, humanitarinių mokslų daktarė, Vilniaus universiteto Rusų filologijos katedros profesorė Ala Lichačiova, humanitarinių mokslų daktarė, Vilniaus universiteto Rusų filologijos katedros profesorė 142
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Qualidade de vida dos indivíduos expostos ao césio-137, em Goiânia, Goiás, Brasil
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1 Programa de Pós-graduação Ciências da Saúde, Universidade Federal de Goiás, Goiânia, Brasil. 2 Instituto de Patologia Tropical e Saúde Pública, Universidade Federal de Goiás, Goiânia, Brasil. 3 Faculdade de Medicina, Universidade Federal de Goiás, Goiânia, Brasil. 4 Faculdade de Farmácia, Universidade Federal de Goiás, Goiânia, Brasil. Correspondência S. C. Fuini Programa de Pós-graduação Ciências da Saúde, Universidade Federal de Goiás. Rua 17-A, 1419, Setor Aeroporto, Goiânia, GO 74075-160, Brasil. silvana.fuini@gmail.com Qualidade de vida dos indivíduos expostos ao césio-137, em Goiânia, Goiás, Brasil Quality of life in individuals exposed to cesium-137 in Goiânia, Goiás State, Brazil Calidad de vida de las personas expuestas al cesio-137, en Goiânia, Goiás, Brasil Silvana Cruz Fuini 1 Rafael Souto 2 Geraldo Francisco do Amaral 3 Rita Goreti Amaral 4 Silvana Cruz Fuini 1 Rafael Souto 2 Geraldo Francisco do Amaral 3 Rita Goreti Amaral 4 Qualidade de Vida; Césio; Impacto Psicossocial; Acidentes 1301 ARTIGO ARTICLE 1301 ARTIGO ARTICLE 1301 ARTIGO ARTICLE Qualidade de vida dos indivíduos expostos ao césio-137, em Goiânia, Goiás, Brasil Quality of life in individuals exposed to cesium-137 in Goiânia, Goiás State, Brazil Calidad de vida de las personas expuestas al cesio-137, en Goiânia, Goiás, Brasil Quality of Life; Cesium; Psychosocial Impact; Accidents Introdução nesta direção 10,11. Sendo assim, a avaliação da qualidade de vida do paciente é reconhecida co- mo uma importante área do conhecimento cien- tífico, em razão do seu conceito se interpor ao de saúde. Tal avaliação tem a vantagem de incluir aspectos subjetivos geralmente não abordados por outros critérios de avaliação 12. O acidente radioativo ocorrido em setembro de 1987, na cidade de Goiânia, Estado de Goiás, Bra- sil, trouxe problemas sérios que perduram até os dias de hoje 1. Esse evento foi provocado por meio da ruptura de um aparelho radioterápico abandonado em uma clínica médica desativada, e posteriormente agravado pelo manuseio incor- reto da cápsula que continha o césio-137 (isóto- po radioativo) por pessoas leigas no assunto 2. O interesse pelo conhecimento da qualidade de vida dos radioacidentados configura-se como uma preocupação constante da população do Estado de Goiás e especialmente do corpo clíni- co do C.A.R.A., sobretudo, devido à recorrência no atendimento psicossocial e à busca frequente pelos serviços médicos e por respostas às dúvi- das que ainda persistem. Na época, em torno de 112 mil pessoas foram envolvidas no acidente, cerca de uma centena diretamente e outras centenas indiretamente, incluindo familiares, vizinhos e agentes públi- cos 2,3. Desde então é realizado regularmente por uma unidade de saúde específica, atualmente denominada Centro de Assistência aos Radioaci- dentados (C.A.R.A.) 3,4, que faz parte da estrutura da Secretaria de Saúde de Goiás, o monitoramen- to das pessoas envolvidas. Até os dias de hoje, os resultados do monitoramento não apontam dados estatisticamente significantes para morbi- mortalidade associada aos efeitos da radiação ionizante. Entretanto, pesquisadores recomen- dam o prosseguimento dos estudos, tendo em vista os efeitos tardios decorrentes do acidente radioativo 1,5. O referido fato corrobora com estudos que consideram o impacto na saúde mental como o maior problema de saúde pública desencadeado por acidentes dessa natureza e que, muitas ve- zes, esses transtornos vêm acompanhado de um grande número de queixas somáticas inespecífi- cas 8,9,13,14. Nesse aspecto, é reconhecido pelo cor- po clínico do C.A.R.A. que desde a ocorrência do acidente radioativo com o césio-137 foi dada ên- fase aos aspectos físicos das vítimas do acidente, em detrimento ao gerenciamento dos problemas sociais e psicológicos 15. Assim, este trabalho teve como objetivo ava- liar a qualidade de vida dos indivíduos expos- tos ao césio-137, durante acidente ocorrido em Goiânia, e sua associação com fatores sociode- mográficos. Abstract Estudo observacional transversal realizado em Goiânia, Goiás, Brasil, em uma amostra de 56% de 111 pessoas, objetivando avaliar a qualida- de de vida dos indivíduos expostos ao césio-137 e sua associação com fatores sociodemográfi- cos. Utilizou-se o WHOQOL-BREF e o Sistema de Monitoramento dos Radioacidentados. Os participantes foram divididos segundo critérios internacionais em: Grupo I – radiodermites e/ ou dosimetria citogenética acima de 20 rads (n = 33); e Grupo II – dosimetria citogenética ≤ 20 rads (n = 29), totalizando 62 sujeitos. Dentre os domínios do WHOQOL-BREF, o meio ambiente apresentou a média de escores mais alta (59,88; DP = 20,39) e o psicológico a média mais baixa (53,02; DP = 17,98). As associações entre os do- mínios físico, psicológico e relações sociais foram significativas para a variável idade. Não houve diferença entre grupos. A associação entre os fa- tores sociodemográficos e qualidade de vida não foi significativa. Os radioacidentados sofrem considerável impacto na qualidade de vida, com persistência de problemas psicossociais, espe- cialmente para aqueles com mais de 41 anos. This cross-sectional observational study in Goiânia, Goiás State, Brazil in a sample of 56% of 111 individuals exposed to cesium-137 aimed to evaluate their quality of life and associations with socio-demographic factors. The study used the WHOQOL-BREF and the Monitoring System for Radiation Victims. Participants were divided according to international criteria: Group I – ra- diation dermatitis and / or cytogenetic dosimetry above 20 rads (n = 33) and Group II - cytogenetic dosimetry ≤ 20 rads (n = 29), totaling 62 subjects. Among the WHOQOL-BREF domains, environ- ment showed the highest mean scores (59.88, SD = 20.39) and psychological the lowest (53.02, SD = 17.98). Associations between the physical, psychological, and social domains were signifi- cant for the age variable. There was no difference between groups. The association between socio- demographic factors and quality of life was not significant. Radiation victims suffer consider- able impact on quality of life, with persistent psychosocial problems, especially among those older than 41 years. Correspondência S. C. Fuini Programa de Pós-graduação Ciências da Saúde, Universidade Federal de Goiás. Rua 17-A, 1419, Setor Aeroporto, Goiânia, GO 74075-160, Brasil. silvana.fuini@gmail.com Quality of Life; Cesium; Psychosocial Impact; Accidents Quality of Life; Cesium; Psychosocial Impact; Accidents Qualidade de Vida; Césio; Impacto Psicossocial; Acidentes Cad. Saúde Pública, Rio de Janeiro, 29(7):1301-1310, jul, 2013 Fuini SC et al. 1302 Introdução Dessa forma, os diversos aspectos relacio- nados à saúde e fatores psicossociais devem ser investigados nas populações potencialmente expostas à radioatividade, sobretudo, tomando- se em conta o trauma, o medo generalizado e o estresse inerentes aos efeitos desse tipo de even- to 6. Estudos apontam que aproximadamente 75% dos indivíduos expostos a contaminações por acidentes nucleares apresentam alguma for- ma de sintomas psicológicos, desde a incapaci- dade de dormir à dificuldade de concentração e isolamento social. Entre aqueles com maior risco de efeitos significativos encontram-se as crianças, mulheres (grávidas e mães de crianças pequenas), idosos e pessoas com um histórico de transtorno psiquiátrico 7,8. Além disso, os indi- víduos expostos têm uma alta taxa de transtorno do estresse pós-traumático (TEPT), o que cola- bora para a percepção de que alguns aspectos re- lacionados à qualidade de vida estão afetados 9. Cad. Saúde Pública, Rio de Janeiro, 29(7):1301-1310, jul, 2013 Métodos Trata-se de um estudo observacional transver- sal realizado no período de janeiro a agosto de 2011, cuja população-alvo foi composta por indivíduos expostos à radiação pelo césio-137, na cidade de Goiânia, devidamente cadastrados no C.A.R.A., unidade da Secretaria de Estado da Saúde de Goiás. Desde a época do acidente radioativo, os pa- cientes cadastrados no C.A.R.A. foram categori- zados por grupos (I, II e III) de acordo com as normas da International Atomic Energy Agency (IAEA), levando-se em conta critérios de classi- ficação conforme a gravidade das lesões cutâne- as e da intensidade de contaminação interna e externa. O Grupo I é composto por indivíduos com radiodermites e/ou dosimetria citogenética acima de 20rads. O II é formado por indivíduos com dosimetria citogenética ≤ 20rads. O Grupo III é constituído por agentes públicos, voluntá- rios, familiares das vítimas e vizinhos 2,3. Nesse contexto, a qualidade de vida é entendi- da a partir de novos paradigmas que têm influen- ciado as práticas do setor saúde, levando-se em conta que o processo saúde/doença é complexo e abrange aspectos econômicos, socioculturais, experiência pessoal e estilos de vida. De acordo com esses novos aspectos, a melhoria da quali- dade de vida passa a ser um dos resultados espe- rados pelas práticas assistenciais promovidas por todas as iniciativas de saúde que movem esforços Foram incluídos os sujeitos dos Grupos I e II com mais de 18 anos. Excluiu-se os indivíduos QUALIDADE DE VIDA DOS INDIVÍDUOS EXPOSTOS AO CÉSIO-137 1303 software desenvolvido para informatizar e orga- nizar o monitoramento e acompanhamento das vítimas do acidente 3. As variáveis analisadas fo- ram idade, sexo, grau de escolaridade e religião. Para a análise tomou-se por base duas faixas etá- rias (18 a 40 anos e 41 ou mais), de acordo com o protocolo clínico/laboratorial de monitoramen- to desses indivíduos, definido pela IAEA, que di- vide os mesmos nestas duas faixas etárias para a realização dos procedimentos estabelecidos. com prejuízo cognitivo e os integrantes do Grupo III, em razão de que a maioria destes não foi mo- nitorada na época do acidente, portanto há pou- cos registros da dosimetria correspondente ao nível de exposição ao césio-137 3. Ainda, o Grupo III encontra-se em processo de cadastramento no C.A.R.A., na medida em que consegue o re- conhecimento (seja administrativo ou judicial) de seu direito de receber os benefícios (pensões) concedidos por força de lei 3. Métodos A análise estatística foi realizada pelo pro- grama SPSS for Windows, versão 15.0 (SPSS Inc., Chicago, Estados Unidos). Foi empregada a sin- taxe WHOQOL-BREF no SPSS para avaliação dos escores do instrumento de qualidade de vida e posteriores correlações, baseando-se no coefi- ciente de Pearson. Para a análise da comparação das variáveis sociodemográficas e os domínios do WHOQOL-BREF foi utilizado o teste t de Stu- dent e Anova para os dados normais. Para verifi- car a frequência das variáveis sexo e idade com os domínios do WHOQOL-BREF foi utilizado o teste qui-quadrado. Considerou-se como nível de significância o valor de 5% (p < 0,05). Inicialmente, os indivíduos dos Grupos I e II foram convidados por carta-convite e conta- tos telefônicos para participarem do estudo. Do total de 111 sujeitos que atendiam aos critérios de inclusão, apenas 62 aceitaram participar da pesquisa (Grupo I n = 33; Grupo II n = 29). Após essa etapa, o questionário foi autoaplicado sob a orientação da pesquisadora responsável, em encontros realizados nas residências dos sujeitos ou nas dependências do C.A.R.A. O instrumento de coleta de dados para ava- liar a qualidade de vida foi o questionário World Health Organization Quality of Life (WHOQOL- BREF), desenvolvido pela Organização Mundial da Saúde (OMS) e validado no Brasil por Fleck et al. 10. Contém 26 perguntas, sendo duas questões gerais de qualidade de vida – a primeira refere-se à autopercepção da qualidade de vida e a ou- tra sobre a satisfação com a saúde. As demais 24 questões são distribuídas em quatro domínios: físico, psicológico, relações sociais e meio am- biente. Cada domínio é representado por facetas e suas perguntas foram formuladas para uma escala de respostas do tipo Likert (1 a 5), que é um tipo de escala de resposta psicométrica usa- da habitualmente em questionários, mais uti- lizada em pesquisas de opinião. Nessa escala é solicitado aos entrevistados que indiquem o seu grau de concordância ou discordância com uma informação que está sendo medida. Atribui-se valores numéricos e/ou sinais às respostas para refletir a força e a direção da reação do entrevis- tado à declaração. As declarações de concordân- cia devem receber valores positivos ou altos, e as declarações das quais discordam devem receber valores negativos ou baixos. A pontuação total da atitude de cada respondente é dada pela so- matória das pontuações obtidas para cada afir- mação 16,17. Métodos Este trabalho foi aprovado pelo Comitê de Ética em Pesquisa Dr. Henrique Santillo da Secre- taria de Estado da Saúde de Goiás, sob protocolo no 0016.0.177.000-10. Cad. Saúde Pública, Rio de Janeiro, 29(7):1301-1310, jul, 2013 Resultados Aspecto social Grupo não investigado Grupo investigado Grupo I (n = 29) Grupo II (n = 20) Valor de p Grupo I (n = 33) Grupo II (n = 29) Valor de p n % n % n % n % Sexo Masculino 18 62,1 9 45,0 18 54,5 14 48,3 Feminino 11 37,9 11 55,0 0,187 15 45,5 15 51,7 0,799 * Idade (anos) 18 |–| 40 11 37,9 13 65,0 14 42,4 17 58,6 > 41 18 62,1 7 35,0 0,058 19 57,6 12 46,4 0,309 ** Escolaridade Ensino Fundamental 17 58,6 7 35,0 9 36,0 15 51,7 Ensino Médio 8 27,6 12 60,0 11 44,0 12 41,4 Ensino Superior 4 13,8 1 5,0 0,071 5 20,0 2 6,9 0,047 * Religião Não possui 3 10,3 1 5,0 6 18,2 2 6,9 Católico 5 17,2 6 30,0 18 54,5 19 65,5 Espírita 6 20,7 13 65,0 1 3,0 1 3,4 Evangélico 15 51,7 0 0,0 0,001 8 24,2 7 24,1 0,606 * * Teste qui-quadrado; ** Teste exato de Fisher. meio ambiente indicou a média de escores mais alta e o domínio psicológico a menor pontuação. Quando analisamos os dois grupos de forma ge- ral, verificou-se que o domínio psicológico obte- ve a menor média e o domínio meio ambiente a maior média. II apresentaram os maiores escores na primeira e os menores na segunda questão, como se segue: Grupo I com 61,36 e 50,00 e o Grupo II com 62,93 e 56,90, respectivamente. II apresentaram os maiores escores na primeira e os menores na segunda questão, como se segue: Grupo I com 61,36 e 50,00 e o Grupo II com 62,93 e 56,90, respectivamente. Dentre o universo de indivíduos que avalia- ram sua qualidade de vida de modo geral como boa/muito boa e ruim/muito ruim, consideran- do a média nos domínios maior que 70 e menor que 30, destaca-se que no Grupo I cerca de 50% eram homens e 50% mulheres, também apre- sentaram o mesmo porcentual nas duas faixas etárias (18-40 anos e 41 ou mais). Não houve predominância de algum grau de escolaridade e religião. Já no Grupo II a maioria era de homens e com idades entre 18 a 40 anos. Também, as va- riáveis escolaridade e religião encontram-se pul- verizadas. Resultados A população analisada foi composta por 62 sujei- tos, correspondendo a 56% do universo de radio- acidentados cadastrados no C.A.R.A, distribuídos por Grupos I e II. Do total de 111 indivíduos, 49 se negaram a participar da pesquisa, como pode ser observado na Tabela 1. Verificou-se que na comparação da distribuição de todas as variáveis analisadas no grupo não investigado houve dife- rença significativa na variável religião (p = 0,001) e no grupo investigado na variável escolaridade (0,047). Os resultados sobre a percepção da qualidade de vida relacionada às duas questões genéricas do WHOQOL-BREF apresentaram na primeira questão – em que os indivíduos avaliaram a pró- pria qualidade de vida – 44,9% como “boa”; 6,5% “muito boa”; e 9,5% “ruim” e “muito ruim”. Des- ses, 39,9% responderam como “nem ruim, nem boa”. Quanto à segunda questão, que mensura a satisfação dos sujeitos em relação à própria saúde, 35,4% disseram estar “satisfeitos”; 11,4% “muito satisfeitos”; e 37,7% consideravam-se “insatisfeitos” e “muito insatisfeitos”. O restante, 16,4%, disse estar “nem satisfeito, nem insatisfei- to”. Ao separar os grupos, observou-se na média geral de cada questão que tanto o Grupo I como o O valor mínimo dos escores de cada domínio do WHOQOL-BREF é zero e o máximo é 100. O escore de cada domínio é obtido em uma escala positiva, isto é, quanto mais alto o escore, melhor a qualidade de vida naquele domínio 11,18. Para obtenção das informações sociodemo- gráficas utilizou-se o banco de dados do Siste- ma de Monitoramento dos Radioacidentados (SISRAD) disponível no C.A.R.A. O SISRAD é um Fuini SC et al. 1304 Distribuição das variáveis de acordo com cada aspecto social por Grupos (I e II) na população de radioacidentados, nos grupos não investigado e investigado. Goiânia, Goiás, Brasil, 2011. Distribuição das variáveis de acordo com cada aspecto social por Grupos (I e II) na população de radioacidentados, nos grupos não investigado e investigado. Goiânia, Goiás, Brasil, 2011. Cad. Saúde Pública, Rio de Janeiro, 29(7):1301-1310, jul, 2013 Resultados Nesses dois grupos, as menores mé- dias (< 30) concentraram-se nos domínios físico e psicológico e as maiores (> 70) nos domínios relações sociais e meio ambiente, com predomi- nância neste último. A Tabela 3 mostra a comparação dos dados gerais separados por duas faixas etárias de 18 a 40 anos e com mais de 41. Os resultados apresenta- ram diferença para a variável idade referente aos domínios físico, psicológico e relações sociais (p < 0,05). Somente o domínio meio ambiente não foi estatisticamente significativo (p > 0,05). A Tabela 3 mostra, ainda, que não houve di- ferença (p > 0,05) na variável sexo entre os domí- nios do WHOQOL-BREF. Apesar de não haver di- ferença, as mulheres apresentaram escores mais baixos em comparação aos homens em todos os domínios. Ao analisar a variável idade separada por gru- po, foi verificada diferença no Grupo I nas duas faixas etárias, referentes aos domínios psicológi- co (p = 0,017) e relações sociais (p = 0,004). Quan- to à variável sexo separada por grupo, constatou- se que não foi significante em todos os domínios, cujos escores ficaram assim representados nos Grupos I e II, respectivamente: domínio físico 52,86/53,81; domínio psicológico 50,56/53,06; A Tabela 2 mostra os valores da distribuição dos índices de qualidade de vida em cada do- mínio do WHOQOL-BREF, indicando que não houve diferença. Para o Grupo I, o domínio meio ambiente apresentou a maior média de escores, enquanto que a menor média foi para o domínio relações sociais. Quanto ao Grupo II, o domínio QUALIDADE DE VIDA DOS INDIVÍDUOS EXPOSTOS AO CÉSIO-137 1305 Tabela 2 Média e Desvio Padrão (DP) dos domínios do WHOQOL-BREF de acordo com o Grupo (I e II) e no geral, na amostra de indivíduos expostos ao césio-137. Goiânia, Goiás, Brasil, 2011. Resultados Domínios WHOQOL-BREF n Média DP Mínimo * Máximo ** Valor de p Físico Grupo I 33 53,68 16,06 17,86 82,14 Grupo II 29 55,91 17,45 25,00 92,86 Total 62 54,72 16,62 17,86 92,86 0,602 Psicológico Grupo I 33 50,88 17,61 12,50 87,50 Grupo II 29 55,46 18,40 25,00 87,50 Total 62 53,02 17,98 12,50 87,50 0,322 Relações sociais Grupo I 33 49,75 21,60 0,00 83,33 Grupo II 29 58,05 17,74 16,67 83,33 Total 62 53,63 20,17 0,00 83,33 0,106 Meio ambiente Grupo I 33 61,27 19,75 25,00 93,75 Grupo II 29 58,30 21,33 15,63 100,00 Total 62 59,88 20,39 15,63 100,00 0,571 Teste t de Student. * Mínimo: 0; ** Máximo: 100. Média e Desvio Padrão (DP) dos domínios do WHOQOL-BREF de acordo com o Grupo (I e II) e no geral, na amostra de indivíduos expostos ao césio-137. Goiânia, Goiás, Brasil, 2011. Esses resultados são consistentes com os apresentados por Miranda et al. 19 em um estudo constituído por um follow up da população de radioacidentados de Goiânia em dois períodos (3 e 15 anos após o evento), cujo objetivo foi anali- sar a percepção das pessoas diretamente atingi- das sobre os efeitos do acidente. Os resultados apontaram que a percepção de problemas psico- lógicos e de saúde geral permaneceu elevada nos dois períodos estudados, tanto para os indivídu- os do Grupo I como para os do Grupo II. domínio relações sociais 46,67/55,56; domínio meio ambiente 59,79/51,88). A Tabela 4 mostra a correlação entre os domí- nios do WHOQOL-BREF. Verifica-se que todos os domínios foram significativos e apresentaram re- lação moderada. Os maiores coeficientes foram encontrados nos domínios psicológico e físico. O domínio das relações sociais apresentou os coe- ficientes de correlação mais baixos. Cad. Saúde Pública, Rio de Janeiro, 29(7):1301-1310, jul, 2013 Discussão Os resultados encontrados no presente tra- balho são semelhantes aos verificados na po- pulação envolvida no acidente de Chernobyl, Ucrânia. Havenaar et al. 9, em um levantamen- to epidemiológico realizado após o desastre de Chernobyl, revelaram que os envolvidos tive- ram substancialmente maiores taxas de queixas de saúde e sofrimento psíquico do que os habi- tantes de uma região não afetada. Além disso, Rahu 20 ressaltou que o desastre de Chernobyl teve um impacto de longo prazo sobre o bem- estar físico e psicológico, doenças e na qualida- de de vida das pessoas envolvidas. Os resultados apresentados pelos indivíduos ex- postos ao césio-137 no questionário WHOQOL- BREF, considerando uma escala de 0 a 100, apre- sentaram, de forma crescente, para os domínios psicológico, relações sociais, físico e meio am- biente valores entre 53,02 e 59,88. Sendo que, as pessoas com mais de 41 anos, mulheres e os sujeitos do Grupo I, indicaram menores escores, principalmente quanto à avaliação geral da pró- pria saúde e nos domínios psicológico e relações sociais. Fuini SC et al. 1306 Tabela 3 Tabela 3 Comparação dos domínios do WHOQOL-BREF em relação ao sexo e faixa etária na amostra de indivíduos expostos ao césio-137. Goiânia, Goiás, Brasil, 2011. Domínios WHOQOL-BREF n Média Valor de p Sexo Físico Masculino 32 56,03 Feminino 30 53,33 Total 62 54,72 0,528 Psicológico Masculino 32 54,17 Feminino 30 51,81 Total 62 53,02 0,610 Relações sociais Masculino 32 55,99 Feminino 30 51,11 Total 62 53,63 0,345 Meio ambiente Masculino 32 63,67 Feminino 30 55,84 Total 62 59,88 0,131 Idade (anos) Físico 18-40 32 59,68 41 ou mais 30 49,77 Total 62 54,72 0,018 Psicológico 18-40 32 59,81 41 ou mais 30 46,24 Total 62 53,02 0,002 Relações sociais 18-40 32 61,56 41 ou mais 30 45,70 Total 62 53,63 0,001 Meio ambiente 18-40 32 63,81 41 ou mais 30 55,95 Total 62 59,88 0,130 Teste t Student. Comparação dos domínios do WHOQOL-BREF em relação ao sexo e faixa etária na amostra de indivíduos expostos ao césio-137. Goiânia, Goiás, Brasil, 2011. das pessoas, assim como ao estigma e à discrimi- nação. Nesse contexto, o acidente ocorrido em Goiânia constituiu-se em um evento bastante negativo, gerando, enquanto evento social, as- pectos relacionados à segregação, desorgani- zação e migração. Ao mesmo tempo, enquanto evento psicológico, o choque, o estresse, o medo e o trauma 13,15,19. Cad. Saúde Pública, Rio de Janeiro, 29(7):1301-1310, jul, 2013 Discussão Especificamente quanto aos menores esco- res apresentados neste estudo referentes aos do- mínios psicológicos e relações sociais, sabe-se que nos últimos anos os acidentes com subs- tâncias radioativas trouxeram com eles graves efeitos psicossociais, que em combinação com outros fatores produziram sérios impactos nas populações atingidas. Essas consequências são bastante complexas pelo fato de não estarem re- lacionadas somente com o acidente em si, mas aos efeitos de deslocamento e remanejamento Com relação ao aspecto físico, estudos ante- riores a respeito da população de radioaciden- tados pelo césio-137 em Goiânia demonstraram QUALIDADE DE VIDA DOS INDIVÍDUOS EXPOSTOS AO CÉSIO-137 1307 Tabela 4 Correlação entre os domínios do WHOQOL-BREF na amostra de indivíduos expostos ao césio-137. Goiânia, Goiás, Brasil, 2011. Domínios WHOQOL-BREF Psicológico Relações Sociais Meio Ambiente Físico r 0,738 0,712 0,736 p < 0,001 < 0,001 < 0,001 Psicológico r - 0,737 0,638 p - < 0,001 < 0,001 Relações Sociais r - - 0,617 p - - < 0,001 Teste Pearson. p: diferem signifi cativamente para p < 0,001; r: relação. Correlação entre os domínios do WHOQOL-BREF na amostra de indivíduos expostos ao césio-137. Goiânia, Goiás, Brasil, Correlação entre os domínios do WHOQOL-BREF na amostra de indivíduos expostos ao césio-137. Goiânia, Goiás, Brasil, 2011. formas mais intrapessoais, como a reflexão e o isolamento social 21,22. que o impacto na saúde física desta população não foi significativo 3,5. É provável que os impac- tos negativos da exposição à radiação foram evi- denciados na percepção dos indivíduos quan- to à sua qualidade de vida e permaneceram ao longo do tempo. Segundo Miranda et al. 19, os problemas de saúde física relatados pelos radio- acidentados de Goiânia não se referem direta- mente aos efeitos esperados em acidentes dessa natureza, mas atuam de forma indireta entre o aspecto orgânico e o mental, por meio de sinto- mas somáticos. No entanto, apesar do instrumento WHOQOL-BREF requerer uma autoavaliação considerando as duas últimas semanas da da- ta de preenchimento do questionário, deve-se considerar o fato de que na época do acidente ra- dioativo de Goiânia alguns eram crianças e ado- lescentes. Nesse aspecto, Compas et al. Cad. Saúde Pública, Rio de Janeiro, 29(7):1301-1310, jul, 2013 Discussão 1308 bastante complexas pelo fato de não estarem re- lacionadas somente com o acidente em si, mas por preocupações com doenças físicas em longo prazo e pelo impacto decorrente de deslocamen- tos, segregação, estigma e discriminação 8,19,29. que são mães e por isto preocupam-se com os fi- lhos 8,25. A vertente de gênero tem sido identifica- da como uma questão bastante relacionada com a avaliação da qualidade de vida; especialmente nos casos de eventos traumáticos pode-se veri- ficar que as mulheres geralmente desenvolvem doenças e apresentam prejuízo na saúde mental mais do que os homens 9,25. Nessa perspectiva, uma das principais carac- terísticas do acidente com o césio-137 é que ele não fazia parte da experiência habitual e carre- gava uma enorme capacidade para gerar pressão física, econômica, social e psicológica sobre os envolvidos. Sendo assim, os resultados demons- traram que as questões preocupantes no início do acidente ainda perduram no presente e no futuro das pessoas afetadas. Essa constatação corrobora com os resultados de outros estudos, confirmando que pelo fato de se tratar de um acidente radioativo, a condição de vítimas per- manece, tornando-se crônica 19,20,29. Em outro estudo de avaliação da qualidade de vida com pessoas expostas a doses baixas e prolongadas pela radiação ionizante por Cobalto 60 em uma região da cidade de Taiwan, Repúbli- ca da China, onde foi utilizado o WHOQOL-BREF, observou-se que os indivíduos mais jovens, as mulheres e as pessoas com maior tempo de ex- posição apresentaram os menores escores em to- dos os domínios, com exceção do domínio meio ambiente 26. Esses resultados são semelhantes aos do presente estudo, excetuando-se a variável idade em que as pessoas mais velhas tiveram os escores mais baixos. Talvez isso se deva ao fato de que o acidente de Goiânia foi ocasionado por características bem diferentes em comparação ao de Taiwan, pois aconteceu de forma mais trau- mática, repentina e impactante e, por sua vez, foi mais presenciado e experimentado pelas pessoas que hoje estão com mais de 41 anos. Dentre as limitações deste estudo pode-se destacar a preferência de alguns radioaciden- tados em se manter na condição de “vítimas”, o que pode ter contribuído para uma tendência em uma pior avaliação da qualidade de vida. Discussão 23 apon- tam a necessidade de alterações para aplicar as noções de estresse e coping para as crianças, já que precisam ser consideradas a dependência da criança em relação ao adulto e as caracterís- ticas básicas do seu desenvolvimento cognitivo e social, pois a idade e as pessoas envolvidas na situação estressante são fatores determinantes na escolha da estratégia a ser utilizada. Geral- mente, as estratégias utilizadas por crianças e adolescentes estão mais relacionadas às respos- tas correspondentes às demandas situacionais, procurando uma adaptação ao ambiente social, indicando que coping é um processo mais si- tuacional do que disposicional, apesar de que não existem respostas adaptativas universais, adequadas para todos os indivíduos, em todas as situações e em todo o tempo 24. Ainda no presente estudo, observou-se que os maiores escores concentraram-se no domínio meio ambiente, talvez porque os aspectos que influenciam uma maior satisfação podem estar relacionados com a salubridade no ambiente fí- sico (clima, ruído, poluição), o que coincide com a maioria das respostas dos sujeitos. Sabe-se que as estratégias de coping (estilo de enfrentamento) são definidas como um con- junto de esforços, cognitivos e comportamentais, utilizado pelos indivíduos com o objetivo de lidar com demandas específicas, internas ou exter- nas, que surgem em situações de estresse e são avaliadas como sobrecarregando ou excedendo seus recursos pessoais. Nesta pesquisa a variável idade apresentou um considerável índice de sig- nificância, mostrando que as pessoas com mais de 41 anos autoavaliaram sua qualidade de vi- da com escores menores do que as mais jovens. Alguns estudos têm sugerido que os padrões de coping estão associados à idade, ou seja, pessoas mais jovens usam formas de coping mais intera- tivas, tais como a expressão de emoções e a busca de informações, enquanto as mais velhas usam Considerando os aspectos relacionados ao gênero, foi observado neste estudo que a percep- ção da qualidade de vida para as mulheres foi inferior à dos homens em todas as faixas etárias, nos dois grupos e em todos os domínios, exce- tuando-se o domínio físico, porém não signifi- cativas. Pesquisas têm revelado que as mulheres tendem ao sofrimento psíquico mais do que os homens em situações relacionadas com a saúde e questões sociais, com destaque para aquelas Fuini SC et al. Discussão Isso se justifica, tendo em consideração que os radio- acidentados pelo césio-137 de Goiânia sofrem as consequências da violência provocada pelo imprevisível e inesperado, bem como os efeitos prolongados resultantes de acidentes com subs- tâncias radioativas. Geralmente esse efeito se ca- racteriza pela ameaça crônica e exacerbada por acidentes dessa natureza, gerando necessidades de suporte em áreas de subsistência de suas vi- das, tais como: econômica, social, saúde física e mental 17. Sendo assim, manter a condição de “vítima” pode significar a garantia da sociedade e do governo em gerar respostas a essas neces- sidades para que as dificuldades sentidas sejam superadas e/ou minimizadas. Como foi demonstrado, a avaliação dos es- cores médios dos domínios de qualidade de vi- da mensurados nesta pesquisa estiveram entre 49,75 e 61,27. Apesar de não terem sido encon- trados estudos em populações semelhantes re- alizados no Brasil, torna-se interessante compa- rar com trabalhos que utilizaram o instrumento WHOQOL-BREF em populações diversas. Entre eles destaca-se um estudo sobre a Qualidade de Vida e Trauma Psíquico em Vítimas da Violência por Arma de Fogo, cujos resultados demonstra- ram médias baixas que variaram entre 44,71 e 52,84, em que o escore médio do domínio meio ambiente foi menor que o dos domínios físico, relações sociais e psicológico 27. Outra limitação foi a dificuldade de conven- cimento dos radioacidentados, pois, a maioria manifestou revolta com os serviços públicos oferecidos e que não mais serviria de “cobaias” de pesquisa, se negando de forma agressiva a participar do presente estudo. Nesse caso, foi ne- cessário uma articulação com a Associação das Vítimas do Césio-137 (AVCESIO) para ajudar na adesão dos mesmos, até alcançar o quantitativo mínimo do cálculo amostral. Outro trabalho que avaliou a Percepção de Qualidade de Vida de Pessoas com Lesão Medu- lar Traumática, apresentou escores entre 49,61 e 55,81, sendo o menor escore representado no domínio meio ambiente e o maior no domínio relações sociais 28. Percebe-se nos estudos citados que os me- nores escores concentraram no domínio meio ambiente, contrariando os resultados da presen- te pesquisa, cujo escore mais alto foi nesse domí- nio. Cad. Saúde Pública, Rio de Janeiro, 29(7):1301-1310, jul, 2013 Discussão Talvez isso se deva aos efeitos comumente chamados “indiretos”, os quais podem ser cau- sados por desastres sem ter uma relação direta com o evento, como é o caso dos impactos psi- cológicos e sociais, sobretudo em eventos com substâncias radioativas, cujas consequências são Concluindo, os resultados deste estudo mos- traram que os indivíduos expostos à radiação pe- lo césio-137 sofrem considerável impacto na sua qualidade de vida. Observou-se a persistência de problemas psicossociais, na medida em que, os menores escores foram associados ao domínio psicológico, seguido de relações sociais, sobretu- do para os indivíduos do Grupo I, as mulheres e pessoas com mais de 41 anos. Ou seja, enquanto evento psicossocial, esse acidente pode ter pro- QUALIDADE DE VIDA DOS INDIVÍDUOS EXPOSTOS AO CÉSIO-137 1309 duzido trauma, medo, estresse, choque, entre outras possíveis reações, até os dias de hoje. Os resultados apontados mostraram que não houve associação ente as variáveis sociodemográficas (religião e escolaridade) com os domínios do WHOQOL-BREF. condizentes e influenciar as decisões e condutas terapêuticas, voltadas a esse segmento da po- pulação, incluindo o fortalecimento da atenção psicossocial no intuito de reduzir o sofrimento das mesmas. Faz-se necessário, também, a realização de novas pesquisas, principalmente porque são muito escassos os estudos que tratam das conse- quências indiretas em populações de vítimas de acidentes com substâncias radioativas. Enfim, há muito o que se fazer para responder efetivamen- te às demandas das pessoas que sofrem com os efeitos da radiação ou, pelo menos, minimizar a potencialidade do desenvolvimento de efeitos futuros. Contudo, mesmo com a manutenção regular do monitoramento dos indivíduos dos Grupos I e II em Goiânia, verifica-se ainda a existência de incertezas quanto aos efeitos do acidente, pois essas dúvidas desempenham um papel negativo na saúde e no bem-estar dessas pessoas. Espera- se com este trabalho que a compreensão sobre a qualidade de vida dos radioacidentados pos- sa subsidiar a elaboração de políticas públicas Resumen Estudio transversal observacional realizado en Goiâ- nia, Goiás, Brasil, con un 56% de 111 individuos para evaluar la calidad de vida de las personas expuestas al cesio-137 y su asociación con factores sociodemo- gráficos. Se utilizó el WHOQOL-BREF y el Sistema de Monitoreo de Radio accidentados. Los participantes fueron divididos: Grupo I - Dosimetría radiodermitis y/o citogenética mayor de 20 rads (n = 33) y Grupo II - dosimetría citogenética ≤ 20 rad (n = 29), de un total de 62 sujetos. Entre los dominios del WHOQOL-BREF, el medio ambiente tenía los puntos más altos de prome- dio (59,88; SD = 20,39) y el promedio más bajo fue el psicológico (53,02; SD = 17,98). Las asociaciones entre las relaciones físicas, psicológicas y sociales fueron sig- nificativas para la variable edad. No hubo diferencias entre los grupos y entre los factores sociodemográficos y de calidad de vida. Los radioaccidentados sufren un impacto considerable en la calidad de vida, con la per- sistencia de los problemas psicosociales, especialmente para aquellos con más de 41 años. S. C. Fuini contribuiu na concepção e design, aquisição de dados e análise e interpretação dos dados, redação do artigo e revisão crítica do conteúdo intelectual. R. Souto fez contribuições substanciais para a concepção e design, análise e interpretação dos dados, e revisão crítica do conteúdo intelectual do artigo. G. F. Amaral e R. G. Amaral fizeram contribuições substanciais para a concepção e design, análise e interpretação dos dados, revisão crítica do conteúdo intelectual e aprovação final da versão a ser publicada. Calidad de Vida; Cesio; Impacto Psicosocial; Accidentes Cad. Saúde Pública, Rio de Janeiro, 29(7):1301-1310, jul, 2013 Referências 17. Mattar FN. Pesquisa de marketing. Edição Compac- ta. 3a Ed. São Paulo: Editora Atlas; 2001. 1. Oliveira AR, Valverde NJL, Brandão-Mello CE, Almei- da CEV. Revisiting the Goiânia accident: medical and dosimetric aspects. Radiat Prot Dosimetry 1998; 77:107-11. 1. Oliveira AR, Valverde NJL, Brandão-Mello CE, Almei- da CEV. Revisiting the Goiânia accident: medical and dosimetric aspects. Radiat Prot Dosimetry 1998; 77:107-11. 18. Fleck MPA, Louzada S, Xavier M, Chachamovich E, Vieira G, Santos L, et al. Aplicação da versão em por- tuguês do instrumento abreviado de avaliação da qualidade de vida "WHOQOL-BREF". Rev Saúde Pú- blica 2000; 34:178-83. 2. International Atomic Energy Agency. The radiologi- cal accident in Goiânia. Vienna: International Atom- ic Energy Agency; 1988. 2. International Atomic Energy Agency. The radiologi- cal accident in Goiânia. Vienna: International Atom- ic Energy Agency; 1988. 3. Superintendência Leide das Neves Ferreira. Mo- nitoramento dos radioacidentados. http://www. cesio137goiania.go.gov.br/ (acessado em 20/Dez/ 2009). 19. Miranda FJ, Pasquali L, Costa Neto SBC, Barreto MQ, David Filho G, Rosa TV. Acidente radioativo de Goiânia: "O tempo cura todos os males?” Arq Bras Psicol 2005; 57:58-87. 3. Superintendência Leide das Neves Ferreira. Mo- nitoramento dos radioacidentados. http://www. cesio137goiania.go.gov.br/ (acessado em 20/Dez/ 2009). 4. Goiás. Lei no 17.430, de 05 de outubro de 2011. Cria as unidades administrativas complementares des- centralizadas de saúde que especifica, na Secretaria de Estado da Saúde de Goiás. Diário Oficial do Esta- do de Goiás 2011; 14 out. 20. Rahu M. Health effects; of the Chernobyl accident: fears, rumours and the truth. Eur J Cancer 2000; 39:295-9. 21. Folkman S. Coping across the life span: theoretical issues. In: Cummings EM, Greene AL, Karraker KH, editors. Life span developmental psychology: per- spectives on stress and coping. Hillsdale: Lawrence Erlbaum Associates; 1991. p. 3-19. 5. Koifman S. Os 20 anos do acidente do césio em Goiás – vigilância ambiental e os riscos para a saú- de. http://www.cesio137goiania.go.gov.br/index. php?idEditoria=3801 (acessado em 7/Nov/2009). 22. Baum A, Fleming R, Singer JE. Coping with victim- ization by technological disaster. J Soc Issues 1983; 39:117-38. 6. Walker RI, Cerveny RJ. Medical consequences of nuclear warfare. Falls Church: Office of the Surgeon General; 1989. 23. Compas BE, Banez GA, Malcarne V, Worsham N. Perceived control and coping with stress: a develop- mental perspective. J Soc Issues 1991; 47:23-34. 7. American Psychiatric Association. Diagnostic and stastistical manual of mental disorders. 4th Ed. Washington DC: American Psychiatric Press; 1994. 24. Cad. Saúde Pública, Rio de Janeiro, 29(7):1301-1310, jul, 2013 Agradecimentos À equipe do Centro de Assistência aos Radioacidenta- dos (C.A.R.A.), aos colegas do Centro de Ensino, Pesqui- sa e Projetos – Leide das Neves Ferreira (CEEPP-LNF) e à Associação das Vítimas do Césio-137, pelo auxílio durante a etapa de coleta de dados deste estudo. Cad. Saúde Pública, Rio de Janeiro, 29(7):1301-1310, jul, 2013 Fuini SC et al. 1310 Referências Referências Dell’Aglio DD, Hutz CS. Estratégias de coping de crianças e adolescentes em eventos estressantes com pares e adultos. Psicologia USP 2002; 13:203-25. 8. Bromet EJ, Semyon G, Joseph ES, Dmitry G. Somatic symptoms in women 11 years after the Chernob- yl accident: prevalence and risk factors. Environ Health Perspect 2002; 110 Suppl 4:625-9. 25. Fullerton CS, Ursano RJ. The other side of chaos: understanding the patterns of post-traumatic re- sponses. In: Fullerton CS, Ursano RJ, editors. Post- trumatic stress disorder: acute and long-term re- sponses to trauma and disaster. Washington DC: American Psychiatric Press; 1997. p. 3-18. 9. Havenaar J, Rumyantzeva G, Kasyanenko A, Kaa- suager K, Westermann N, Brink WVD, et al. Health effects of the Chernobyl disaster: illness or illness behavior: a comparative general health survey in two former Soviet Regions. Environ Health Perspect 1997; 105:1533-7. 26. Pimei NY, Chen-Chang Y, Peter WC, Jing-Shiang H, Hui-Chen L, Kuan-Liang K, et al. Perception of qual- ity of life of a cohort population years after reloca- tion from previous low-dose radiation exposure in Co-60 contaminated buildings in Taiwan. Int J Ra- diat Biol 2011; 87:453-60. 10. Fleck MPA. A avaliação da qualidade de vida: guia para profissionais de saúde. Porto Alegre: Editora Artmed; 2008; 11. The WHOQOL Group. Development of the World Health Organization WHOQOL-BREF Quality of life assessment. Psychol Med 1998; 28:551-8. 27. Mello-Silva ACC, Brasil VV, Minamisava R, Olivei- ra LMAC, Cordeiro JABL, Barbosa MA. Qualidade de vida e trauma psíquico em vítimas da violência por arma de fogo. Texto Contexto-Enferm 2012; 21: 558-65. 12. Seidl EMF, Zannon CMLC. Qualidade de vida e saúde: aspectos conceituais e metodológicos. Cad Saúde Pública 2004; 20:580-8. 13. Leon GR. Special issue: overview of the psychoso- cial impact of disasters. Prehosp Disaster Med 2011; 26:1-127. 28. Bampi LNS, Guilhem D, Lima DD. Qualidade de vi- da em pessoas com lesão medular traumática: um estudo com o WHOQOL-BREF. Rev Bras Epidemiol 2008; 11:67-77. 14. Likh IA, Kovgan LN, Jabob P, Anspaugh LP. Cher- nobyl accident retrospective and prospective esti- mates of external dose of the population of Ukraine. Health Phys 2002; 3:290-303. 29. Loganovsky KN, Loganovoskaya TK. Shizophrenia spectrum disorders in persons exposed to ioniz- ing radiation as a result of the Chernobyl accident. Shizophr Bull 2000; 26:751-73. 15. Helou S, Costa Neto SB. Conseqüências psicossoci- ais do acidente de Goiânia. Goiânia: Editora UFG; 1995. Referências Recebido em 17/Set/2012 Versão final reapresentada em 08/Fev/2013 Aprovado em 11/Mar/2013 Recebido em 17/Set/2012 Versão final reapresentada em 08/Fev/2013 Aprovado em 11/Mar/2013 16. Backer P. Gestão ambiental: administração verde. Rio de Janeiro: Qualitymark; 1995. 16. Backer P. Gestão ambiental: administração verde. Rio de Janeiro: Qualitymark; 1995. Cad. Saúde Pública, Rio de Janeiro, 29(7):1301-1310, jul, 2013
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Control region sequences indicate that multiple externae represent multiple infections by Sacculina carcini (Cirripedia: Rhizocephala)
Ecology and evolution
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university of copenhagen university of copenhagen Control region sequences indicate that multiple externae represent multiple infections by Sacculina carcini (Cirripedia: Rhizocephala) university of copenhagen Control region sequences indicate that multiple externae represent multiple infections by Sacculina carcini (Cirripedia: Rhizocephala) Citation for published version (APA): Rees, D., & Glenner, H. (2014). Control region sequences indicate that multiple externae represent multiple infections by Sacculina carcini (Cirripedia: Rhizocephala). Ecology and Evolution, 4(16), 3290-3297. https://doi.org/10.1002/ece3.1177 Download date: 24. Oct. 2024 ª 2014 The Authors. Ecology and Evolution published by John Wiley & Sons Ltd. This is an open access article under the terms of the Creative Commons Attribution License, which permits use, distribution and reproduction in any medium, provided the original work is properly cited. Control region sequences indicate that multiple externae represent multiple infections by Sacculina carcini (Cirripedia: Rhizocephala) Rees, David; Glenner, Henrik Document version Publisher's PDF, also known as Version of record Citation for published version (APA): Rees, D., & Glenner, H. (2014). Control region sequences indicate that multiple externae represent multiple infections by Sacculina carcini (Cirripedia: Rhizocephala). Ecology and Evolution, 4(16), 3290-3297. https://doi.org/10.1002/ece3.1177 Download date: 24. Oct. 2024 Funding Information Funding Information The study was finaced by Carlsberg Foundation (grant no. 2008-01-0491). Received: 17 January 2014; Revised: 4 July 2014; Accepted: 5 July 2014 Ecology and Evolution 2014; 4(16): 3290– 3297 Ecology and Evolution 2014; 4(16): 3290– 3297 doi: 10.1002/ece3.1177 2001; for further details of the development of Sacculina carcini and its morphological and behavioral impacts on Carcinus maenas see, e.g., Kristensen et al. 2012; Høeg 1995; Høeg and Lutzen 1995). Control region sequences indicate that multiple externae represent multiple infections by Sacculina carcini (Cirripedia: Rhizocephala) 1Department of Biology, Marine Biodiversity, University of Bergen, Thormøhlensgate 53a, Box 7800, Bergen N-5020, Norway 2Center for Macroecology, Evolution and Climate, University of Copenhagen, Universitetsparken 15, 2100 Copenhagen, Denmark Correspondence Henrik Glenner Correspondence Henrik Glenner Department of Biology, Marine Biodiversity, University of Bergen, Thormøhlensgate 53a, Box 7800, Bergen N-5020, Norway. Tel: +47 45 86 46 47; Fax: +47 55 58 44 00; E-mail: henrik.glenner@bio.uib.no Funding Information The study was finaced by Carlsberg Foundation (grant no. 2008-01-0491). Keywords y Carcinus maenas, control region, mitochondrial DNA, parasitism, population genetics, rhizocephala. Carcinus maenas, control region, mitochondrial DNA, parasitism, population genetics, rhizocephala. The rhizocephalan barnacle, Sacculina carcini, is a common parasite of the European shore crab, Carcinus maenas, in which it causes significant detrimen- tal physical and behavioral modifications. In the vast majority of cases, the external portion of the parasite is present in the form of a single sac-like exter- na; in rare cases, double or even triple externae may occur on the same individ- ual host. Here, we use a highly variable DNA marker, the mitochondrial control region (CR), to investigate whether multiple externae in S. carcini rep- resent infection by multiple parasites or asexual cloning developed by a single parasite individual. Sequences for multiple externae from C. maenas hosts from the Danish inlet, Limfjorden, and from the mud flates at Roscoff, France, were compared. In almost all cases, double or triple externae from an individual host yielded different haplotypes. In the few cases where identical haplotypes were identified from externae on a multiple-infected host, this always represented the most commonly found haplotype in the population. This indicates that in Sac- culina carcini, the presence of multiple externae on a single host reflects infec- tion by different individual parasites. A haplotype network of CR sequences also suggests a degree of geographical partitioning, with no shared haplotypes between the Limfjorden and Roscoff. Our data represent the first complete CR sequences for a rhizocephalan, and a unique gene order was also revealed. Although the utility of CR sequences for population-level work must be investi- gated further, the CR has proved a simple to use and highly variable marker for studies of S. carcini and can easily be applied to a variety of studies in this important parasite. Introduction The rhizocephalan barnacle, Sacculina carcini, is a para- sitic castrator of the European shore crab, Carcinus mae- nas. The adult parasite consists of an external sac-like structure located at the ventral side of the host abdomen at a position where adult female crabs carry their eggs (see Fig. 1A, B). This sac, the externa, contains the repro- ductive organs of the parasite and communicates with an internal root-like structure, the interna, via a stalk that penetrates the abdominal cuticle of the host. The interna is an extensive structure that infiltrates most of the larger blood sinuses of the crab and serves as a trophic organ extracting nutrients from the crab hemolymph (Glenner Occasionally, more than one externa occurs on the same crab (Rainbow et al. 1979). This can be interpreted as either a double infection, where two individual parasite specimens are competing for the same food resource entailing that each externa is feeding from two indepen- dent internas (see Fig. 1C). Alternatively, the phenome- non can be regarded as asexual budding of two externae connected to, and feeding from, the same interna. Which of the two alternatives is in play is of crucial importance in order to understand the biology of the parasite and the 3290 Multiple Infections of C. maenas by S. carcini D. Rees & H. Glenner (A) (B) (C) Figure 1. Comparison of a gravid female crab carrying its egg m under the abdomen and mimicking rhizocephalan parasites situa at the same position. (A) Gravid female Carcinus maenas; (B) fem C. maenas with a single mature S. carcini parasite; (C) m C. maenas with three recently emerged Sacculina carcini extern The abdomen has been considerable broadened (feminized) as ef of the presence of the parasites. (A) collection of an unprecedented number of crabs with mul- tiple externae. This allowed a comprehensive examination of whether the observed multiple externae were caused by a single infection, multiple infections, or a combination of both alternatives. Multiple externae are documented as being developed by clonal, or asexual reproduction, in one sacculinid genus, Polyascus, where multiple externae on the same host is the rule (Glenner et al. 2003). Introduction In other sacculi- nid genera, the presence of multiple externae on the same host is rare, but notable exceptions do exist, as in Heteros- accus dolfusi, which parasitizes the swimming crab, Charyb- dis longicollis, a Lessepsian invader of the Mediterranean sea (Galil and Lutzen 1995). In this parasite–host system, parasitized crabs predominantly have more than one exter- na and 3–5 per host is common. With an extraordinary prevalence of 60–90%, the chances of a cypris larva finding a host already occupied by another parasites are much higher than finding an un-parasitized host, and it is there- fore believed that the intensity observed in most cases is due to multiple infections – not asexual reproduction (Glenner and Hebsgaard 2006). However, since morpho- logical inspections of the internal parasite is unable to dis- tinguish the presence of more than one externa, the question of asexual budding or multiple infection can only be addressed by developing, and employing, high-resolu- tion molecular markers. (A) (B) (B) (C) (C) (C) Mitochondrial (mt) DNA is a popular marker for phylo- genetic, phylogeographic, and population genetic studies at a wide range of taxonomic and geographical scales. Notable benefits of mtDNA markers include relative ease of amplifi- cation, presence of variable regions flanked by conserved stretches suitable for primer design, and predominantly nonrecombinant inheritance. The mtDNA control region (CR) is a noncoding portion of the mt genome, responsible for replication and transcription, and is usually the fastest evolving mtDNA region in invertebrates (Avise 2000; Bil- lington 2003). The CR exhibits an evolutionary rate three- fold to fivefold higher than other regions of the mt genome (Brown et al. 1993), making it a popular marker for genetic studies involving a wide range of taxa. This marker has been widely utilized in vertebrates and insects but also been applied to studies of genetic variability and population structure in a number of commercially important marine crustaceans, for example, the swimming crab Portunus tri- tuberculatus (Guo et al. 2012), the scalloped lobster Panuli- rus homarus (Farhadi et al. 2013), and the mantis shrimp Oratosquilla oratoria (Lui et al. 2010). Figure 1. Comparison of a gravid female crab carrying its egg mass under the abdomen and mimicking rhizocephalan parasites situated at the same position. (A) Gravid female Carcinus maenas; (B) female C. maenas with a single mature S. carcini parasite; (C) male C. maenas with three recently emerged Sacculina carcini externae. ª 2014 The Authors. Ecology and Evolution published by John Wiley & Sons Ltd. Introduction The three most common haplotypes are also indicated, as in Fig. 2, by a, b, and c. 2007; Tsang et al. 2007; Dawson et al. 2010). No studies involving Rhizocephala, and no complete control region sequences for this group, have been published to date. The high variability associated with the CR made this an ideal candidate for our work involving Sacculina carcini, both for assessing population – and individual-level variation. An initial test of control region sequence data for Sacculina carcini, aimed at assessing suitability for population studies, indicated surprisingly high levels of variability. This pre- sented the opportunity to investigate another aspect of S. carcini biology: whether (rare) double and triple infec- tions (indicated by multiple externae) are the result of emergence by multiple individuals of S. carcini, or whether multiple externae can emerge from a single parasite. In this study, we present new primers and a simple method for tar- geting the CR in Sacculina carcini and examine DNA sequence variation in multiple externa from two disjunct populations. Externa Host No. Introduction Location Haplotype GenBank accession SA01 1 Roscoff R01 KF649275 SA02 2 Roscoff R02 KF649276 SA03 3 Roscoff R03 KF649277 SA04 4 Limfjorden L08 b KF649263 SA05 5 Limfjorden L17 c KF649272 SA06 6 Limfjorden L18 KF649273 TC01A 7 Limfjorden L17 c TC01B Limfjorden L09 KF649264 TC02A 8 Limfjorden L16 KF649271 TC02B Limfjorden L11 a KF649266 TC03A 9 Limfjorden L11 a TC03B Limfjorden L13 KF649268 TC04A 10 Limfjorden L06 KF649261 TC04B Limfjorden L01 KF649256 TC05A 11 Limfjorden L19 KF649274 TC05B Limfjorden L08 b TC06A 12 Limfjorden L11 a TC06B Limfjorden L08 b 502A 13 Roscoff R04 KF649278 502B Roscoff R05 KF649279 503A 14 Roscoff R06 KF649280 503B Roscoff R07 KF649281 504A 15 Roscoff R08 KF649282 504B Roscoff R09 KF649283 512A 16 Roscoff R10 KF649284 512B Roscoff R11 KF649285 513A 17 Roscoff R12 KF649286 513B Roscoff R13 KF649287 SAC11A 18 Limfjorden L05 KF649260 SAC12B Limfjorden L17 c SAC13C Limfjorden L11 a SAC14A 19 Limfjorden L14 KF649269 SAC15B Limfjorden L11 a SAC16C Limfjorden L03 KF649258 SAC17A 20 Limfjorden L17 c SAC18B Limfjorden L04 KF649259 SAC19C Limfjorden L15 KF649270 SAC20A 21 Limfjorden L01 SAC21B Limfjorden L08 b SAC22A 22 Limfjorden L17 c SAC23B Limfjorden L08 b SAC24A 23 Limfjorden L02 KF649257 SAC25B Limfjorden L08 b SAC26A 24 Limfjorden L10 KF649265 SAC27B Limfjorden L08 b SAC28A 25 Limfjorden L17 c SAC29B Limfjorden L17 c SAC30A 26 Limfjorden L07 KF649262 SAC31B Limfjorden L09 SAC32A 27 Limfjorden L17 c SAC33B Limfjorden L08 b Sample collection and DNA extraction Sacculina carcini externa were collected from the shore crab/green crab Carcinus maenas from Limfjorden, Den- mark, and from near Roscoff, north-west France, and pre- served in 96% ethanol prior to DNA extraction. Genomic DNA extraction was performed for a total of 57 externa comprising six single infections, 21 double, and three triple infections (double and triple infections being characterized by two or three externa from a single host specimen. Five double and three single externae were collected from C. maenas near Roscoff and the remainder (three triple, 16 double, and three single externae) came from Limfjorden (see the data accessibility section, Table 1 for sample details). Approximately 1 mm3 of mantle tissue was care- fully excised from individual Sacculina carcini externae for DNA extraction. Alternatively, eggs or nauplii larvae from the mantle cavity of the female externae were DNA extracted. Tissue from the receptacle region were carefully omitted to avoid DNA contamination from a dwarf male. All extractions were carried out using the Qiagen DNeasy Blood and Tissue kit (QIAGEN Inc., Valencia, CA, USA) or a GeneMole extraction robot, following the manufacturers’ standard protocols. Multiple parallel extractions were also performed for individual externa, along with PCR amplifi- cation and sequencing, to confirm reproducibility and specificity of DNA sequence data. Introduction The abdomen has been considerable broadened (feminized) as effect of the presence of the parasites. Figure 1. Comparison of a gravid female crab carrying its egg mass under the abdomen and mimicking rhizocephalan parasites situated at the same position. (A) Gravid female Carcinus maenas; (B) female C. maenas with a single mature S. carcini parasite; (C) male C. maenas with three recently emerged Sacculina carcini externae. The abdomen has been considerable broadened (feminized) as effect of the presence of the parasites. host–parasite interaction. Due to the delicacy of the interna, which consists of a dense network of extremely fine root extensions, it is impossible to distinguish morphologi- cally whether two externae are connected to a common interna (asexual externa budding), or to separate internas (independent specimens). As part of a 4-years, large-scale study on the population dynamics and biology of the Euro- pean shore crab, Carcinus maenas, in the Danish inlet Limf- jorden (see www.Carcinus.com), specimens with the rare occurrence of multiple externae were collected. The large quantity of crabs examined during the project allowed host–parasite interaction. Due to the delicacy of the interna, which consists of a dense network of extremely fine root extensions, it is impossible to distinguish morphologi- cally whether two externae are connected to a common interna (asexual externa budding), or to separate internas (independent specimens). As part of a 4-years, large-scale study on the population dynamics and biology of the Euro- pean shore crab, Carcinus maenas, in the Danish inlet Limf- jorden (see www.Carcinus.com), specimens with the rare occurrence of multiple externae were collected. The large quantity of crabs examined during the project allowed Use of CR sequences in published studies of Cirripedia is few, and its use seems to be limited mainly to population and taxonomic studies involving a small number of Tho- racican barnacles; Chthamalus stellatus (Sasson et al. 2012), Semibalanus balanoides (Flight et al. 2012), Catomerus poly- merus (York et al. 2008), and Tetraclita spp. (Chan et al. 3291 ª 2014 The Authors. Ecology and Evolution published by John Wiley & Sons Ltd. Multiple Infections of C. maenas by S. carcini D. Rees & H. Glenner D. Rees & H. Glenner Table 1. Sacculina carcini host, location, and haplotype information. Individual hosts are numbered and double or triple externa denoted by A/B/C. Associated haplotypes indicated for each externa and Gen- Bank accession numbers are listed for each unique haplotype. ª 2014 The Authors. Ecology and Evolution published by John Wiley & Sons Ltd. Sequence characteristics The final aligned dataset, comprising DNA sequence data for 57 individual externa, was 812 base-pair (bp) long. The presence of insertions and deletions (indels) resulted in unaligned individual sequence lengths ranging from 795 to 803 bp. BLAST searches confirmed partial matches to S. carcini 12S and COI genes (http://blast.ncbi.nlm.nih. gov/Blast.cgi) and subsequent alignment of the sequenced region with the complete mitochondrial genome sequence for S. carcini (unpublished data) confirmed the relative positions of the flanking regions and the presence of the putative control region. Annotation of the sequenced region using the MITOS package indicated that the sequenced region comprised approximately 175 bp of 12S and 35 bp of COI, flanking a 600-bp segment of noncoding A+T-rich sequence (84% A+T). Analyses also indicated that the noncoding region between the 12S and COI genes was split by one 56-bp tRNA (tRNA Cysteine; gca), resulting in a 145 bp of noncoding sequence adjacent to 12S and 400 bp adjacent to COI. The overall length of the amplified fragment was significantly shorter than expected (see below) but with primers located in conserved, coding flanking regions, we minimized potential problems with amplification (or nondetection) of nuclear mitochondrial copies (Numts) or CR paralogs (see, e.g., Walther et al. 2011). Comparison of our coding sequence data indicates complete congruence with those held in GenBank, as well as complete agreement for our total sequence reads with an independently generated full mitochondrial S. carcini gen- ome sequence (unpublished data). Analysis of this full mtDNA genome sequence identified all 13 protein coding genes, two rRNA genes, and 22 tRNA genes. sequences; the forward primer was designed from the single 12S rRNA sequence available in GenBank (AY520690), and the reverse primer was designed from an alignment of multiple GenBank cytochrome c oxidase 1 gene (COI) sequences. Primer design was performed using the Pri- mer3Plus web interface (Untergasser et al. (2007); http:// www.bioinformatics.nl/cgi-bin/primer3plus/primer3plus. cgi) and the following primers were selected: 12SF_Sacc (50-TGAATTCAGATTAGGTGCAAAGA-30) and COIR_ Sacc (50-CCCCCACTAAACCTGATCATA-30). PCR ampli- fications were carried out in 25 lL volumes containing 19 PCR buffer, 1.2-lL 2-mmol/L dNTPs, 0.4 lmol/L of each primer, 0.75 units of Takara polymerase, and 1 lL of tem- plate (and ddH2O up to 25 lL). Primer design and PCR Specific polymerase chain reaction (PCR) primers were designed from flanking regions using published S. carcini ª 2014 The Authors. Ecology and Evolution published by John Wiley & Sons Ltd. 3292 Multiple Infections of C. maenas by S. carcini D. Rees & H. Glenner Table 1. Continued. Externa Host No. Location Haplotype GenBank accession SAC34A 28 Limfjorden L11 a SAC35B Limfjorden L17 c SAC36A 29 Limfjorden L12 KF649267 SAC37B Limfjorden L17 c SAC38A 30 Limfjorden L17 c SAC39B Limfjorden L17 c Table 1. Continued. genome for S. carcini (unpubl. data Podsiadlowski , L., Hecht, J., Rees D., Noever, D., Glenner, H.) using the MI- TOS web server (Bernt et al. 2012; http://mitos.bioinf. uni-leipzig.de/). Sequence characteristics PCRs were performed on a Bio-Rad C1000 Thermal with the following cycling pro- file: initial denaturation at 94°C for 5 min, then 35 cycles of 94°C for 30 sec, annealing at 54°C for 30 sec, and exten- sion at 72°C for 2 min, followed by a final 72°C extension for 7 min. Amplification products were visualized on 1.5% agarose gels to confirm fragment size and quality. PCR purification was carried out by the addition of 1 unit each of exonuclease I and shrimp alkaline phosphatase (plus 0.9 lL ddH2O) to 8 lL of each reaction, with reactions subsequently heated to 37°C for 30 min and then 85°C for 15 min. In a small number of cases, PCR products were gel-purified using the Qiagen MinElute gel extraction kit, with the target band excised directly from the gel prior to sequencing. ª 2014 The Authors. Ecology and Evolution published by John Wiley & Sons Ltd. Sequencing and data analysis With our primers located in the 12S and CO1 genes, we had expected to amplify the NADH dehydrogenase subunit 2 (ND2) gene along with the control region, since the gene order for all Cirripedia for which data is avail- able (with one exception, see below), as well as for the majority of crustaceans, is 12S, CR, ND2, and CO1. This also applies to ancestral pancrustaceans and arthropods (Lavrov et al. 2004). However, our sequence data indi- cated that for Sacculina carcini, ND2 was not present in this position (and the fragment was therefore ~1 kb shorter than expected). Both strands of all PCR products were sequenced on an ABI 3730 capillary sequencer using the BigDye v3.1 cycle sequencing kit (Applied Biosystems, Inc., Norwalk, CT, USA) and the same primers as in the initial PCR. Forward and reverse sequences were aligned and edited in Sequen- cher v.5.0.1 (Gene Codes) and a contig of all sequences exported to eBioX v.1.5.1 (www.ebioinformatics.org) for final alignment using MUSCLE (Edgar 2004). A haplotype network was subsequently estimated using the TCS pro- gram (v.1.21; Clement et al. 2000) using the default 95% connection limit. In addition, annotation of the sequenced region was accomplished by analyses of the full mitochondrial A survey of full mitochondrial genome sequences held in GenBank for crustaceans (18 August 2013) indicated that 3293 ª 2014 The Authors. Ecology and Evolution published by John Wiley & Sons Ltd. Multiple Infections of C. maenas by S. carcini D. Rees & H. Glenner the majority of taxa present the expected order for genes flanking the control region (12S, CR, ND2, COI). This gene order exists in 49 of 78 noncirriped crustaceans in which the position of the control region has been annotated (an additional 23 full crustacean mitochondrial genomes have no control region specified). Different flanking genes are reported for 27 taxa (including ten species in a single amphipod genus), but all Cirripedia for which data are available conform to the majority pattern, with the excep- tion of the single rhizocephalan barnacle for which a near- complete mitochondrial genome has been published: Pol- yascus gregaria (Yan et al. 2012). Although Yan et al. were unable to present full sequence for the control region in P. Sequencing and data analysis gregaria due to problematic repetitive elements, the gene order presented by the authors is consistent with other cir- ripeds except for the additional presence of ND1 between the control region and ND2. appears variable in some taxa and if a few cases, two control regions have been annotated. Position and percent- age A+T content support the assumption that the region sequenced for this study is likely to be the control region. However, there is a possibility that Sacculina carcini also possesses a second control region, since annotation of the full mitochondrial sequence for S. carcini (unpublished data) indicates an A+T-rich (85%) 655-bp noncoding region between the cytochrome b (cob) and 12S genes. Haplotype diversity The initial group of S. carcini externa examined included three single externa from two locations; Roscoff, north-west France, and Limfjorden in Denmark. These six individual externa yielded six haplotypes, with 0.4–0.6% pairwise divergence among the Roscoff samples (mean = 0.5%) and 0.1–1.1% among those from Limfjorden (mean = 0.8%). Together with sequences from double or triple externa, the final dataset of 57 externa yielded 32 haplotypes, shown as a TCS haplotype network in Fig. 2. Representatives of all haplotypes shown in the TCS network have been deposited As such, the flanking gene order for Sacculina carcini is, at present, unique among Cirripedia and also Crusta- cea. Although a predominant flanking gene order was evi- dent from the survey of crustacean full mitochondrial genome sequences, the position of the control region L04 L05 L18 L02 L06 L10 L08 L14 L13 L09 L12 L01 L15 L07 L03 L11 L16 L19 L17 R12 R13 R07 R08 R02 R10 R04 R01 R11 R03 R09 R05 R06 α γ β n = 2 n = 2 n = 6 n = 12 n = 8 Figure 2. Statistical parsimony network inferred from mtDNA sequence data, with haplotypes coded “R” for Roscoff or “L” for Limfjorden. Ovals represent sampled haplotypes, branches connecting haplotypes represent nucleotide substitution steps, and small circles represent missing haplotypes. The square represents the haplotype (L17/C) inferred by TCS to be ancestral. Oval sizes are relative to haplotype frequency; details of individuals represented, along with single, double, or triple externa status and GenBank accession numbers, are presented in Table 1. The bold bar indicates the division between parts of the network corresponding to Limfjorden and Roscoff samples. Figure 2. Statistical parsimony network inferred from mtDNA sequence data, with haplotypes coded “R” for Roscoff or “L” for Limfjorden. Ovals represent sampled haplotypes, branches connecting haplotypes represent nucleotide substitution steps, and small circles represent missing haplotypes. The square represents the haplotype (L17/C) inferred by TCS to be ancestral. Oval sizes are relative to haplotype frequency; details of individuals represented, along with single, double, or triple externa status and GenBank accession numbers, are presented in Table 1. The bold bar indicates the division between parts of the network corresponding to Limfjorden and Roscoff samples. Figure 2. Statistical parsimony network inferred from mtDNA sequence data, with haplotypes coded “R” for Roscoff or “L” for Limfjorden. ª 2014 The Authors. Ecology and Evolution published by John Wiley & Sons Ltd. Life history conclusions extracted from the study Observations of multiple externae are extraordinarily rare in crab populations infested by Sacculina carcini. This is true even in crab populations with high prevalence of the parasite, as in this study. The dataset is based on the examination of 24878 crabs collected between May 2011 and April 2012 in the Danish inlet, Limfjorden. On average, 9% of these crabs were visibly infested by Sacculina carcini due to the presence of externae. Of these 2239 (9%), 40 (1.79%) were double infected, and of those two (5%) were triple infected (female crabs were slightly but significantly more parasitized than male crabs). The small number of multiple-infected crabs is dramatically lower than would be expected if cypris infection on parasitized and un-parasit- ized crabs were indiscriminate and random. This is in accordance with the study of (Rainbow et al. 1979). The absence of shared haplotypes among the initial set of single externa samples from Roscoff and Limfjorden populations was also consistent in the full dataset. No haplotypes were shared by individuals from these two locations and on examination of the haplotype network (Fig. 2) suggested that a degree of geographic structuring was present in the data. All samples (single and double infections) from Roscoff form one part of the network, distinct from the Limfjorden samples (Fig. 2). The most similar haplotypes from the two populations are still sep- arated by five substitutions and the mean pairwise dis- tance between externa from Limfjorden and Roscoff is 0.9%. Although preliminary, this is in contrast to the findings of Sasson et al. (2012) who reported no phyloge- ographic pattern among CR sequences from populations of the thoracican barnacle Chthamalus stellatus. Sasson et al. (2012) also reported a higher proportion of single- ton haplotypes (68%) among sequenced individuals than we observed in S. carcini (47%). Considering the rarity of multiple S. carcini externae on infected hosts, it is worth speculating as to possible mecha- nisms that might be involved in the observed pattern, that is, why multiple infections are so rare. The relative rarity of multiple externa may be linked to a possible preference of infecting cypris larvae for uninfected crabs, with chemi- cal cues involved in signaling infection status. In other barnacles, attraction of conspecifics via chemical and other cues has been documented (see, e.g., Clare et al. 1994; Dreanno et al. 2007) but signaling of infection status by S. carcini remains uninvestigated. Haplotype diversity Ovals represent sampled haplotypes, branches connecting haplotypes represent nucleotide substitution steps, and small circles represent missing haplotypes. The square represents the haplotype (L17/C) inferred by TCS to be ancestral. Oval sizes are relative to haplotype frequency; details of individuals represented, along with single, double, or triple externa status and GenBank accession numbers, are presented in Table 1. The bold bar indicates the division between parts of the network corresponding to Limfjorden and Roscoff samples. ª 2014 The Authors. Ecology and Evolution published by John Wiley & Sons Ltd. ª 2014 The Authors. Ecology and Evolution published by John Wiley & Sons Ltd. 3294 Multiple Infections of C. maenas by S. carcini D. Rees & H. Glenner in GenBank under the accession numbers KF649256– KF649287. All 13 externa from Roscoff and one-third of those from Limfjorden gave unique haplotypes. Within the Limfjorden sample, three haplotypes were found to be more common; one haplotype was shared by six individual exter- na, one by eight and one by 12 samples (denoted by in the TCS network (Fig. 2) by A, B, and C, respectively). The most common Limfjorden haplotype (C) contained sequences from two pairs of externa (i.e., two double infec- tions with identical haplotypes). Although these could be viewed as possibly resulting from asexual budding in single S. carcini individuals, the fact that distinct haplotypes were found in all other double and triple externa suggest that these are more likely to be the result of a shared, relatively common haplotype in the Limfjorden population. Pairwise divergences among the 57 externa ranged from 0% (the two pairs of externa sharing the common Limfjorden “C” hap- lotype) to 1.5%, and overall pairwise divergence was 0.7%. in studies of genetic differentiation at finer scales, as in the case of multiple externa. Combined with specific primers and ease of amplification, the control region offers a great deal of promise as a marker that bridges the gap between phylogenetics and population genetics and is a welcome additional tool to studies of S. carcini. ª 2014 The Authors. Ecology and Evolution published by John Wiley & Sons Ltd. Conflict of Interest None declared. Flight, P. A., M. A. O’Brien, P. S. Schmidt, and D. M. Rand. 2012. Genetic structure and the North American postglacial expansion of the barnacle, Semibalanus balanoides. J. Hered. 103:153–165. Life history conclusions extracted from the study Active avoidance of infected hosts in favor of searching for an uninfected crab may be unlikely due to the small size (250 lm) and limited energy resources (5 days; Glenner et al. 1989; Glenner and Werner 1998) of cypris larvae. However, if two (or in even rarer cases, three) cypris larvae successfully infect a new host at the same time, then this could occasionally lead to simultaneous establishment and later emergence of multi- ple externae. Unfortunately, detailed geographical information regarding specific sites of individual samples was not available for the material used in this study, but this will be addressed in forthcoming work. Large-scale sampling of S. carcini, involving multiple localities including Ro- scoff and Limfjorden, is underway and material will be analyzed both with microsatellites and sequencing of the control region. This will allow us to assess congruence between the two datasets and to properly determine what degree of geographic resolution can be achieved from analyses of control region sequences. Alternatively, rarity of multiple externae might be explained by the fact that once a virgin externa has emerged through the abdominal cuticle of the host, host molting is arrested and competing parasites are unable to emerge (Høeg 1995; Høeg and Lutzen 1995). The result of this “first past the post” scenario would be that although multi- ple cypris larvae might initially infect a host, external emer- gence by one parasite would prevent others from utilizing the hosts’ resources for developing their reproductive Our work with the mitochondrial control region in Sac- culina carcini has shown this A+T-rich region, flanked by the 12S rRNA and COI genes, to be highly variable. The level of variability observed makes this marker suitable for population-level studies and has also demonstrated utility 3295 Multiple Infections of C. maenas by S. carcini D. Rees & H. Glenner apparatus. Again, in rare cases, two or more parasites might simultaneously emerge, giving rise to multiple externae. single, double (A/B), or triple (A/B/C) externa present on a single C. maenas host. Haplotype codes are the same as those used in Fig. 2; GenBank accession numbers are indicated for each of the 32 unique haplotypes. References The most widely used methods of genotyping compromises restriction-fragment-length polymorphism (RFLP), ran- dom-amplified polymorphism detection (RAPD), ampli- fied-fragment-length polymorphism (AFLP), microsatellite genotyping, and single-nucleotide polymorphisms (SNP). For nonmodel species, with no previously existing proce- dure protocols, the time and money spent for developing and optimizing the methods to a new species are often dis- proportionally high compared to the outcome. Our study demonstrates that DNA sequence data from the highly vari- able mitochondrial control region is an inexpensive, easy, and robust alternative method to genetically differentiate specimens of the parasitic barnacles, Sacculina carcini. This marker also possesses broad utility; as well as testing for the presence/absence of asexual reproduction in adult parasites, we have successfully used Sacculina-specific CR amplifica- tion to screen for endoparasitic internas in potentially infected hosts without visible externae, and we will be fur- ther testing the resolution of CR sequences for small-scale population studies. Ease of use, along with the level of vari- ation observed in S. carcini, makes the application of this marker for genotyping studies in other rhizocephalan spe- cies an attractive and exciting proposition. Avise, J. C. 2000. Phylogeography: the history and formation of species, P. 464. Harvard Univ. Press, Cambridge, MA. Bernt, M., A. Donath, F. J€uhling, F. 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On the antennular secretion of the cyprid of Balanus amphitrite, and its role as a settlement pheromone. J. Mar. Biol. Assoc. U.K. 74:243–250. Acknowledgments Clement, M., D. Posada, and K. A. Crandall. 2000. TCS: a computer program to estimate gene genealogies. Mol. Ecol. 9:1657–1660. The authors wish to thank the Carlsberg Foundation for covering all expenses connected with the study (grant no. 2008-01-0491). We are also grateful to the staff at the Dan- ish Shellfish Center (DSC), Nykøbing Mors, Denmark, for collecting some of the material and providing laboratory facilities. In addition Jørgen L€utzen, Christoffer Grub, Tommy Christiensen, Jefferey Shields, and Jens Høeg assisted with sampling infected crabs. Teresa Costa partici- pated in part of the molecular work, which was carried out at the biodiversity laboratories (BDL, DNA section) at the Department of Biology, University of Bergen. 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Regulatory mechanisms of microRNAs in endocrine disorders and their therapeutic potential
Frontiers in genetics
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Abbreviations: miRNA, microRNA; DM, Diabetes mellitus; T1D, Type 1 diabetes; T2D, Type 2 diabetes; Treg Regulatory T cells AITDs, Autoimmune thyroid disorders; GD, Graves’ disease; HT, Hashimoto’s thyroiditis; BMD, Bone mineral density; MSC, Mesenchymal stem cells; APA, Aggressive pituitary adenoma; CAB, Cabergoline. KEYWORDS miRNAs, endocrine disorders, diabetes mellitus, thyroid diseases, osteoporosis, pituitary tumors OPEN ACCESS OPEN ACCESS EDITED BY Lingling Wang, Hainan Normal University, China REVIEWED BY Zhongxin Lu, Huazhong University of Science and Technology, China Kinga Fulopne Nemeth, University of Texas MD Anderson Cancer Center, United States *CORRESPONDENCE Luis M. Ruiz-Manriquez, a01701195@tec.mx Asim K. Duttaroy, a.k.duttaroy@medisin.uio.no Sujay Paul, spaul@tec.mx †These authors have contributed equally to this work SPECIALTY SECTION This article was submitted to RNA, a section of the journal Frontiers in Genetics RECEIVED 03 January 2023 ACCEPTED 09 February 2023 PUBLISHED 21 February 2023 CITATION Ledesma-Pacheco SJ, Uriostegui-Pena AG, Rodriguez-Jacinto E, Gomez-Hernandez E, Estrada-Meza C, Banerjee A, Pathak S, Ruiz-Manriquez LM, Duttaroy AK and Paul S (2023), Regulatory mechanisms of microRNAs in endocrine disorders and their therapeutic potential. Front. Genet. 14:1137017. doi: 10.3389/fgene.2023.1137017 S. Janin Ledesma-Pacheco1†, Andrea G. Uriostegui-Pena 1†, Estefania Rodriguez-Jacinto1, Elizabeth Gomez-Hernandez1, Carolina Estrada-Meza 1, Antara Banerjee2, Surajit Pathak2, Luis M. Ruiz-Manriquez1,3*, Asim K. Duttaroy4* and Sujay Paul1* 1Tecnologico de Monterrey, School of Engineering and Sciences, Queretaro, Mexico, 2Chettinad Academy of Research and Education (CARE), Chettinad Hospital and Research Institute (CHRI), Department of Medical Biotechnology, Faculty of Allied Health Sciences, Chennai, India, 3Tecnologico de Monterrey, Escuela de Medicina, Monterrey, Mexico, 4Department of Nutrition, Institute of Basic Medical Sciences, Faculty of Medicine, University of Oslo, Oslo, Norway MicroRNAs (miRNAs) are small endogenous non-coding RNA molecules capable of regulating gene expression at the post-transcriptional level either by translational inhibition or mRNA degradation and have recently been importantly related to the diagnosis and prognosis of the most relevant endocrine disorders. The endocrine system comprises various highly vascularized ductless organs regulating metabolism, growth and development, and sexual function. Endocrine disorders constitute the fifth principal cause of death worldwide, and they are considered a significant public health problem due to their long-term effects and negative impact on the patient’s quality of life. Over the last few years, miRNAs have been discovered to regulate various biological processes associated with endocrine disorders, which could be advantageous in developing new diagnostic and therapeutic tools. The present review aims to provide an overview of the most recent and significant information regarding the regulatory mechanism of miRNAs during the development of the most relevant endocrine disorders, including diabetes mellitus, thyroid diseases, osteoporosis, pituitary tumors, Cushing’s syndrome, adrenal insufficiency and multiple endocrine neoplasia, and their potential implications as disease biomarkers. OPEN ACCESS CITATION Ledesma-Pacheco SJ, Uriostegui-Pena AG, Rodriguez-Jacinto E, Gomez-Hernandez E, Estrada-Meza C, Banerjee A, Pathak S, Ruiz-Manriquez LM, Duttaroy AK and Paul S (2023), Regulatory mechanisms of microRNAs in endocrine disorders and their therapeutic potential. Front. Genet. 14:1137017. doi: 10.3389/fgene.2023.1137017 © 2023 Ledesma-Pacheco, Uriostegui- Pena, Rodriguez-Jacinto, Gomez- Hernandez, Estrada-Meza, Banerjee, Pathak, Ruiz-Manriquez, Duttaroy and Paul. 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. TYPE Review PUBLISHED 21 February 2023 DOI 10.3389/fgene.2023.1137017 TYPE Review PUBLISHED 21 February 2023 DOI 10.3389/fgene.2023.1137017 TYPE Review PUBLISHED 21 February 2023 DOI 10.3389/fgene.2023.1137017 1 Introduction ( , ) Recently, miRNAs have been demonstrated to regulate various processes associated with endocrine disorders, such as high-glucose- induced apoptosis, insulin secretion, and proliferation (Wang et al., 2020), as well as glycolipid metabolism (Zhang et al., 2020) in diabetes mellitus; abnormal ciliogenesis (Martínez-Hernández et al., 2019) in thyroid diseases; osteoclast and osteoblast differentiation (Li et al., 2018; Wang et al., 2021) in osteoporosis; proliferation and apoptosis (He et al., 2018; He et al., 2020) in pituitary tumors; proliferation (Vetrivel et al., 2022) in Cushing’s syndrome; steroidogenesis (Bitetto et al., 2022) in adrenal insufficiency; and adrenocortical proliferation (Li et al., 2021) in multiple endocrine neoplasia; among others. The association of these small RNA molecules in endocrine disorders could be advantageous in developing novel diagnostic and therapeutic tools. The present review aims to illuminate the most recent and relevant information about the potential role of microRNAs in endocrine disorders such as diabetes mellitus, thyroid diseases, osteoporosis, pituitary tumors, Cushing’s syndrome, adrenal insufficiency, multiple endocrine neoplasia, as well as their diagnostic and therapeutic potential. Recently, miRNAs have been demonstrated to regulate various processes associated with endocrine disorders, such as high-glucose- induced apoptosis, insulin secretion, and proliferation (Wang et al., 2020), as well as glycolipid metabolism (Zhang et al., 2020) in diabetes mellitus; abnormal ciliogenesis (Martínez-Hernández et al., 2019) in thyroid diseases; osteoclast and osteoblast differentiation (Li et al., 2018; Wang et al., 2021) in osteoporosis; proliferation and apoptosis (He et al., 2018; He et al., 2020) in pituitary tumors; proliferation (Vetrivel et al., 2022) in Cushing’s syndrome; steroidogenesis (Bitetto et al., 2022) in adrenal insufficiency; and adrenocortical proliferation (Li et al., 2021) in multiple endocrine neoplasia; among others. The association of these small RNA molecules in endocrine disorders could be advantageous in developing novel diagnostic and therapeutic tools. Alternatively, numerous non-canonical miRNA biogenesis pathways have been identified and thoroughly revised (Treiber et al., 2018; Stavast and Erkeland, 2019). These pathways employ various combinations of the canonical pathway proteins, primarily Drosha, Dicer, exportin 5, and AGO2 (Kim et al., 2016; O’Brien et al., 2018). The non-canonical miRNA biogenesis can be divided into Dicer-independent and Drosha/DGCR8-independent pathways (Annese et al., 2020). Pre-miRNAs produced by the Drosha/DGCR8-independent pathway resemble Dicer substrates. Mirtrons, which are created during splicing from the introns of mRNA, is an illustration of such pre-miRNAs. The 7- methylguanosine (m7G)-capped pre-miRNA serves as another illustration. 1 Introduction The main function of the endocrine system’s highly vascularized ductless organs is to produce hormones that can control metabolism, growth, development, and sexual function (Thi et al., 2017; Perle, 2021). The major glands that comprise the endocrine system are the adrenal, hypothalamus, pituitary, parathyroids, pineal body, thyroid, and ovaries or testes Frontiers in Genetics 01 frontiersin.org 10.3389/fgene.2023.1137017 Ledesma-Pacheco et al. 10.3389/fgene.2023.1137017 human genome (Zhang and Wang, 2017), and currently, 1,917 precursors (hairpin portion of a miRNA transcript) and 2,654 mature human miRNA sequences are deposited to the microRNA database/miRbase (miRBase, 2022; Kozomara et al., 2019; Kozomara and Griffiths-Jones, 2014). human genome (Zhang and Wang, 2017), and currently, 1,917 precursors (hairpin portion of a miRNA transcript) and 2,654 mature human miRNA sequences are deposited to the microRNA database/miRbase (miRBase, 2022; Kozomara et al., 2019; Kozomara and Griffiths-Jones, 2014). (Malespin & Nassri, 2019). The epithelial cells of the organs secrete their specific hormone product into the bloodstream, which binds to specific receptors in the target organs, affecting cellular functions (Perle, 2021). Endocrine disorders occur with the dysregulation of hormone levels or if the body does not respond to hormones appropriately (Lause et al., 2017) and constitute the fifth principal cause of death worldwide. Moreover, they are a significant public health issue since they can substantially decrease patients’ quality of life and cause long-term disabilities (Crafa et al., 2021). (Malespin & Nassri, 2019). The epithelial cells of the organs secrete their specific hormone product into the bloodstream, which binds to specific receptors in the target organs, affecting cellular functions (Perle, 2021). Endocrine disorders occur with the dysregulation of hormone levels or if the body does not respond to hormones appropriately (Lause et al., 2017) and constitute the fifth principal cause of death worldwide. Moreover, they are a significant public health issue since they can substantially decrease patients’ quality of life and cause long-term disabilities (Crafa et al., 2021). Over the past decades, it has been demonstrated that miRNAs can regulate hormone production, activity, and target cell responsiveness (Peng and Wang, 2018). 1 Introduction Without Drosha cleavage, these developing RNAs are exported directly to the cytoplasm by exportin 1. Conversely, Dicer- independent miRNAs are processed by Drosha from endogenous short hairpin RNA (shRNA) transcripts. These pre-miRNAs demand AGO2 terminate their maturation within the cytoplasm since they are of insufficient length to be Dicer-substrates. The present review aims to illuminate the most recent and relevant information about the potential role of microRNAs in endocrine disorders such as diabetes mellitus, thyroid diseases, osteoporosis, pituitary tumors, Cushing’s syndrome, adrenal insufficiency, multiple endocrine neoplasia, as well as their diagnostic and therapeutic potential. Frontiers in Genetics frontiersin.org 1 Introduction For instance, miRNAs can directly target genes encoding hormones or enzymes involved in hormone production or metabolism, affecting hormone concentrations; and proteins that modulate hormone actions, such as antagonists, can also be regulated by miRNAs; also, many studies have demonstrated that hormones regulate miRNAs, and such regulation can occur at the level of transcription or processing resulting in a complex regulatory network that ultimately impacts cell homeostasis (Peng and Li, 2022). Moreover, miRNAs can target hormone receptors and intracellular signaling molecules to alter target cell responses (Derghal et al., 2016; Bayraktar et al., 2017). Recently, evidence postulated that miRNAs could be secreted to the extracellular milieu and act as endocrine factors, performing endocrine and paracrine crosstalk between cells and tissues (Wu et al., 2022). MiRNAs are small (21–24 nucleotides long) endogenous non- coding RNA molecules that post-transcriptionally regulate gene expression either by degradation of mRNA or translational inhibition (Xiang et al., 2017; de Sousa et al., 2019). Biogenesis and processing of the miRNA molecules start in the cell’s nucleus, where RNA polymerase II transcribes the genes that codify the miRNA into the hairpin-structured primary miRNA, which is converted into a shorter stem-loop precursor miRNA by the microprocessor complex that consists of the enzymes Drosha and DGCR8. Afterward, the precursor miRNA is exported to the cytoplasm due to an interaction with Exportin 5, to be further processed into a mature miRNA/miRNA* duplex by the action of RNase III Dicer and trans-activation responsive RNA-binding protein. Next, a helicase separates the duplex so that the guide strand can be integrated into the RNA-induced silencing complex, also known as RISC, which is guided by the AGO2 protein. Finally, the RISC-miRNA complex identifies specific mRNA targets by base complementarity leading to translational inhibition or mRNA degradation (Paul et al., 2021; Ruiz-Manriquez et al., 2022). 1.1 Diabetes mellitus Diabetes mellitus (DM) is a chronic metabolic disorder characterized by persistently elevated blood glucose levels or hyperglycemia (Wang et al., 2020). Hyperglycemia can be caused by inefficiency in insulin action (also known as insulin resistance), secretion (also known as insulin deficiency), or both, as well as perturbations in the metabolism of carbohydrates, fats, or proteins (Cannata et al., 2020). The progression and development of DM are known to be a consequence of malfunctioned pancreatic β-cells, given that these cells are involved in insulin production (Wang et al., 2020). Type 1 (T1D) and type 2 diabetes (T2D) are the most common types of diabetes (Wang et al., 2020). T1D is caused by the autoimmune destruction of β-cells, leading to complete insulin According to most studies, miRNAs induce translational repression, mRNA deadenylation, and decapping by binding to a specific sequence at the 3′UTR of their target mRNAs (Dexheimer and Cochella, 2020). Nevertheless, miRNA binding sites have also been found in the 5′UTR, coding sequence, promoter regions, and other mRNA regions (Treiber et al., 2018). While the interaction of miRNAs with the promoter region has been reported to induce transcription, miRNA binding to the 5′UTR and coding regions has been shown to silence the expression of specific genes (O’Brien et al., 2018; Liu et al., 2021; Xiang et al., 2022). Interestingly, these molecules are found to regulate about 60% of genes of the 02 frontiersin.org Biological mechanism Source References Oxidative stress, high-glucose-induced β-cell apoptosis, insulin secretion, proliferation, β-cell protection Pancreatic and lung mice tissue Wang et al. (2018) Proliferation in acute pancreatitis and apoptosis Pancreatic rat tissue Ling et al. (2020) Proliferation, insulin secretion, β-cell apoptosis Blood from diabetic patients Li et al. (2016) Treg cell function Peripheral blood and lymph nodes of T1D patients Sebastiani et al. (2017) Treg cell induction and stability, autoimmune activation, and progression Human and mice CD4+ cells during the onset of islet autoimmunity and T1D Scherm et al. (2019) Glycogen synthesis, hepatic gluconeogenesis and lipogenesis T2D mice and rat liver tissue Xu et al. (2018) Hepatocytes Insulinitis, insulin resistance and secretion, pancreatic inflammation β cells of βTG mice Sun et al. (2021) R1, EXT1, MTPN, B2, RHOQ, SOX5, Insulin sensitivity, preservation of β-cell function Serum exosomes of obese mice and humans Xu et al. (2020) Immune response Thyroid tissue from AITD patients Martínez-Hernández et al. (2018) Ciliogenesis Thyroid tissue from AITD patients Martínez-Hernández et al. 1.1 Diabetes mellitus (2019) (Continued on following page) Biological mechanism Source References Immune response Peripheral blood mononuclear cells from HT and GD patients Kagawa et al. (2016) Hormone regulation, tumor proliferation migration and agiogenesis Plasmatic cells from GD patients Yao et al. (2019) Osteoclastogensis Serum samples and lumbar spine BMD of post-menopausal osteoporotic patients Li et al. (2018) Osteoclast differentiation and apoptosis Blood and bone tissue from patients with osteoporosis Li et al. (2020) Cell viability, apoptosis and osteoblast differentiation Human osteoblasts Wang et al. (2021) Formation of adipocytes, osteogenic and macrophage differentiation, bone development Mice bone marrow MSCs and stromal ST2 cell lines during adipogenesis Tian et al. (2017) Osteogenic differentiation Osteoporosis patients Chen et al. (2019) Osteoblast differentiation Primary rat MSC cells Li et al. (2019) Osteogenesis, osteoblastogenesis. Osteoporosis dignosis Serum from post-menopausal osteoporotic patients Du et al. (2015); Kerschan-Schindl et al. (2021); Lei et al. (2019); Sun et al. (2017) (Continued on following page) Biological mechanism Source References Obsteoblast proliferation, osteoclast, and osteoblast differentiation, oxidative stress Mice PBMC Lu et al. (2021) Metastasis Human pituitary tumor tissue He et al. (2020) Proliferation, migration, apoptosis HP75 and MMQ tumor cell lines Metastasis, glycolysis Human pituitary tumor tissue He et al. (2018) Proliferation, apoptosis HP75 and MMQ tumor cell lines APA metastasis, tumor size, temozolomide resistance Human APA tissues Lou et al. (2021) (Continued on following page) Biological mechanism Source References Metastasis, apoptosis, proliferation, migration, invasion, temozolomide resistance, cancer cell differentiation, survival and movement GH3 tumor cell line Cabergoline resistance DA-resistant human prolactinoma tissues Wu et al. (2019) Apoptosis, autophagy MMQ and GH3 tumor cell lines DNA synthesis, proliferation, cell cycle progression GH-producing human pituitary tumor cells and GH3 cell line He et al. (2019) Tumor cell’s invasiveness Patients with non-functional pituitary neuroendocrine tumors Wang et al. (2020) Cell cycle transition, viability, proliferation aT3-1 cell line Tumor progression Plasma from pituitary adenomas Németh et al. (2019) Proliferation, metastasis Peripheral blood samples from untreated NFPA patients Lin et al. (2018); Zhang et al. (2021) Proliferation, metastasis, prognosis You et al. (2019); Zhang et al. (2021) Proliferation, migration Park et al. (2018); Zhang et al. (2021) Tumor growth Han et al. (2018); Zhang et al. (2021) Autoimmune response modulation Serum from CS patients Vetrivel et al. (2021) Androstenedione production, proliferation Adrenal tissue from CS patients Vetrivel et al. (2022) ENTPD1, RY2, HRB, Immune response Peripheral blood samples from Adisson’s disease patients Fichna et al. Frontiers in Genetics 1.1 Diabetes mellitus (2021) Steroidogenesis Adrenal gland tissue from patients with adrenal insufficiency Bitetto et al. (2022) Adrenocortical proliferation, adrenal tumorogenesis Men1+/−mice adrenal glands Li et al. (2021) Growth induction, growth arrest Serum from MEN1 patients Kooblall et al. (2022) 10.3389/fgene.2023.1137017 Ledesma-Pacheco et al. FIGURE 1 miRNAs expression profiles observed in diabetes mellitus type I, type II, and unspecified, and the biological mechanisms involved. FIGURE 1 miRNAs expression profiles observed in diabetes mellitus type I, type II, and unspecified, and the biological mechanisms involved. GURE 1 miRNAs expression profiles observed in diabetes mellitus type I, type II, and unspecified, and the biological mechanisms involved. FIGURE 2 Dysregulation of miRNA expression profiles in Hashimoto’s thyroiditis and Grave’s disease, and the biological mechanism involved. FIGURE 2 Dysregulation of miRNA expression profiles in Hashimoto’s thyroiditis and Grave’s disease, and the biological mechanism involved 2019). According to the World Health Organization (2021), 1.5 million deaths were estimated to be caused directly by diabetes, placing this disease as the ninth leading cause of death worldwide. Given the high occurrence of DM and its complication, deficiency. In contrast, T2D is attributed to a continuous loss of β- cell insulin secretion, often in the context of insulin resistance (Association, 2019). Diabetes prevalence increases yearly, affecting around 500 million people worldwide (Saeedi et al., 07 frontiersin.org 10.3389/fgene.2023.1137017 Ledesma-Pacheco et al. FIGURE 3 miRNAs expressions profile involved in osteoblast, osteoclast, and osteogenic differentiation, and apoptosis that disrupts bone homeostasis, ultimately leading to osteoporosis. FIGURE 3 miRNAs expressions profile involved in osteoblast, osteoclast, and osteogenic differentiation, and apoptosis that disrupts bone homeostasis, ultimately leading to osteoporosis. xpressions profile involved in osteoblast, osteoclast, and osteogenic differentiation, and apoptosis that disrupts bone homeostasis, leading to osteoporosis. SOCS3 axis dysregulation in T2D development. Furthermore, they noticed that the expression of miR-19-3p was notably downregulated in diabetic patients’ blood and that there was an inverse correlation between blood glucose concentration and plasma miR-19a-3p level (Li et al., 2016). Further, the authors demonstrated that miR-19a-3p promotes cell proliferation and insulin secretion while inhibiting pancreatic β-cell apoptosis by directly targeting SOCS3 (Li et al., 2016). several investigations have been carried out to establish the role of miRNAs during the development of this disease to propose novel therapeutic approaches (Table 1) (Figure 1). Frontiers in Genetics frontiersin.org 1.1 Diabetes mellitus According to the American Thyroid Association (2016), about 20 million Americans suffer from thyroid problems, and up to 60% of them are unaware of their condition. MiRNAs have been discovered to regulate numerous biological processes, including immune activation, making them promising candidates for the management of thyroid problems (Table 1) (Figure 2) (Martínez-Hernández et al., 2019). miR-142-3p was upregulated in both human and murine models with islet autoimmunity and T1D. Furthermore, miR-142-3p was identified to affect FOXP3 CNS2-induced DNA methylation and directly target TET2, a modulator of DNA methylation, and is suggested to be crucial to T cell activation and Treg induction. Overall, it has been concluded that the miR142-3p/TET2/ FOXP3 axis hampers efficient Treg cell induction, compromising Treg stability and contributing to autoimmune activation and progression (Scherm et al., 2019). Dysregulated glycolipid metabolism is one of the crucial causes of T2D development (Zhang et al., 2020). In this sense, Xu et al. (2018) showed that the enhancement of miR-125a-5p expression appears advantageous for glycolipid metabolism in T2D mice. Putatively, the authors evidenced a downregulation of miR-125a- 5p in mouse and rat livers with T2D. Also, they demonstrated that miR-125a-5p targets the STAT3 in hepatocytes. Due to the involvement of STAT3 in glycolipid metabolism, it was revealed that the inhibition of miR-125a-5p increased hepatic gluconeogenesis and lipogenesis, decreased glycogen synthesis, and aggravated hyperglycemia and hyperlipidemia. Conversely, the elevation of miR-125a-5p mitigated glucose and lipid metabolic disorders. This information suggests a critical association between miR-125a-5p and glycolipid metabolism (Xu et al., 2018). Martínez-Hernández et al. (2018) found that individuals with AITDs have severely dysregulated levels of a variety of miRNAs connected to critical immunological mechanisms that might be involved in thyroid disorders. Eight miRNAs were found to be differentially expressed (miR-21-5p, miR-142-3p, miR-146a-5p, miR-146b-5p, miR-155-5p, miR-338-5p, miR-342-5p, and miR- 766-3p) in patients with AITD. Furthermore, when the expression of these miRNAs was compared to additional miRNAs previously linked to AITD in peripheral blood, the expression of four of them (miR-Let7d-5p, miR-21-5p, miR-96- 5p, miR-142-3p, and miR-301a-3p) was found to be notably higher in AITDs and, in patients with GD. Only miR-Let-7d was downregulated. Besides, the expression level of those miRNAs was associated with higher disease severity, including active ophthalmopathy, higher antibody titers, goiter, and/or higher recurrence rates. Interestingly, prior research has linked miR- Let7d-5p, miR-126-3p, miR-142-5p, miR-223-3p, and miR-301a- 3p to autoimmune illnesses, including in AITD. 1.1 Diabetes mellitus Furthermore, the authors found a link between miR-Let7d-5p, miR-21-5p, miR-96- 5p, miR-142-3p, and miR-301-3p and the levels of all three thyroid autoantibodies, suggesting that these miRNAs could be used to predict the likelihood of getting AITD (Martínez-Hernández et al., 2018). Pancreatic inflammatory diseases, including acute or chronic pancreatitis, could result in diabetes (Gál et al., 2021). Pancreatic inflammation has been found to be promoted by the overexpression of miR-29 in β cells of βTG mice (Sun et al., 2021). Explicitly, miR-29 overexpression causes the suppression of the TRAF3, whose axis regulates the response to metabolic stress associated with inflammation. Additionally, the inhibition of miR-29 improves insulitis, insulin resistance, and dysfunction of glucose-stimulates insulin secretion, causing the attenuation of inflammation and diabetes (Sun et al., 2021). Primary cilia are a sensory organelle that reacts to mechanical and chemical stimuli in the environment and transmits that information to the inside of the cell. Martínez-Hernández et al. (2018) examined the distribution and length of primary cilia, which act as sensory regulators in the thyroid to modulate hormone secretion in thyroid tissues from AITD patients, finding that both the number and length were significantly lower in AITDs than in healthy tissue. It was noticed that certain miRNAs, including miR- 21-5p, miR-146b-3p, miR-5571-3p, and miR-6503-3p, were upregulated in the cilia, therefore, associated with these conditions. Therefore, the study suggests the potential use of miRNAs to detect AITD (Martínez-Hernández et al., 2019). Given the importance of insulin secretion and sensitivity in maintaining normal glucose levels, their disruption results in DM (Bełtowski et al., 2018). In this regard, miR-26a has been shown to improve peripheral insulin sensitivity and preservation of β cell function (Xu et al., 2020). Putatively, miR-26a expression was downregulated in the serum exosomes of obese mice and humans. Interestingly, by triggering miR-26a expression, glucose dysregulation (hyperinsulinemia) could be averted since target genes of miR-26a such as CACNA1C, CTGF, CREBRF, ESR1, EXT1, MTPN, ONECUT2, PJA2, PLCB1, PFKFB2, RHOQ, and SOX5, are the regulators involved in insulin secretion and β cell proliferation/survival (Xu et al., 2020). Additionally, other target genes such as INSR, PDX1, AKT1, and IGF1R (β cell hyperplasia activators) were downregulated in the islet of RIP TG mice (Xu et al., 2020). IL-10 is a cytokine directly involved in reducing inflammation induced by macrophages and T cells. 1.1 Diabetes mellitus One of the essential functions of IL-10 is inhibiting Th1 cells, a type of T cell that synthesizes inflammatory cells (Ouyang and O’Garra, 2019). Interestingly, Let- 7e has been found to target the interleukin IL-10 genes (Kagawa et al., 2016). Precisely, Kagawa et al. (2016) observed that IL-10 production was inhibited in peripheral blood mononuclear cells of patients with HT and that serum levels of Th1 cytokines were elevated. Importantly, let-7e expression was considerably higher in HT patients compared to GD patients or control subjects. However, there were no significant variations in Let-7e expression levels between GD patients and control subjects. Moreover, Let-7e expression levels in severe HT patients were Altogether, the above information proves that the dysregulation of a great number of miRNAs contributes to the development and progression of diabetes. Therefore, these miRNAs could be potential biomarkers and therapeutic tools for treating diabetic patients. Frontiers in Genetics 1.1 Diabetes mellitus For example, it has been shown that myeloid KLF2 overexpression exerts a protective effect in mice against insulin resistance; therefore, it is considered a crucial regulator of obesity and its sequelae, such as diabetes (Sweet et al., 2020). Interestingly, Wang et al., 2018 suggested that miRNA-92a can negatively regulate the expression of KLF2, which is thought to function via modulating the Notch signaling pathway. The authors also revealed that miRNA-92a was predominantly expressed in the pancreas and lungs in mice models, which was an indicator of the involvement of miRNA-92a in pancreatic function. Moreover, they validated that miRNA-92a might modulate oxidative stress, inhibit high-glucose-induced apoptosis of β-cells, increase insulin secretion and proliferation, and protect pancreatic β-cell function (Wang et al., 2018). Consistent with these findings, another analogous study highlighted that the downregulation of miRNA-92a-3p enhances KLF2 expression and inhibits apoptosis, thus promoting proliferation in acute pancreatitis in pancreatic rat tissue (Ling et al., 2020). It is well known that Regulatory T cells (Treg) are important regulators of peripheral immune tolerance and that the development of autoimmunity disorders, including T1D, can be due to their insufficiency (Visperas and Vignali, 2016). In this context, an upregulation of miR-125a-5p in Treg cells obtained from T1D patients’ peripheral blood and lymph nodes was noted, suggesting that this overexpression could impede Treg-cell function (Sebastiani et al., 2017). Moreover, miR-125a-5p targets TNFR2 and CCR2. Remarkably, CCR2 has been implicated in T1D pathogenesis, and its expression is thought to modulate Treg cell function. This data suggests that the upregulation of miR-125a-5p in Treg cells of T1D patients leads to a decrease in CCR2 expression, which might hinder the migration of critical immune cells to the pancreas required for the maintenance of peripheral tolerance (Sebastiani et al., 2017). Similarly, Scherm et al., 2019 demonstrated that by inhibiting miR-142-3p, Treg induction and stability could be enhanced. More precisely, the authors showed that Over the past years, SOCS3 has been associated with glucose metabolism and diabetes progression, given that it is a crucial negative regulator of insulin signaling (Iqbal et al., 2020). Notably, Li et al. (2016) found an inverse correlation between SOCS3 plasma levels and miR-19a-3p, which led them to suggest the role of the miR-19a-3p/ 08 frontiersin.org 10.3389/fgene.2023.1137017 Ledesma-Pacheco et al. 10.3389/fgene.2023.1137017 metabolic processes (Kyritsi and Kanaka-Gantenbein, 2020). Thyroid diseases include hypothyroidism, hyperthyroidism, and autoimmune thyroid disorders (AITDs), such as Graves’ disease (GD) and Hashimoto’s thyroiditis (HT). frontiersin.org 1.2 Thyroid diseases Thyroid diseases are conditions in which the thyroid gland is affected. This gland plays an essential role in regulating various 09 frontiersin.org frontiersin.org 10.3389/fgene.2023.1137017 Ledesma-Pacheco et al. FIGURE 4 Oncogenic and tumor suppressor miRNAs expression profile involved in pituitary tumors, ultimately affecting cabergoline resistance, proliferation, metastasis, apoptosis, and migration. FIGURE 4 Oncogenic and tumor suppressor miRNAs expression profile involved in pituitary tumors, ultimately affecting cabergoline resistance, proliferation, metastasis, apoptosis, and migration. have proven to be a valuable tool for the early detection of thyroid diseases such as HT and GD; however, further research is required to use them as potential disease biomarkers. Moreover, investigating the roles of miRNAs in hyper and hypothyroidism might open a new arena of thyroid disease management. found to be considerably lower than in mild HT patients (Kagawa et al., 2016). These findings lead to the understanding of Let-7e in the regulation of the immune response of people with HT, which might eventually help with HT diagnosis and treatment. Graves’ disease is an autoimmune condition known as the most common cause of hypothyroidism (Yao et al., 2019). It is characterized by an activated immune system, which results in the production of thyroid-stimulating antibodies (TSAs); consequently, it makes the thyroid gland overactive, producing more hormones than usual (Razmara et al., 2021). GD is a complex condition resulting from environmental and genetic factors (including mutations in the TSHR and CD40 genes). Yao et al. (2019) demonstrated that various plasmatic miRNAs are associated with GD and might serve as biomarkers for diagnosing this condition. They found that five miRNAs were substantially differentially expressed in GD. Among them, miR- 16-1-3p, miR-122-5p, miR-221-3p, and miR-762 were upregulated, while miR-144-3p was downregulated. Intriguingly, miR-762 was positively associated with FT3, a thyroid hormone, and TRab (Yao et al., 2019), which is considered the gold standard diagnostic test for the autoimmunity of GD (Bell et al., 2018). Moreover, miR-144-3p has been shown to be downregulated in many kinds of carcinoma and is associated with tumor proliferation, migration, and angiogenesis (He et al., 2020). Frontiers in Genetics frontiersin.org 1.3 Osteoporosis (2019a) consistently studied miR-449b-5p expression in primary rat MSC cells, and miR-449b-5p was reported to be underexpressed in osteogenic differentiation; moreover, the differentiation could be inhibited by the miRNA overexpression, thus revealing the mechanism of miR-449b-5p in suppressing bone formation. In addition, miR-449b-5p was proved to target the SATB2 gene, which enhances osteoblast differentiation, regulates skeletal development, and promotes bone formation. Therefore, miR-449b-5p is thought to repress the osteogenic differentiation of MSCs via SATB2, therefore promoting the progression of osteoporosis (Li et al., 2019). Similarly, miR-483-5p has also been suggested to promote osteoclast differentiation in osteoporosis. Putatively, in samples of blood and bone tissue from osteoporotic patients, miR-483-5p was found to be significantly upregulated, whereas its predicted target IGF2 was underexpressed. Interestingly, the upregulation of miR- 483-5p promoted the differentiation of the M-CSF, a relevant cytokine for osteoclast differentiation in mammals, and RANKL- induced CD14+ peripheral blood mononuclear cells into osteoclasts. Moreover, overexpression of IGF2 was discovered to revert the miRNA effects on osteoclast differentiation and thus enhance osteoclast apoptosis. Therefore, a dysregulation in miR-483-5p expression might contribute to osteoclast differentiation by targeting IGF2, causing dysregulation in the balance between bone formation and resorption, ultimately causing osteoporosis (Li et al., 2020). Kerschan-Schindl et al. (2021) used a signature panel of nineteen circulating miRNAs to serve as biomarkers to diagnose osteoporosis in post-menopausal patients based on the WHO criteria and with the ability to identify patients that had previously suffered fractures. The miRNAs in the panel represent different independent factors related to bone disease and the risk of fractures. The panel includes miR-127-3p, miR-133b, miR-203a, miR-214-3p, miR-31-5p, and miR-375 (all of which the effects of hemolysis did not bias measurement); miR-143-3p, miR-188-5p, and miR-582-5p (these miRNAs along with miR-375 strongly contributed with the WHO criteria); miR-550a-3p and miR-152-3p (which contributed to the major osteoporotic fractures); and miR-141-3p, miR-144-5p, miR- 17-5p, miR-19b-3p, miR-29-3p, miR-320a, miR-335-5p, and let-7b- 5p. Out of these, the upregulation of serum miR-375 indicated the presence of osteoporosis according to the WHO criteria and has been associated with reducing the bone-formation effect of teriparatide, a drug to treat severe osteoporosis (Lei et al., 2019). MiR-375 has been found to negatively modulate osteogenesis The bone formation also relies on osteoblast differentiation (Wang et al., 2021); therefore, an alteration in this process may lead to skeletal disorders such as osteoporosis. 1.3 Osteoporosis MiRNAs have an important role in the regulation of bone formation and resorption (Li et al., 2018; Chen et al., 2019) that allows a balance to maintain the healthy state of the bones, and their dysregulation is associated with osteoporosis; however, the precise underlying mechanism is still poorly understood. Hence, there is a current need to elucidate the regulatory pathways of miRNAs in osteoporosis to prevent and treat this global disease (Table 1) (Figure 3). It is estimated that one in three 50 + aged women will suffer a fracture in their remaining lifetime (International Osteoporosis Foundation, 2022), strongly influenced by estrogen deficiency and aging (Li et al., 2018). One of the hallmarks of post- menopausal osteoporosis is a low bone mineral density (BMD) derived from the altered activity of osteoblasts and osteoclasts. Notably, miR-133a has been significantly upregulated in serum samples and showed a negative correlation with lumbar spine BMD of post-menopausal patients with osteoporosis. Interestingly, this miRNA was upregulated during osteoclastogenesis, thus promoting the differentiation of RAW264.7 and THP-1 cells into osteoclasts, which would be induced by RANKL, a crucial cytokinin for osteoclast development (Li et al., 2018). Consistently, an in vivo analysis in OVX rats demonstrated that a knockdown of miR-133a augmented lumbar spine BMD, dysregulated the serum levels of factors related to osteoclastogenesis, and modified the histomorphology of the bone mainly by perforation, connectivity loss, and thinning of the trabecular bone. Altogether, it was demonstrated that miR-133a promotes osteoclast differentiation, thus contributing to the regulation of osteoporosis in post-menopausal women (Li et al., 2018). Although bone mass recovery strategies are limited, miRNA- mediated osteogenic differentiation has become a target of interest for osteoporosis therapeutics. In that matter, Chen et al. (2019) discovered that serum levels of miR-19a-3p were underexpressed in patients with osteoporosis. Furthermore, the levels of miR-19a-3p, RUNX2, and OCN (these last being osteogenesis-related genes) would be gradually upregulated as the osteogenic differentiation of MSCs prolongated. miR-19a-3p was found to target the HDAC4 gene, a critical factor in bone formation, bone metabolism, and osteoblast differentiation. Thus, the analyzed miRNA was suggested to reduce osteoporosis progression by accelerating the osteogenic differentiation of MSCs by inhibiting HDAC4 (Chen et al., 2019). Li et al. 1.3 Osteoporosis Osteoporosis is a multifactorial condition considered as the most prevailing metabolic bone disorder in humans (Scimeca et al., 2017). It is caused by complex interactions between genetic and environmental factors (Li et al., 2018) and is characterized by an imbalance between the bone formation mediated by osteoblasts and the bone resorption mediated by osteoclasts. Bone homeostasis is commonly altered by dysregulation in osteoblasts and osteoclasts’ proliferation, differentiation, or apoptosis (Wang et al., 2021), which leads to bone mass and strength decrease, degenerative change of bone microarchitecture, and non- or low-trauma fractures (Cosman et al., 2014; Scimeca et al., 2017). Osteoporosis usually has no symptoms prior to a first fracture, which is associated with a risk increment of a subsequent fracture by 86% (International Osteoporosis Foundation, 2022). Osteoporosis has become a serious health concern, affecting around 50% of post-menopausal females and 20% of males over 50 (Gennari et al., 2016). Furthermore, it causes over 8.9 million fractures annually (Hu et al., 2018), many of which lead to mortality (International Since thyroid diseases affect a large number of people, most of whom are unaware of it, it is crucial to find new ways to detect thyroid diseases to implement an adequate early treatment. MiRNAs 10 frontiersin.org 10.3389/fgene.2023.1137017 10.3389/fgene.2023.1137017 Ledesma-Pacheco et al. MSCs generates a bone mass decrease associated with osteoporosis (Hu et al., 2018; Li et al., 2019), the regulation mechanism of miRNAs in these processes has been extensively studied in order to develop potential target treatments for this skeletal disorder. In that context, miR-148a-3p was overexpressed in mice bone marrow MSCs and stromal ST2 cell lines during adipogenesis (Tian et al., 2017). miR-148a-3p potentiated the formation of adipocytes in the adipogenic medium while inhibiting osteoblast differentiation. Further study revealed KDM6b as the direct target of miR-148a- 3p. In the investigation, KDM6b was found to influence the differentiation into mature adipocytes; however, it has also been related to the modulation of osteogenic commitment of MSCs, osteogenic and macrophage differentiation, and bone development. Altogether it was concluded that miR-148a-3p has a positive adipogenic and negative osteogenic function by directly targeting and negatively regulating KDM6B, affecting the MSCs fate decision (Tian et al., 2017). Osteoporosis Foundation, 2022). Frontiers in Genetics frontiersin.org Ledesma-Pacheco et al. 1.3 Osteoporosis A study in human osteoblasts revealed the downregulation of miR-133b and subsequent overexpression of its target GNB4 in osteoporotic patients (Wang et al., 2021). Intriguingly, the induction of miR- 133b led to higher cell viability, diminished apoptosis, and promotion of osteoblast differentiation (Wang et al., 2021); hence, the underlying mechanism of the miR-133b/GNB4 might be of use for osteoporosis treatment. Correspondingly, miRNAs can also influence bone marrow mesenchymal stem cells (MSCs), known to be osteoblast and adipocyte progenitor cells (Hu et al., 2018). Since an imbalance of osteoblast and adipogenic differentiation of 11 frontiersin.org 10.3389/fgene.2023.1137017 Ledesma-Pacheco et al. (Kerschan-Schindl et al., 2021), possibly by targeting RUNX2 (Du et al., 2015; Lei et al., 2019), and suppressing WNT pathways by targeting LRP5 and ß-catenin (Sun et al., 2017). Moreover, the upregulation of miR-203a-3p was a potential marker for fragility fractures and has been identified to negatively regulate osteoblastogenesis via BMP-2 and DLX5, which activate the transcription factors for osteoblast differentiation: RUNX2 and Osterix. Altogether, the analyzed panel could serve as a novel and non-invasive tool for osteoporosis diagnosis and identifying patients with fracture history (Kerschan-Schindl et al., 2021). decreased levels of vimentin were also noticed in HP75 and MMQ cells overexpressing miR-448 (He et al., 2020). Caspase-3 and PARP are pro-apoptotic proteins (Hsu et al., 2018), while E-cadherin and vimentin are biomarkers that, in this case, suggest the inhibition of epithelial to mesenchymal transition of pituitary tumor cells (Liao et al., 2019; He et al., 2020). Hence, the elucidated miR-448–BCL2 pathway might be of use as a therapeutic strategy to regulate the malignant behavior of pituitary tumor cells. (Kerschan-Schindl et al., 2021), possibly by targeting RUNX2 (Du et al., 2015; Lei et al., 2019), and suppressing WNT pathways by targeting LRP5 and ß-catenin (Sun et al., 2017). Moreover, the upregulation of miR-203a-3p was a potential marker for fragility fractures and has been identified to negatively regulate osteoblastogenesis via BMP-2 and DLX5, which activate the transcription factors for osteoblast differentiation: RUNX2 and Osterix. Altogether, the analyzed panel could serve as a novel and non-invasive tool for osteoporosis diagnosis and identifying patients with fracture history (Kerschan-Schindl et al., 2021). He et al. (2018) also observed the downregulation of miR-1 in human pituitary tumor tissues. Moreover, a much lower abundance of miR-1 was found in the presence of lymphatic metastasis, associated with a worse prognosis in patients. 1.3 Osteoporosis Other assessments showed that the overexpression of miR-1 in HP75 and MMQ cell lines suppresses the proliferation and increases the apoptosis rate of the cells. Also, it was demonstrated that miR-1 negatively regulates the protein expression of its target gene G6PD, limiting its function of generating ribose-5-phosphate and NADPH, which are promoters of glycolysis and nucleotide biosynthesis in tumor cells; therefore, pituitary tumor tissues under expressing miR-1 showed an augmentation of G6PD (C. He et al., 2018), suggesting miR-1 as a target to diagnose and treat pituitary tumors. Currently, microRNA biomarkers for osteoporosis are found to be very promising since conventional protein osteoporosis biomarkers are not very efficient and often produce false positive results. In this matter, Lu et al. (2021) found thirteen significantly consistent overexpressed miRNAs (miR-1271-5p, miR-132-3p, miR-153a-3p, miR-153b-3pA, miR-153-3p, miR-15a-5p, miR- 15b-5p, miR-182-5p, miR-223-3p, miR-27a-3p, miR-370-3p, miR-486-5p, and miR-9-5p) in mice PBMC that were positively correlated with osteoporosis progression, therefore elucidating their potential role as biomarkers for this disease. The mentioned miRNAs were FOXO1 regulators, an important molecule in bone metabolism that activates osteoblasts’ proliferation and differentiation, suppresses the differentiation and viability of osteoclasts, and diminishes the negative effects of oxidative stress to favor bone cell function (Lu et al., 2021). gg g g g p y Pituitary tumors are referred to as aggressive pituitary adenomas (APAs) when they exhibit aggressive behaviors such as solid invasiveness and resistance to conventional therapies (Lou et al., 2021). The role of miRNA-146b-5p in APA was studied by Lou et al. (2021). MiRNA-146b-5p was found to be downregulated in human APA tissues and pituitary tumor cell lines (AtT-20, HP75, GH3, GT1-1) compared to non-tumor tissues and normal human astrocytes, respectively. Furthermore, the downregulation of miRNA-146b-5p in APA patients was associated with poor prognosis, poor survival rate, and features indicative of advanced tumor stage related to APA metastasis, such as temozolomide resistance, larger tumor size, poorer Hardy grade, and poorer Knosp grade (Lou et al., 2021); these last grades refer to classification systems indicating local invasion and cavernous sinus invasion (Araujo-Castro et al., 2021). Subsequent assessments demonstrated that the induced overexpression of miRNA-146b-5p in GH3 cells suppressed metastasis by triggering cell apoptosis and negatively regulating cell proliferation, migration, and invasion. The direct target of miRNA-146b-5p, EPHA7 was also found to be overexpressed in APA tissues as well as in the pituitary tumor cell lines previously mentioned (Lou et al., 2021). 1.3 Osteoporosis EPHA7 mediates the inflammatory response to cell injury, and its low expression has been found in different carcinomas (Gajdzis et al., 2020). The anti-metastatic properties of miRNA-146b-5p in GH3 cells were reversed by the induced overexpression of EPHA7 (Lou et al., 2021). Interestingly, compared to chemosensitive tissues, chemoresistant tissues showed overexpression of EPHA7 and underexpression of miRNA-146b-5p. The same results were found in a temozolomide-resistant cell line of GH3 compared to the parental cell line. It was speculated that miRNA-146b-5p might regulate autophagy since treatment with this miRNA decreased the autophagic activity of temozolomide-resistant cells, while Lv- EPHA7 treatment enhanced it. Thus, it was suggested that abnormal expressions of miRNA-146b-5p and EPHA7 might be involved in the temozolomide resistance in APA. Finally, IRAK4 and TRAF6 protein expression and NF-κB phosphorylation were suppressed in GH3 cells overexpressing Although osteoporosis is a common concern in the elderly and post-menopausal population, bone restoration strategies are currently limited. Recently, several investigations have elucidated the role of miRNAs in osteoporosis which could be promising in discovering novel miRNA-based diagnostic and therapeutic strategies that could increase the patient’s quality of life. Frontiers in Genetics frontiersin.org 1.4 Pituitary tumors Pituitary tumors, also called pituitary neuroendocrine tumors, are adenomas that arise from the anterior pituitary gland and constitute around 10 to 15 percent of intracranial tumors (Asa et al., 2017; Lou et al., 2021). Pituitary tumors arise sporadically from any of the five hormone-producing cell types of the adenohypophysis, but despite being monoclonal, they contain plenty of multi-responsive and multi-hormonal cells whose role is not yet fully understood (Fiordelisio et al., 2021). Many neoplasms, including pituitary tumors, have exhibited altered miRNA expression, thus being a significant field of research for understanding, diagnosing, and treating pituitary tumors (Table 1) (Figure 4). Although pituitary tumors are mostly benign, they have the potential to develop into cancer. He et al. (2020) noticed a reduced expression of miR-448 in human pituitary tumor tissues and an even lower expression in tissues from patients with metastasis. The induced overexpression of miR-448 was evaluated in HP75 and MMQ cells (pituitary adenoma cell lines), showing inhibition of proliferation and migration of both cell lines and an increase in the apoptosis rate. It was suggested that miR-448 negatively regulates its target gene, BCL2, an integral outer mitochondrial membrane protein that inhibits apoptosis in HP75 and MMQ cells. In addition, increased levels of PARP, E-cadherin, caspase-3, and 12 frontiersin.org Ledesma-Pacheco et al. 10.3389/fgene.2023.1137017 10.3389/fgene.2023.1137017 miRNA-146b-5p; however, restoration of EPHA7 expression reversed these inhibitory effects. The IRAK4/TRAF6/NF-κB signaling pathway is essential in regulating cancer cell differentiation, survival, and movement, and EPHA7 is required. Hence, miRNA-146b-5p may inhibit invasion and metastasis by directly targeting EPHA7 and suppressing the IRAK4/TRAF6/NF- κB signaling pathway (Lou et al., 2021). These findings suggest that miRNA-146b-5p could be used as a molecular marker to evaluate and predict pituitary tumor aggressiveness and chemotherapeutic drug resistance. since it modulates the cell cycle via SKP2/p27Kip1, thus revealing a novel mechanism that might have significant therapeutic implications. miRNA-146b-5p; however, restoration of EPHA7 expression reversed these inhibitory effects. The IRAK4/TRAF6/NF-κB signaling pathway is essential in regulating cancer cell differentiation, survival, and movement, and EPHA7 is required. Hence, miRNA-146b-5p may inhibit invasion and metastasis by directly targeting EPHA7 and suppressing the IRAK4/TRAF6/NF- κB signaling pathway (Lou et al., 2021). These findings suggest that miRNA-146b-5p could be used as a molecular marker to evaluate and predict pituitary tumor aggressiveness and chemotherapeutic drug resistance. 1.4 Pituitary tumors Afterward, the miRNA-93-5p inhibitor was used to transfect MMQ cells which were then injected subcutaneously into nude mice; the results showed an enhancement of the therapeutic effectiveness of CAB, a diminution in the prolactin expression in mice, an upregulation of LC3-II protein, and the overproduction of autophagosomes. Therefore, the investigation suggests that miRNA-93-5p inhibition enhances CAB efficiency in vivo by upregulating autophagy. With the objective of identifying circulating miRNAs that could serve as biomarkers for pituitary adenomas, Nemeth et al. (2019) investigated the dysregulation of several miRNAs in plasma and extracellular vesicles from patients with pituitary adenomas at a preoperative, as well as early and late postoperative stages. A total of 29 miRNAs were found promising to distinguish preoperative plasma samples from normal controls. Interestingly, the study demonstrated that miR-143-3p was substantially differentially expressed (downregulated) in late postoperative samples of FSH/ LH + adenomas compared to preoperative samples, indicating a successful surgery. Nevertheless, no significant alteration of this miRNA has been noticed when comparing early postoperative samples with preoperative ones. Although the results are promising, further investigation is needed to determine the potential role of this miRNA as a marker for tumor recurrence (Németh et al., 2019). Growth hormone (GH) pituitary tumors are another subtype of functioning pituitary tumors characterized by excessive production of GH by the pituitary gland leading to acromegaly and gigantism (Ji et al., 2022). He et al. (2019) reported a reduced expression of miR-186 in GH-producing human pituitary tumors. The miR-186 regulates the expression of its direct target, the SKP2, an oncogene that targets p27Kip1 to inhibit its expression (He et al., 2019). The p27Kip1 is a tumor suppressor that inhibits the transition of the cell cycle from the G1 to the S phase (Li et al., 2021). The role of miR-186 was tested in human GH-secreting pituitary tumor cells and GH3 cells, and its inhibition resulted in the overexpression of SKP2 and underexpression of p27Kip1, causing an induction in the DNA synthesis and cell proliferation. Consistently, the overexpression of miR-186 had the opposite effect on the cell lines and induced G0/G1 cell cycle arrest. In addition, miR-186 and p27Kip1 were downregulated in GH-producing human pituitary tumor tissues compared to normal human pituitary tissues, while SKP2 was upregulated (He et al., 2019). 1.4 Pituitary tumors Non-functional pituitary neuroendocrine tumors remain a challenge since the only first-line treatment choices are surgery and radiotherapy, and early detection of invasiveness is scarce due to a lack of diagnostic biomarkers (Bao et al., 2021). Wang et al. (2020) analyzed specimens of patients with non-functional pituitary neuroendocrine tumors and reported that miR-134 might inhibit the tumor cell’s invasiveness and expression of Ki-67, a marker of cellular proliferation. The verified target of miR-134 was the VEGFA (Wang et al., 2020), the most functional isoform of the proangiogenic factors VEGF, which contribute to angiogenesis, successively helping the generation and development of cancer (Zhang et al., 2019). Additionally, they found that by influencing the expression of miR-134, SDF-1α could promote the development of tumors. SDF-1α and VEGFA expression levels were relatively high in the analyzed invasive specimens, defining invasiveness as resistance to conventional treatment and numerous recurrences. It was confirmed in mouse pituitary aT3-1 cells that due to miR-134 upregulation, VEGFA expression and transition of the cell cycle from the G1 to the S phase are inhibited. Interestingly, SDF-1α treatment reduces miR-134 expression and boosts VEGFA expression, which promotes cell viability and proliferation by allowing cell cycle transition (Wang et al., 2020). For that reason, the SDF-1α/miR-134/VEGFA axis might be a potential target for detecting and treating non-functional pituitary neuroendocrine tumors. Pituitary tumors can be functioning (which involves the overproduction of certain hormones) or non-functioning (low hormone production) (Butt and Srinivasan, 2022). The most common functioning pituitary tumors are prolactin-secreting tumors called prolactinomas (Kim et al., 2022). In most cases, cabergoline (CAB) or other dopamine agonists are used for the treatment, but there are dopamine agonist-resistant prolactinomas whose management remains a challenge (Wu et al., 2019). Wu et al. (2019) proposed that miRNA-93-5p, a miRNA overexpressed in dopamine agonists-resistant prolactinoma tissues, targets ATG7 and intervenes in CAB resistance. Autophagy is a crucial pathway for cell survival under stress that degrades large structures such as organelles and proteins (Li et al., 2019), and ATG7 is an essential protein downregulated in dopamine agonists-resistant human prolactinoma tissues (Wu et al., 2019). Assays performed with MMQ and GH3 rat prolactinoma cell lines indicated that the upregulation of miRNA-93-5p may decrease the CAB-induced autophagic cell death by inhibiting the expression level of cleaved caspase-8 and LC3-II, proteins related with CAB-induced apoptosis and autophagy, respectively. Frontiers in Genetics frontiersin.org 1.5.3 Multiple endocrine neoplasia Multiple endocrine neoplasia type 1 (MEN1) is an autosomal dominant disorder characterized by various endocrine and non- endocrine tumors. It is a rare disorder, given that it affects between 3–20 in every 100,000 people (Brandi et al., 2021). Recently, miR- 486-3p was found to be notably downregulated in Men1+/−mice’s adrenal glands (Li et al., 2021). miR-486-3p was proven to target fatty acid synthase (FASN), which is involved with adrenocortical proliferation. Thus, it is theorized that miR-486-3p downregulation might play a crucial role in adrenal tumorigenesis (Li et al., 2021). Moreover, a significant downregulation of miR-3156-5p was observed in serum samples of MEN1 patients compared to controls. This downregulation may be the result of a reduction in MEN1 expression (Kooblall et al., 2022). It was also found that miR- 3156-5p directly targets MORF4L2, given that miR-3156-5p upregulation causes a decrease of MORF4L2 expression, which plays an important role in the activation of oncogene and proto- oncogene-mediated growth induction, as well as in tumor suppressor-mediated growth arrest. Additionally, MORF4L2 is part of the NETest, a useful tool for neuroendocrine tumor (NET) subtype management and diagnosis, therefore, it might be of utility for MEN1 diagnosis (Kooblall et al., 2022). 1.4 Pituitary tumors Similarly, miRNA-455 and miRNA-125a were significantly downregulated in the adrenal gland of Allgrove syndrome patients with adrenal insufficiency (Bitetto et al., 2022). These miRNAs are known to be involved in the regulation of Scavenger receptor class B-1 (SCARB1), an important factor in steroidogenesis found to be downregulated in the samples. Due to the downregulation of miR- 455 and miR-125a, there is an induction of SCARB1 on the cell surface (Bitetto et al., 2022). proliferation, metastasis, and prognosis of colon cancer cells (You et al., 2019); miR-206 has been reported to increase the migration and proliferation of colorectal cancer cells (Park et al., 2018); and miR- let-7e has been reported to diminish tumor growth (Han et al., 2018). Altogether this information could imply using these four miRNAs as novel targets for the clinical management of NFPA (Zhang et al., 2021). Because of their essential roles in regulating the development of pituitary tumors, miRNAs have significant clinical value. More specifically, miRNAs could be used to diagnose the presence and progression of a tumor and classify it more accurately, predict invasiveness or aggressiveness, treat or regulate the behavior of pituitary tumors, and evaluate the success of an operation. 1.5.1 Cushing’s disease Cushing’s syndrome (CS) is a rare disease caused by an overproduction of cortisol in most cases due to adrenocorticotropic hormone (ACTH)-producing pituitary adenomas (Belaya et al., 2020). In a study where circulating miRNA expression profiles were compared in serum samples of patients with and without CS, miR-182-5p was identified as a potential biomarker due to its persistent considerable differential expression between the samples (Vetrivel et al., 2021). Along with miR-182-5p, other members of the miR-183 cluster, including miR- 96-5p and miR-183-5p, were found to be substantially downregulated in the samples. This cluster is associated with auto-immune, neurological, and psychiatric disorders, as well as with cancer (Vetrivel et al., 2021). In a recent study, upregulation of miR-1247-5p and miR-379-5p was observed in the adrenal tissues of patients with different forms of CS in comparison to controls (Vetrivel et al., 2022). One target of miR-1247-5p and miR-379- 5p was found to be Cytochrome b5 (CYB5A), which was notably downregulated in all forms of CS and is known to intervene in the regulation of androstenedione production. However, other target genes were identified as involved in the WNT signaling pathway, whose dysregulation promotes proliferation in ACTH-secreting pituitary adenomas (Vetrivel et al., 2022). 2 Discussion Endocrine disorders are a significant health issue that decreases the quality of patients’ life and sometimes could be fatal. To date, none of the conventional diagnoses or therapies for endocrine disorders are efficient or precise, which has outstood the urge to develop novel and more functional techniques. Recently, miRNAs have been shown to regulate various vital processes associated with endocrine disorders such as diabetes mellitus, thyroid diseases, osteoporosis, pituitary tumors, Cushing’s syndrome, adrenal insufficiency, and multiple endocrine neoplasia. Although the precise roles of several miRNAs in various signaling pathways during the development of endocrine disorders have yet not been fully elucidated, these small molecules have drawn the attention of global researchers to be used for novel therapeutic purposes. Furthermore, miRNAs have shown promising results as disease biomarkers to detect the disease at an early stage, distinguish among different conditions of the patients, as well as to determine the disease’s severity. Altogether, miRNAs have revealed their potential for developing novel tools and techniques that allow the precise detection and management of endocrine disorders, resulting in a general improvement in the patient’s life quality. 1.4 Pituitary tumors The above-mentioned information indicates that miR-186 has a tumor-suppressive role in pituitary tumors Dysregulation of miRNAs has also been associated with tumor incidence and progression, although the regulatory mechanism of these tiny molecules behind non-functional pituitary adenomas (NFPA) has not yet been clarified. In this context, Zhang et al. (2021) explored the correlation between miRNAs expression levels and NFPA by comparing miRNA expression levels in peripheral blood samples from untreated NFPA patients and healthy controls. Four specific miRNAs: miR-26b, miR-138, miR-206, and miR-let-7e were significantly downregulated in NFPA patients than healthy controls, suggesting the involvement of these molecules in the occurrence and progression of NFPAs. Relevantly, miR-26b has been previously associated with the inhibition of proliferation and metastasis in hepatocellular carcinoma (Lin et al., 2018); miR-138 has been reported as a tumor-suppressor gene related to 13 frontiersin.org 10.3389/fgene.2023.1137017 10.3389/fgene.2023.1137017 Ledesma-Pacheco et al. SEMA4C, SIRPA, SLC11A, THRB, and VGLL3. These genes are thought to be involved in immune response, primary cellular component regulation, and metabolic processes (Fichna et al., 2021). Similarly, miRNA-455 and miRNA-125a were significantly downregulated in the adrenal gland of Allgrove syndrome patients with adrenal insufficiency (Bitetto et al., 2022). These miRNAs are known to be involved in the regulation of Scavenger receptor class B-1 (SCARB1), an important factor in steroidogenesis found to be downregulated in the samples. Due to the downregulation of miR- 455 and miR-125a, there is an induction of SCARB1 on the cell surface (Bitetto et al., 2022). proliferation, metastasis, and prognosis of colon cancer cells (You et al., 2019); miR-206 has been reported to increase the migration and proliferation of colorectal cancer cells (Park et al., 2018); and miR- let-7e has been reported to diminish tumor growth (Han et al., 2018). Altogether this information could imply using these four miRNAs as novel targets for the clinical management of NFPA (Zhang et al., 2021). Because of their essential roles in regulating the development of pituitary tumors, miRNAs have significant clinical value. More specifically, miRNAs could be used to diagnose the presence and progression of a tumor and classify it more accurately, predict invasiveness or aggressiveness, treat or regulate the behavior of pituitary tumors, and evaluate the success of an operation. SEMA4C, SIRPA, SLC11A, THRB, and VGLL3. These genes are thought to be involved in immune response, primary cellular component regulation, and metabolic processes (Fichna et al., 2021). Frontiers in Genetics References MicroRNA-375 inhibits osteogenic differentiation by targeting runt-related transcription factor 2. Exp. Ther. Med. 10 (1), 207–212. doi:10.3892/etm.2015.2477 Bao, X., Wang, G., Yu, S., Sun, J., He, L., Zhao, H., et al. (2021). Transcriptomic analysis identifies a tumor subtype mRNA classifier for invasive non-functioning pituitary neuroendocrine tumor diagnostics. Theranostics 11 (1), 132–146. doi:10. 7150/THNO.47525 Fichna, M., Zurawek, M., Fichna, J. P., and Ruchała, M. (2021). 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A significant upregulation of miR-7977 was found in CD4+ T cells obtained from the peripheral blood of patients with autoimmune Addison’s disease compared to healthy controls (Fichna et al., 2021). Additionally, a positive correlation was found between the amount of co-occurring autoimmune disorders and the abundance of miR-7977, as well as with the expression of miR- 7977 and circulating autoantibodies against thyroid peroxidase (aTPO). Although the particular target of miR-7977 is still unknown, 18 potential targets involved in autoimmunity were identified, and they are ARRB1, CD80, CEBPG, CMKLR1, CRP, ENTPD1, HIPK2, HRH4, IL16, JAG1, MAVS, P2RY2, SEMA3G, 14 Frontiers in Genetics frontiersin.org Ledesma-Pacheco et al. 10.3389/fgene.2023.1137017 Conflict of interest The Supplementary Material for this article can be found online at: https://www.frontiersin.org/articles/10.3389/fgene.2023.1137017/ full#supplementary-material 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. Publisher’s note SJL-P and AGU-P conceived, performed the literature search and wrote the manuscript. ER-J, EG-H, CE-M, and LR-M performed the literature search and contributed to writing the manuscript. AB, SRP, AKD, and SJP critically revised the manuscript. All authors have reviewed and approved the final manuscript. 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Selective synthesis and crystal structures of manganese(I) complexes with a bi- or tridentate terpyridine ligand
Acta crystallographica. Section E, Crystallographic communications
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research communications Selective synthesis and crystal structures of manganese(I) complexes with a bi- or tridentate terpyridine ligand Selective synthesis and crystal structures of manganese(I) complexes with a bi- or tridentate terpyridine ligand ISSN 2056-9890 Kosei Wadayama,a Tsugiko Takaseb and Dai Oyamab* aGraduate School of Science and Engineering, Fukushima University, 1 Kanayagawa, Fukushima 960-1296, Japan, and bDepartment of Natural Sciences and Informatics, Fukushima University, 1, Kanayagawa, Fukushima 960-1296, Japan. *Correspondence e-mail: daio@sss.fukushima-u.ac.jp Received 3 June 2020 Accepted 19 June 2020 Edited by M. Weil, Vienna University of Technology, Austria Keywords: crystal structure; manganese(I) complex; terpyridyl ligand; distinct coordination mode; disorder. CCDC references: 2010792; 2010791 Supporting information: this article has supporting information at journals.iucr.org/e Received 3 June 2020 Accepted 19 June 2020 The crystal structures of two manganese(I) complexes with a different coord- ination mode of the supporting ligand are reported: fac-bromidotricarbonyl(40- phenyl-2,20:60,200-terpyridine-2N,N0)manganese(I), [MnBr(C21H15N3)(CO)3], I, and cis-bromidodicarbonyl(40-phenyl-2,20:60,200-terpyridine-3N,N0,N00)mangan- ese(I), [MnBr(C21H15N3)(CO)2], II. In both complexes, the manganese(I) atom is coordinated by terminal carbonyl ligands, a bromide ion, and a 40-phenyl- 2,20:60,200-terpyridine ligand within a distorted octahedral environment. In I, the metal ion is facially coordinated by three carbonyl ligands and the terpyridine ligand binds in a bidentate fashion. The non-coordinating nitrogen atom in the terpyridine ligand is positioned on the side opposite to the bromido ligand. In II, the metal ion is coordinated by two carbonyl ligands in a cis configuration and the terpyridine ligand binds in a tridentate fashion; notably, one carbonyl and the trans bromido ligand are mutually disordered over two positions. In I, the complex molecules are linked by C—H  Br hydrogen bonds. In II, aromatic – contacts are present, as well as pairs of C—H  Br and C—H  O hydrogen bonds. Edited by M. Weil, Vienna University of Technology, Austria Keywords: crystal structure; manganese(I) complex; terpyridyl ligand; distinct coordination mode; disorder. 1. Chemical context 1. Chemical context 1. Chemical context Carbonylmanganese(I) complexes with polypyridyl ligands are of particular interest as novel active molecules that are able to release CO in response to photoirradiation (Carrington et al., 2013; Chakraborty et al., 2014; Jimenez et al., 2015) or as electrocatalysts of CO2 reduction (Grills et al., 2018; Stanbury et al., 2017). Among these compounds, studies have concentrated mainly on tricarbonyl complexes comprising bidentate polypyridyl supporting ligands; by contrast, only few reports exist on dicarbonyl complexes bearing tridentate ligands (Compain et al., 2015; Machan & Kubiak, 2016). In fact, even though the typically tridentate ligands 2,20:60,200-terpyridine and derivatives thereof coordin- ate to an MnI ion, the majority of them bind the metal ion in a bidentate manner (Compain et al., 2014; Moya et al., 2001). As indicated by the results of studies focusing on the comparison between carbonylmanganese complexes containing bidentate and tridentate terpyridines (Compain et al., 2015; Machan & Kubiak, 2016), investigating the rela- tionship between reactivity and molecular structure is a key research objective. However, comparing these two systems experimentally is difficult, particularly considering that avail- able structural data on complexes comprising tridentate terpyridine ligands are quite scarce. https://doi.org/10.1107/S2056989020008178 1139 1139 Acta Cryst. (2020). E76, 1139–1142 Acta Cryst. (2020). E76, 1139–1142 research communications pyridyl rings remains outside the coordination sphere. The corresponding non-coordinating N atom, N3, is positioned on the side opposite to the Br atom. As a result, the torsion angle between the coordinating and non-coordinating pyridyl rings in I (N2—C13—C14—N3) is much smaller [47.9 (3)] than those reported for related MnI complexes with bidentate terpyridine derivatives (Compain et al., 2014, 2015). The non- coordinating N atom is positioned in proximity of the equa- torial carbonyl ligand (C2 O2), with a short value for the interatomic distance between C2 and N3 [2.900 (4) A˚ ]. Since this distance is considerably shorter than the sum of the two atoms’ van der Waals radii (3.25 A˚ ; Bondi, 1964), evidence suggests that an interaction exists between the free pyridine and the adjacent CO ligand. This interaction may explain the observation that the Mn1—C2 distance [1.840 (3) A˚ ] is longer than the other two corresponding distances in I [Mn1—C1 = 1.805 (3) and Mn1—C3 = 1.796 (3) A˚ ]. Herein, we report the structural characterization of complex fac(CO)-[Mn(tpyPh-2N,N0)(CO)3Br] (I; tpyPh = 40- phenyl-2,20:60,200-terpyridine) comprising a bidentate terpyr- idine-based ligand, which has been synthesized by Moya et al. 1. Chemical context (2001), and the synthesis and characterization of the corre- sponding complex cis(CO)-[Mn(tpyPh-3N,N0,N00)(CO)2Br] (II), whereby the same terpyridine-based ligand is tridentate. The crystal structures of MnI dicarbonyl complexes with tridentate terpyridines have very rarely been reported (Compain et al., 2015), because of the instability in solution of compounds of this type. In II, the carbonyl ligands are in cis configuration, again in accordance with IR data. Differently from I, in II the MnI ion is coordinated by a tridentate terpyridyl ligand, as well as two CO ligands and a Br ion. Only the central Mn—N2 bond is slightly shortened (by 0.05 A˚ ) as a result of geometric constraints. In contrast to I, where no disorder is observed, in II one of the CO ligands (C2 O2) and the Br ligand are mutually disordered over two positions. The dihedral angle between the phenyl pendant and the central pyridyl ring in II is slightly larger than the corresponding angle in I. Specifically, the C10—C11—C19— C20 torsion angle has a value of 19.3 (5) in II and 9.9 (4) in I, but both values indicate an essential quasi-coplanarity. Notably, the extended conjugation made possible by the mentioned quasi-planarity may contribute to an increased stability of these compounds. 5. Synthesis and crystallization In the crystal structure of II, weak C—H  Br and C— H  O hydrogen bonding interactions (Table 2) exist between the terpyridyl ligand and the disordered CO/Br ligands. Additional – interactions [Cg3  Cg2iv = 4.000 (2) and Cg1   Cg1i = 4.128 (3) A˚ ; Cg1, Cg2 and Cg3 are the centroids of the N1/C4–C8, N2/C9–C13 and N3/C14–C18 rings, respec- tively; symmetry codes: (i) 1  x, y, 2  z; (iv) x, y + 1 2, z  1 2] consolidate the crystal packing. These interactions lead to the formation of a three-dimensional network structure (Fig. 4). All the manganese(I) complexes were handled and stored in the dark to minimize exposure to light. Compound I was synthesized as described by Moya et al. (2001). The compound thus obtained proved to be analytically and spectroscopically pure (as determined by microanalysis, IR, UV–vis, and 1H NMR data). Crystals suitable for use in X-ray diffraction experiments were grown by vapor diffusion of diethyl ether into an acetone solution of I. For the synthesis of compound II, bromidopentacarbonyl- manganese(I) (30 mg, 0.11 mmol) and 40-phenyl-2,20:60,200- terpyridine (31 mg, 0.10 mmol) were dissolved in an acetone– water mixture (20/30 ml). The solution thus obtained was refluxed for 24 h; the solvent was then evaporated under reduced pressure, and the resulting solid was placed in diethyl ether (50 ml); the resulting mixture was stirred for 30 min to remove the starting materials and subsequently filtered; the isolated residue was washed with diethyl ether to obtain a yield for the desired complex of 43 mg (86%). Single crystals suitable for X-ray diffraction experiments were grown by slow vapor diffusion of n-hexane into an acetone solution of II. FTIR CO (KBr pellet): 1916 (s), 1838 (s) cm1. research communications Table 1 Hydrogen-bond geometry (A˚ , ) for I. D—H  A D—H H  A D  A D—H  A C7—H4  Br1i 0.95 2.83 3.754 (3) 165 C16—H8  Br1ii 0.95 2.88 3.612 (4) 135 C20—H11  Br1i 0.95 2.92 3.844 (2) 163 Symmetry codes: (i) x þ 1; y  1 2; z þ 3 2; (ii) x þ 1; y þ 1 2; z þ 3 2. Table 2 Hydrogen-bond geometry (A˚ , ) for II. D—H  A D—H H  A D  A D—H  A C5—H2  Br1i 0.95 2.84 3.528 (4) 130 C7—H4  Br1ii 0.95 2.86 3.771 (4) 162 C12—H6  Br2iii 0.95 2.75 3.688 (7) 171 C12—H6  O2iii 0.95 2.55 3.491 (7) 173 C15—H7  Br2iii 0.95 2.81 3.759 (7) 175 C15—H7  O2iii 0.95 2.50 3.447 (7) 172 C16—H8  Br2iv 0.95 2.52 3.286 (7) 138 C16—H8  O2iv 0.95 2.57 3.363 (7) 141 C20—H11  Br1ii 0.95 2.81 3.743 (4) 168 C20—H11  O3ii 0.95 2.55 3.446 (18) 158 C24—H15  Br2iii 0.95 2.84 3.611 (7) 139 Symmetry codes: (i) x þ 1; y; z þ 2; (ii) x; y þ 1 2; z þ 1 2; (iii) x; y þ 1 2; z þ 3 2; (iv) x; y þ 1 2; z  1 2. Table 2 Hydrogen-bond geometry (A˚ , ) for II. 3. Supramolecular features In the crystal structure of I, complex molecules display three kinds of C—H  Br hydrogen bonds (i.e., between the Br ligand and the C—H groups in the coordinating pyridyl ring, the free pyridyl ring, and the phenyl pendant), forming a three-dimensional supramolecular structure (Table 1 and Fig. 3). 2. Structural commentary The molecular structures of compounds I and II are displayed in Figs. 1 and 2, respectively. Although I was prepared by Moya et al. (2001), its structure has not previously been determined. In I and II, the manganese(I) atoms exhibit distorted octahedral coordination environments, similar to those reported for other structurally related complexes (Compain et al., 2014, 2015). In I, the fac configuration of the three CO ligands around the central manganese(I) atom is in agreement with the IR data of the complex and similar to those previously reported for complexes of this type (Compain et al., 2014, 2015). As can be evinced from Fig. 1, the terpyridine ligand exhibits a bidentate coordination with respect to the central MnI atom, so that one of the outer Figure 1 The molecular structure of compound I, with atom labeling and displacement ellipsoids drawn at the 50% probability level. Figure 2 The molecular structure of compound II, with atom labeling and displacement ellipsoids drawn at the 50% probability level. Only the major components (Br1/C2 O2) of the disordered groups are shown. Figure 1 The molecular structure of compound I, with atom labeling and displacement ellipsoids drawn at the 50% probability level. Figure 2 The molecular structure of compound II, with atom labeling and displacement ellipsoids drawn at the 50% probability level. Only the major components (Br1/C2 O2) of the disordered groups are shown. i Figure 2 Figure 1 The molecular structure of compound I, with atom labeling and displacement ellipsoids drawn at the 50% probability level. Figure 2 The molecular structure of compound II, with atom labeling and displacement ellipsoids drawn at the 50% probability level. Only the major components (Br1/C2 O2) of the disordered groups are shown. g The molecular structure of compound II, with atom labeling and displacement ellipsoids drawn at the 50% probability level. Only the major components (Br1/C2 O2) of the disordered groups are shown. 1140 Wadayama et al.  [MnBr(C21H15N3)(CO)3] and [MnBr(C21H15N3)(CO)2] 1140 Wadayama et al.  [MnBr(C21H15N3)(CO)3] and [MnBr(C21H15N3)(CO)2] Acta Cryst. (2020). E76, 1139–1142 4. Database survey With respect to manganese(I) complexes with a tridentate terpyridine derivative ligand of the form cis(CO)- [Mn(tpyR)(CO)2Br], only a single structure, whereby R = p- tolyl, has been reported (Compain et al., 2015). In contrast, some structures of bidentate terpyridine derivative-coordi- nated manganese(I) complexes have been reported by Compain et al. (2014, 2015). Figure 3 The crystal packing of compound I with C—H  Br hydrogen bonds shown as dashed lines. Figure 3 The crystal packing of compound I with C—H  Br hydrogen bonds FTIR CO (KBr pellet): 1916 (s), 1838 (s) cm . Figure 3 The crystal packing of compound I with C—H  Br hydrogen bonds shown as dashed lines. Figure 4 The crystal packing of compound II with C—H  Br and C—H  O hydrogen bonds (blue) and – contacts (green) shown as dashed lines; ring centroids are shown as red spheres. Figure 4 The crystal packing of compound II with C—H  Br and C—H  O hydrogen bonds (blue) and – contacts (green) shown as dashed lines; ring centroids are shown as red spheres. Figure 3 The crystal packing of compound I with C—H  Br hydrogen bonds shown as dashed lines. Figure 4 The crystal packing of compound II with C—H  Br and C—H  O hydrogen bonds (blue) and – contacts (green) shown as dashed lines; ring centroids are shown as red spheres. Figure 3 The crystal packing of compound I with C—H  Br hydrogen bonds shown as dashed lines. g The crystal packing of compound II with C—H  Br and C—H  O hydrogen bonds (blue) and – contacts (green) shown as dashed lines; ring centroids are shown as red spheres. Wadayama et al.  [MnBr(C21H15N3)(CO)3] and [MnBr(C21H15N3)(CO)2] 1141 Wadayama et al.  [MnBr(C21H15N3)(CO)3] and [MnBr(C21H15N3)(CO)2] 1141 1141 Acta Cryst. (2020). E76, 1139–1142 II Crystal data Chemical formula [MnBr(C21H15N3)(CO)3] [MnBr(C21H15N3)(CO)2] Mr 528.24 500.23 Crystal system, space group Monoclinic, P21/c Monoclinic, P21/c Temperature (K) 93 93 a, b, c (A˚ ) 11.6630 (3), 11.6691 (3), 15.8892 (4) 10.497 (3), 14.123 (5), 13.504 (4)  () 103.0774 (7) 96.767 (3) V (A˚ 3) 2106.39 (10) 1988.0 (11) Z 4 4 Radiation type Mo K Mo K  (mm1) 2.56 2.71 Crystal size (mm) 0.15  0.08  0.03 0.20  0.08  0.05 Data collection Diffractometer Rigaku Saturn70 Rigaku Saturn70 Absorption correction Multi-scan (REQAB, Rigaku, 1998) Multi-scan (REQAB, Rigaku, 1998) Tmin, Tmax 0.774, 0.926 0.795, 0.873 No. of measured, independent and observed [F 2 > 2.0(F 2)] reflections 21455, 4813, 4253 19872, 4518, 4016 Rint 0.030 0.028 (sin / )max (A˚ 1) 0.649 0.649 Refinement R[F 2 > 2(F 2)], wR(F 2), S 0.035, 0.092, 1.06 0.046, 0.096, 1.27 No. of reflections 4813 4518 No. of parameters 289 289 No. of restraints 0 3 H-atom treatment H-atom parameters constrained H-atom parameters constrained  max,  min (e A˚ 3) 0.96, 0.32 0.83, 0.80 Computer programs: PROCESS-AUTO (Rigaku, 1998), CrystalClear (Rigaku, 2008), SIR97 (Altomare et al., 1999), SHELXL2018/3 (Sheldrick, 2015), Mercury (Macrae et al., 2020), ORTEP-3 for Windows (Farrugia, 2012), CrystalStructure (Rigaku, 2019), PLATON (Spek, 2020) and publCIF (Westrip, 2010). Crystal data Chemical formula Mr Crystal system, space group Temperature (K) a, b, c (A˚ )  () V (A˚ 3) Z Radiation type  (mm1) Crystal size (mm) Data collection Diffractometer Absorption correction Tmin, Tmax No. of measured, independent and observed [F 2 > 2.0(F 2)] reflections Rint (sin / )max (A˚ 1) Computer programs: PROCESS-AUTO (Rigaku, 1998), CrystalClear (Rigaku, 2008), SIR97 (Altomare et al., 1999), SHELXL2018/3 (Sheldrick, 2015), Mercury (Macrae et al., 2020), ORTEP-3 for Windows (Farrugia, 2012), CrystalStructure (Rigaku, 2019), PLATON (Spek, 2020) and publCIF (Westrip, 2010). Computer programs: PROCESS-AUTO (Rigaku, 1998), CrystalClear (Rigaku, 2008), SIR97 (Altomare et al., 1999), SHELXL2018/3 (Sheldrick, 2015), Mercury (Macrae et al., 2020), ORTEP-3 for Windows (Farrugia, 2012), CrystalStructure (Rigaku, 2019), PLATON (Spek, 2020) and publCIF (Westrip, 2010). Funding information Wood, P. A. (2020). J. Appl. Cryst. 53, 226–235. Funding for this research was provided by: Japan Society for the Promotion of Science (grant No. JP17K05799). Moya, S. A., Pastene, R., Le Bozec, H., Baricelli, P. J., Pardey, A. J. & Moya, S. A., Pastene, R., Le Bozec, H., Baricelli, P. J., Pardey, A. J. & Gimeno, J. (2001). Inorg. Chim. Acta, 312, 7–14. Moya, S. A., Pastene, R., Le Bozec, H., Baricelli, P. Gimeno, J. (2001). Inorg. Chim. Acta, 312, 7–14. Rigaku (1998). REQAB and PROCESS-AUTO. Rigaku Corpora- tion, Tokyo, Japan. 6. Refinement Chakraborty, I., Carrington, S. J. & Mascharak, P. K. (2014). ChemMedChem, 9, 1266–1274. Crystal data, data collection, and structure refinement details are summarized in Table 3. All hydrogen atoms were placed at calculated positions (C—H = 0.95 A˚ ) and refined using a riding model with Uiso(H) = 1.2Ueq(C). In compound II, the CO group and the Br atom trans to it were refined as being disordered over two sets of sites, (Br1/C2 O2) and (Br2/ C3 O3), respectively, with an occupancy ratio of 0.807 (2): 0.193 (2). Compain, J.-D., Bourrez, M., Haukka, M., Deronzier, A. & Chardon- Noblat, S. (2014). Chem. Commun. 50, 2539–2542. Compain, J.-D., Bourrez, M., Haukka, M., Deronzier, Compain, J.-D., Stanbury, M., Trejo, M. & Chardon-Noblat, S. (2015). Eur. J. Inorg. Chem. pp. 5757–5766. Farrugia, L. J. (2012). J. Appl. Cryst. 45, 849–854. Grills, D. C., Ertem, M. Z., McKinnon, M., Ngo, K. T. & Rochford, J. (2018). Coord. Chem. Rev. 374, 173–217. Jimenez, J., Chakraborty, I. & Mascharak, P. K. (2015). Eur. J. Inorg. Chem. pp. 5021–5026. Machan, C. W. & Kubiak, C. P. (2016). Dalton Trans. 45, 17179–17186. Macrae, C. F., Sovago, I., Cottrell, S. J., Galek, P. T. A., McCabe, P., Pidcock, E., Platings, M., Shields, G. P., Stevens, J. S., Towler, M. & Wood, P. A. (2020). J. Appl. Cryst. 53, 226–235. research communications Table 3 Experimental details. Table 3 Experimental details. Altomare, A., Burla, M. C., Camalli, M., Cascarano, G. L., Giacovazzo, C., Guagliardi, A., Moliterni, A. G. G., Polidori, G. & Spagna, R. (1999). J. Appl. Cryst. 32, 115–119. Bondi, A. (1964). J. Phys. Chem. 68, 441–451. Carrington, S. J., Chakraborty, I. & Mascharak, P. K. (2013). Chem. Commun. 49, 11254–11256. References y p Rigaku (2008). CrystalClear. Rigaku Corporation, Tokyo, Japan. Altomare, A., Burla, M. C., Camalli, M., Cascarano, G. L., Giacovazzo, C., Guagliardi, A., Moliterni, A. G. G., Polidori, G. & Spagna, R. (1999). J. Appl. Cryst. 32, 115–119. Bondi, A. (1964). J. Phys. Chem. 68, 441–451. Carrington, S. J., Chakraborty, I. & Mascharak, P. K. (2013). Chem. Commun. 49, 11254–11256. Rigaku (2019). CrystalStructure. Rigaku Corporation, Tokyo, Japan. Altomare, A., Burla, M. C., Camalli, M., Cascarano, G. L., Giacovazzo, C., Guagliardi, A., Moliterni, A. G. G., Polidori, G. & Spagna, R. (1999). J. Appl. Cryst. 32, 115–119. Bondi, A. (1964). J. Phys. Chem. 68, 441–451. Carrington, S. J., Chakraborty, I. & Mascharak, P. K. (2013). Chem. Commun. 49, 11254–11256. Sheldrick, G. M. (2015). Acta Cryst. C71, 3–8. Spek, A. L. (2020). Acta Cryst. E76, 1–11. ( ) Stanbury, M., Compain, J.-D., Trejo, M., Smith, P., Goure´, E. & Chardon-Noblat, S. (2017). Electrochim. Acta, 240, 288–299. Stanbury, M., Compain, J.-D., Trejo, M., Smith, P., Goure´, E. & Stanbury, M., Compain, J.-D., Trejo, M., Smith, P., Goure, E. & Chardon-Noblat, S. (2017). Electrochim. Acta, 240, 288–299. Westrip S P (2010) J Appl Cryst 43 920 925 Chardon-Noblat, S. (2017). Electrochim. Acta, 240, 288–299. Westrip, S. P. (2010). J. Appl. Cryst. 43, 920–925. Chardon Noblat, S. (2017). Electrochim. Acta, 2 Westrip, S. P. (2010). J. Appl. Cryst. 43, 920–925. 1142 Wadayama et al.  [MnBr(C21H15N3)(CO)3] and [MnBr(C21H15N3)(CO)2] Acta Cryst. (2020). E76, 1139–1142 supporting information supporting information a Cryst. (2020). E76, 1139-1142 [https://doi.org/10.1107/S2056989020008178] Selective synthesis and crystal structures of manganese(I) complexes with a bi- or tridentate terpyridine ligand Kosei Wadayama, Tsugiko Takase and Dai Oyama Kosei Wadayama, Tsugiko Takase and Dai Oyama Computing details Data collection: PROCESS-AUTO (Rigaku, 1998) for (I); CrystalClear (Rigaku, 2008) for (II). Cell refinement: PROCESS-AUTO (Rigaku, 1998) for (I); CrystalClear (Rigaku, 2008) for (II). Data reduction: PROCESS-AUTO (Rigaku, 1998) for (I); CrystalClear (Rigaku, 2008) for (II). For both structures, program(s) used to solve structure: SIR97 (Altomare et al., 1999); program(s) used to refine structure: SHELXL2018/3 (Sheldrick, 2015); molecular graphics: Mercury (Macrae et al., 2020), ORTEP-3 for Windows (Farrugia, 2012); software used to prepare material for publication: CrystalStructure (Rigaku, 2019), PLATON (Spek, 2020), publCIF (Westrip, 2010). fac-Bromidotricarbonyl(4′-phenyl-2,2′:6′,2′′-terpyridine-κ2N,N′)manganese(I) (I) sup-1 Acta Cryst. (2020). supporting information E76, 1139-1142 Crystal data [MnBr(C21H15N3)(CO)3] Mr = 528.24 Monoclinic, P21/c a = 11.6630 (3) Å b = 11.6691 (3) Å c = 15.8892 (4) Å β = 103.0774 (7)° V = 2106.39 (10) Å3 Z = 4 F(000) = 1056.00 Dx = 1.666 Mg m−3 Mo Kα radiation, λ = 0.71075 Å Cell parameters from 18973 reflections θ = 3.0–27.5° µ = 2.56 mm−1 T = 93 K Platelet, orange 0.15 × 0.08 × 0.03 mm Data collection Rigaku Saturn70 diffractometer Detector resolution: 7.143 pixels mm-1 ω scans Absorption correction: multi-scan (REQAB, Rigaku, 1998) Tmin = 0.774, Tmax = 0.926 21455 measured reflections 4813 independent reflections 4253 reflections with F2 > 2.0σ(F2) Rint = 0.030 θmax = 27.5°, θmin = 3.0° h = −15→15 k = −15→15 l = −20→19 Refinement Refinement on F2 R[F2 > 2σ(F2)] = 0.035 wR(F2) = 0.092 S = 1.06 4813 reflections 289 parameters 0 restraints Primary atom site location: structure-invariant direct methods Secondary atom site location: difference Fourier map Hydrogen site location: inferred from neighbouring sites H-atom parameters constrained Crystal data [MnBr(C21H15N3)(CO)3] Mr = 528.24 Monoclinic, P21/c a = 11.6630 (3) Å b = 11.6691 (3) Å c = 15.8892 (4) Å β = 103.0774 (7)° V = 2106.39 (10) Å3 Z = 4 F(000) = 1056.00 Dx = 1.666 Mg m−3 Mo Kα radiation, λ = 0.71075 Å Cell parameters from 18973 reflections θ = 3.0–27.5° µ = 2.56 mm−1 T = 93 K Platelet, orange 0.15 × 0.08 × 0.03 mm 4813 independent reflections 4253 reflections with F2 > 2.0σ(F2) Rint = 0.030 θmax = 27.5°, θmin = 3.0° h = −15→15 k = −15→15 l = −20→19 supporting information w = 1/[σ2(Fo2) + (0.0416P)2 + 2.6282P] where P = (Fo2 + 2Fc2)/3 (Δ/σ)max = 0.001 Δρmax = 0.96 e Å−3 Δρmin = −0.32 e Å−3 Special details Refinement. Refinement was performed using all reflections. The weighted R-factor (wR) and goodness of fit (S) are based on F2. R-factor (gt) are based on F. The threshold expression of F2 > 2.0 sigma(F2) is used only for calculating R- factor (gt). w = 1/[σ2(Fo2) + (0.0416P)2 + 2.6282P] where P = (Fo2 + 2Fc2)/3 (Δ/σ)max = 0.001 Δρmax = 0.96 e Å−3 Δρmin = −0.32 e Å−3 S i l d t il Δρmax = 0.96 e Å−3 Δρmin = −0.32 e Å−3 p Refinement. Refinement was performed using all reflections. The weighted R-factor (wR) and goodness of fit (S) are based on F2. R-factor (gt) are based on F. The threshold expression of F2 > 2.0 sigma(F2) is used only for calculating R- factor (gt). Refinement. Refinement was performed using all reflections. The weighted R-factor (wR) and goodness of fit (S) are based on F2. R-factor (gt) are based on F. The threshold expression of F2 > 2.0 sigma(F2) is used only for calculating R- factor (gt). Refinement Primary atom site location: structure-invariant direct methods Secondary atom site location: difference Fourier map Hydrogen site location: inferred from neighbouring sites H-atom parameters constrained sup-1 Acta Cryst. (2020). E76, 1139-1142 supporting information supporting information Fractional atomic coordinates and isotropic or equivalent isotropic displacement parameters (Å2) x y z Uiso*/Ueq Br1 0.61476 (2) 0.17999 (2) 0.88742 (2) 0.02812 (9) Mn1 0.78248 (3) 0.17015 (3) 0.81054 (2) 0.02513 (10) O1 0.99149 (17) 0.17222 (19) 0.73649 (14) 0.0400 (5) O2 0.7925 (2) 0.42515 (18) 0.81993 (14) 0.0444 (5) O3 0.95375 (19) 0.1798 (2) 0.97692 (14) 0.0457 (5) N1 0.76212 (17) −0.00325 (19) 0.81015 (13) 0.0252 (4) N2 0.65411 (17) 0.14582 (19) 0.69692 (13) 0.0238 (4) N3 0.7794 (2) 0.3305 (2) 0.62983 (17) 0.0359 (5) C1 0.9075 (2) 0.1690 (2) 0.76117 (18) 0.0320 (6) C2 0.7848 (3) 0.3278 (3) 0.81319 (19) 0.0354 (6) C3 0.8852 (2) 0.1758 (2) 0.91343 (18) 0.0327 (6) C4 0.8291 (2) −0.0776 (2) 0.86473 (17) 0.0313 (6) H1 0.893046 −0.048384 0.907298 0.038* C5 0.8095 (2) −0.1941 (3) 0.86198 (19) 0.0358 (6) H2 0.860275 −0.243958 0.900658 0.043* C6 0.7144 (2) −0.2373 (3) 0.80188 (19) 0.0350 (6) H3 0.698453 −0.317169 0.798723 0.042* C7 0.6431 (2) −0.1615 (2) 0.74643 (18) 0.0313 (6) H4 0.576614 −0.188952 0.705236 0.038* C8 0.6693 (2) −0.0454 (2) 0.75134 (15) 0.0247 (5) C9 0.6038 (2) 0.0406 (2) 0.69157 (15) 0.0232 (5) C10 0.4996 (2) 0.0143 (2) 0.63271 (15) 0.0232 (5) H5 0.466483 −0.060220 0.632112 0.028* C11 0.44359 (19) 0.0971 (2) 0.57461 (15) 0.0214 (5) C12 0.5003 (2) 0.2023 (2) 0.57681 (16) 0.0248 (5) H6 0.467288 0.260028 0.536409 0.030* C13 0.6042 (2) 0.2244 (2) 0.63698 (16) 0.0254 (5) C14 0.6644 (2) 0.3362 (2) 0.63063 (17) 0.0280 (5) C15 0.6033 (2) 0.4378 (2) 0.62349 (19) 0.0326 (6) H7 0.522062 0.438538 0.624227 0.039* C16 0.6619 (3) 0.5393 (3) 0.6152 (2) 0.0418 (7) H8 0.621545 0.610530 0.610537 0.050* C17 0.7798 (3) 0.5349 (3) 0.6138 (2) 0.0446 (7) H9 0.822144 0.602872 0.607979 0.054* C18 0.8345 (3) 0.4297 (3) 0.6212 (2) 0.0403 (7) H10 0.915543 0.426919 0.620071 0.048* C19 0.3301 (2) 0.0749 (2) 0.51219 (15) 0.0215 (5) Fractional atomic coordinates and isotropic or equivalent isotropic displacement parameters (Å2) l atomic coordinates and isotropic or equivalent isotropic displacement parameters (Å2) Acta Cryst. (2020). supporting information E76, 1139-1142 sup-2 supporting information C20 0.2826 (2) −0.0347 (2) 0.49944 (17) 0.0316 (6) H11 0.324220 −0.097276 0.530401 0.038* C21 0.1756 (3) −0.0542 (3) 0.44231 (19) 0.0389 (7) H12 0.144475 −0.129640 0.434142 0.047* C22 0.1145 (2) 0.0361 (3) 0.39740 (19) 0.0373 (6) H13 0.039539 0.023966 0.359983 0.045* C23 0.1626 (3) 0.1429 (3) 0.4072 (2) 0.0453 (8) H14 0.122516 0.204709 0.374184 0.054* C24 0.2688 (3) 0.1626 (2) 0.4644 (2) 0.0402 (7) H15 0.300073 0.238042 0.470790 0.048* Atomic displacement parameters (Å2) U11 U22 U33 U12 U13 U23 Br1 0.02474 (13) 0.03228 (14) 0.02610 (14) 0.00130 (9) 0.00315 (10) −0.00370 (10) Mn1 0.01744 (18) 0.0327 (2) 0.0221 (2) −0.00087 (14) −0.00207 (14) −0.00159 (15) O1 0.0209 (9) 0.0603 (14) 0.0372 (11) −0.0046 (9) 0.0033 (8) −0.0054 (10) O2 0.0517 (13) 0.0352 (11) 0.0424 (12) −0.0087 (10) 0.0023 (10) −0.0043 (9) O3 0.0325 (11) 0.0571 (14) 0.0377 (12) −0.0050 (9) −0.0125 (9) −0.0002 (10) N1 0.0163 (9) 0.0359 (11) 0.0211 (10) 0.0032 (8) −0.0007 (8) −0.0008 (8) N2 0.0168 (9) 0.0321 (11) 0.0209 (10) 0.0015 (8) 0.0005 (8) −0.0036 (8) N3 0.0263 (11) 0.0384 (13) 0.0427 (14) −0.0042 (9) 0.0072 (10) −0.0009 (10) C1 0.0241 (13) 0.0399 (15) 0.0271 (13) −0.0049 (10) −0.0045 (10) −0.0031 (11) C2 0.0300 (14) 0.0417 (16) 0.0323 (15) −0.0015 (11) 0.0028 (11) −0.0017 (12) C3 0.0303 (14) 0.0343 (14) 0.0312 (14) −0.0039 (11) 0.0019 (11) −0.0009 (11) C4 0.0212 (12) 0.0407 (15) 0.0271 (13) 0.0043 (10) −0.0049 (10) −0.0010 (11) C5 0.0266 (13) 0.0432 (16) 0.0323 (15) 0.0071 (11) −0.0041 (11) 0.0091 (12) C6 0.0300 (13) 0.0368 (15) 0.0345 (15) −0.0005 (11) −0.0005 (11) 0.0060 (12) C7 0.0252 (12) 0.0385 (15) 0.0266 (13) −0.0036 (10) −0.0017 (10) 0.0053 (11) C8 0.0178 (11) 0.0354 (13) 0.0198 (11) 0.0014 (9) 0.0019 (9) 0.0013 (10) C9 0.0178 (10) 0.0328 (13) 0.0186 (11) 0.0010 (9) 0.0031 (9) 0.0008 (9) C10 0.0187 (10) 0.0280 (12) 0.0212 (11) −0.0022 (9) 0.0009 (9) 0.0020 (9) C11 0.0165 (10) 0.0282 (12) 0.0184 (11) 0.0000 (9) 0.0016 (8) −0.0003 (9) C12 0.0185 (11) 0.0276 (12) 0.0263 (12) 0.0017 (9) 0.0007 (9) 0.0011 (10) C13 0.0196 (11) 0.0287 (12) 0.0267 (13) 0.0008 (9) 0.0027 (10) −0.0034 (10) C14 0.0246 (12) 0.0308 (13) 0.0276 (13) −0.0025 (10) 0.0038 (10) −0.0005 (10) C15 0.0259 (12) 0.0313 (13) 0.0412 (15) −0.0036 (10) 0.0089 (11) −0.0030 (11) C16 0.0374 (15) 0.0309 (14) 0.0568 (19) −0.0007 (12) 0.0103 (14) 0.0005 (13) C17 0.0380 (16) 0.0354 (15) 0.060 (2) −0.0105 (13) 0.0105 (14) 0.0009 (14) C18 0.0279 (14) 0.0428 (16) 0.0506 (18) −0.0074 (12) 0.0100 (13) −0.0054 (14) C19 0.0179 (10) 0.0283 (12) 0.0172 (11) 0.0023 (9) 0.0018 (9) −0.0002 (9) C20 0.0300 (13) 0.0320 (13) 0.0278 (13) −0.0007 (10) −0.0039 (11) 0.0056 (11) C21 0.0344 (15) 0.0347 (15) 0.0399 (16) −0.0088 (12) −0.0078 (12) 0.0015 (12) C22 0.0246 (13) 0.0417 (16) 0.0368 (15) 0.0031 (11) −0.0115 (11) −0.0062 (12) C23 0.0420 (17) 0.0332 (15) 0.0465 (18) 0.0079 (13) −0.0194 (14) 0.0018 (13) C24 0.0377 (16) 0.0266 (13) 0.0432 (17) −0.0041 (11) −0.0182 (13) 0.0049 (12) supporting information supporting information Atomic displacement parameters (Å2) sup-3 Acta Cryst. (2020). E76, 1139-1142 sup-3 supporting information supporting information supporting information C9—N2—Mn1 113.14 (16) C15—C16—H8 120.6 C13—N2—Mn1 128.74 (17) C18—C17—C16 118.4 (3) C18—N3—C14 117.3 (2) C18—C17—H9 120.8 O1—C1—Mn1 174.2 (2) C16—C17—H9 120.8 O2—C2—Mn1 175.2 (3) N3—C18—C17 123.6 (3) O3—C3—Mn1 177.3 (3) N3—C18—H10 118.2 N1—C4—C5 123.3 (2) C17—C18—H10 118.2 N1—C4—H1 118.4 C24—C19—C20 117.6 (2) C5—C4—H1 118.4 C24—C19—C11 120.9 (2) C4—C5—C6 118.8 (3) C20—C19—C11 121.5 (2) C4—C5—H2 120.6 C21—C20—C19 121.1 (2) C6—C5—H2 120.6 C21—C20—H11 119.5 C7—C6—C5 118.6 (3) C19—C20—H11 119.5 C7—C6—H3 120.7 C22—C21—C20 119.8 (3) C5—C6—H3 120.7 C22—C21—H12 120.1 C6—C7—C8 119.7 (2) C20—C21—H12 120.1 C6—C7—H4 120.2 C23—C22—C21 119.3 (2) C8—C7—H4 120.2 C23—C22—H13 120.3 N1—C8—C7 121.7 (2) C21—C22—H13 120.3 N1—C8—C9 114.5 (2) C22—C23—C24 120.9 (3) C7—C8—C9 123.7 (2) C22—C23—H14 119.6 N2—C9—C10 122.9 (2) C24—C23—H14 119.6 N2—C9—C8 115.1 (2) C19—C24—C23 121.2 (3) C10—C9—C8 122.0 (2) C19—C24—H15 119.4 C9—C10—C11 120.0 (2) C23—C24—H15 119.4 C8—N1—C4—C5 1.8 (4) Mn1—N2—C13—C14 20.3 (3) Mn1—N1—C4—C5 178.9 (2) C11—C12—C13—N2 −1.1 (4) N1—C4—C5—C6 −1.9 (5) C11—C12—C13—C14 175.4 (2) C4—C5—C6—C7 0.4 (4) C18—N3—C14—C15 −0.3 (4) C5—C6—C7—C8 1.0 (4) C18—N3—C14—C13 177.8 (3) C4—N1—C8—C7 −0.3 (4) N2—C13—C14—N3 47.9 (3) Mn1—N1—C8—C7 −177.7 (2) C12—C13—C14—N3 −128.7 (3) C4—N1—C8—C9 −177.3 (2) N2—C13—C14—C15 −134.0 (3) Mn1—N1—C8—C9 5.3 (3) C12—C13—C14—C15 49.5 (4) C6—C7—C8—N1 −1.1 (4) N3—C14—C15—C16 −0.1 (4) C6—C7—C8—C9 175.7 (2) C13—C14—C15—C16 −178.1 (3) C13—N2—C9—C10 −5.6 (3) C14—C15—C16—C17 0.4 (5) Mn1—N2—C9—C10 164.82 (19) C15—C16—C17—C18 −0.3 (5) C13—N2—C9—C8 173.0 (2) C14—N3—C18—C17 0.5 (5) Mn1—N2—C9—C8 −16.7 (3) C16—C17—C18—N3 −0.2 (5) N1—C8—C9—N2 7.9 (3) C12—C11—C19—C24 −10.3 (4) C7—C8—C9—N2 −169.1 (2) C10—C11—C19—C24 170.7 (3) N1—C8—C9—C10 −173.6 (2) C12—C11—C19—C20 169.1 (2) C7—C8—C9—C10 9.4 (4) C10—C11—C19—C20 −9.9 (4) N2—C9—C10—C11 1.4 (4) C24—C19—C20—C21 −1.8 (4) C8—C9—C10—C11 −177.0 (2) C11—C19—C20—C21 178.7 (3) C9—C10—C11—C12 2.9 (3) C19—C20—C21—C22 −0.2 (5) 113.14 (16) C15—C16—H8 120.6 128.74 (17) C18—C17—C16 118.4 (3) 117.3 (2) C18—C17—H9 120.8 174.2 (2) C16—C17—H9 120.8 175.2 (3) N3—C18—C17 123.6 (3) 177.3 (3) N3—C18—H10 118.2 123.3 (2) C17—C18—H10 118.2 118.4 C24—C19—C20 117.6 (2) 118.4 C24—C19—C11 120.9 (2) 118.8 (3) C20—C19—C11 121.5 (2) 120.6 C21—C20—C19 121.1 (2) 120.6 C21—C20—H11 119.5 118.6 (3) C19—C20—H11 119.5 120.7 C22—C21—C20 119.8 (3) 120.7 C22—C21—H12 120.1 119.7 (2) C20—C21—H12 120.1 120.2 C23—C22—C21 119.3 (2) 120.2 C23—C22—H13 120.3 121.7 (2) C21—C22—H13 120.3 114.5 (2) C22—C23—C24 120.9 (3) 123.7 (2) C22—C23—H14 119.6 122.9 (2) C24—C23—H14 119.6 115.1 (2) C19—C24—C23 121.2 (3) 122.0 (2) C19—C24—H15 119.4 120.0 (2) C23—C24—H15 119.4 1.8 (4) Mn1—N2—C13—C14 20.3 (3) 178.9 (2) C11—C12—C13—N2 −1.1 (4) −1.9 (5) C11—C12—C13—C14 175.4 (2) 0.4 (4) C18—N3—C14—C15 −0.3 (4) 1.0 (4) C18—N3—C14—C13 177.8 (3) −0.3 (4) N2—C13—C14—N3 47.9 (3) −177.7 (2) C12—C13—C14—N3 −128.7 (3) −177.3 (2) N2—C13—C14—C15 −134.0 (3) 5.3 (3) C12—C13—C14—C15 49.5 (4) −1.1 (4) N3—C14—C15—C16 −0.1 (4) 175.7 (2) C13—C14—C15—C16 −178.1 (3) −5.6 (3) C14—C15—C16—C17 0.4 (5) 164.82 (19) C15—C16—C17—C18 −0.3 (5) 173.0 (2) C14—N3—C18—C17 0.5 (5) −16.7 (3) C16—C17—C18—N3 −0.2 (5) 7.9 (3) C12—C11—C19—C24 −10.3 (4) −169.1 (2) C10—C11—C19—C24 170.7 (3) −173.6 (2) C12—C11—C19—C20 169.1 (2) 9.4 (4) C10—C11—C19—C20 −9.9 (4) 1.4 (4) C24—C19—C20—C21 −1.8 (4) −177.0 (2) C11—C19—C20—C21 178.7 (3) 2.9 (3) C19—C20—C21—C22 −0.2 (5) sup-5 Acta Cryst. supporting information Geometric parameters (Å, º) Br1—Mn1 2.5325 (5) C10—H5 0.9500 Mn1—C3 1.796 (3) C11—C12 1.390 (3) Mn1—C1 1.805 (3) C11—C19 1.486 (3) Mn1—C2 1.840 (3) C12—C13 1.388 (3) Mn1—N1 2.037 (2) C12—H6 0.9500 Mn1—N2 2.088 (2) C13—C14 1.496 (3) O1—C1 1.135 (4) C14—C15 1.375 (4) O2—C2 1.143 (3) C15—C16 1.388 (4) O3—C3 1.138 (3) C15—H7 0.9500 N1—C4 1.345 (3) C16—C17 1.381 (4) N1—C8 1.353 (3) C16—H8 0.9500 N2—C9 1.355 (3) C17—C18 1.376 (4) N2—C13 1.355 (3) C17—H9 0.9500 N3—C18 1.346 (4) C18—H10 0.9500 N3—C14 1.347 (3) C19—C24 1.374 (3) C4—C5 1.377 (4) C19—C20 1.391 (4) C4—H1 0.9500 C20—C21 1.386 (4) C5—C6 1.384 (4) C20—H11 0.9500 C5—H2 0.9500 C21—C22 1.377 (4) C6—C7 1.385 (4) C21—H12 0.9500 C6—H3 0.9500 C22—C23 1.361 (4) C7—C8 1.387 (4) C22—H13 0.9500 C7—H4 0.9500 C23—C24 1.379 (4) C8—C9 1.471 (3) C23—H14 0.9500 C9—C10 1.390 (3) C24—H15 0.9500 C10—C11 1.393 (3) C3—Mn1—C1 87.58 (13) C9—C10—H5 120.0 C3—Mn1—C2 86.53 (13) C11—C10—H5 120.0 C1—Mn1—C2 90.48 (13) C12—C11—C10 116.6 (2) C3—Mn1—N1 95.30 (10) C12—C11—C19 121.2 (2) C1—Mn1—N1 95.53 (11) C10—C11—C19 122.3 (2) C2—Mn1—N1 173.78 (11) C13—C12—C11 121.2 (2) C3—Mn1—N2 172.86 (11) C13—C12—H6 119.4 C1—Mn1—N2 96.60 (10) C11—C12—H6 119.4 C2—Mn1—N2 99.18 (11) N2—C13—C12 121.9 (2) N1—Mn1—N2 78.58 (8) N2—C13—C14 120.3 (2) C3—Mn1—Br1 89.33 (9) C12—C13—C14 117.7 (2) C1—Mn1—Br1 176.28 (9) N3—C14—C15 122.7 (2) C2—Mn1—Br1 87.27 (9) N3—C14—C13 116.2 (2) N1—Mn1—Br1 86.80 (6) C15—C14—C13 121.0 (2) N2—Mn1—Br1 86.69 (6) C14—C15—C16 119.1 (3) C4—N1—C8 117.9 (2) C14—C15—H7 120.4 C4—N1—Mn1 126.07 (17) C16—C15—H7 120.4 C8—N1—Mn1 115.98 (16) C17—C16—C15 118.9 (3) C9—N2—C13 117.3 (2) C17—C16—H8 120.6 sup-4 Acta Cryst. (2020). E76, 1139-1142 supporting information Acta Cryst. (2020). E76, 1139-1142 supporting information (2020). E76, 1139-1142 supporting information C9—C10—C11—C19 −178.0 (2) C20—C21—C22—C23 2.7 (5) C10—C11—C12—C13 −3.1 (4) C21—C22—C23—C24 −3.1 (5) C19—C11—C12—C13 177.8 (2) C20—C19—C24—C23 1.4 (5) C9—N2—C13—C12 5.3 (3) C11—C19—C24—C23 −179.1 (3) Mn1—N2—C13—C12 −163.30 (18) C22—C23—C24—C19 1.0 (6) C9—N2—C13—C14 −171.0 (2) Hydrogen-bond geometry (Å, º) D—H···A D—H H···A D···A D—H···A C7—H4···Br1i 0.95 2.83 3.754 (3) 165 C16—H8···Br1ii 0.95 2.88 3.612 (4) 135 C20—H11···Br1i 0.95 2.92 3.844 (2) 163 Symmetry codes: (i) −x+1, y−1/2, −z+3/2; (ii) −x+1, y+1/2, −z+3/2. Hydrogen-bond geometry (Å, º) cis-Bromidodicarbonyl(4′-phenyl-2,2′:6′,2′′-terpyridine-κ3N,N′,N′′)manganese(I) (II) cis-Bromidodicarbonyl(4′-phenyl-2,2′:6′,2′′-terpyridine-κ3N,N′,N′′)manganese(I) (II) Crystal data [MnBr(C21H15N3)(CO)2] Mr = 500.23 Monoclinic, P21/c a = 10.497 (3) Å b = 14.123 (5) Å c = 13.504 (4) Å β = 96.767 (3)° V = 1988.0 (11) Å3 Z = 4 F(000) = 1000.00 Dx = 1.671 Mg m−3 Mo Kα radiation, λ = 0.71075 Å Cell parameters from 5160 reflections θ = 3.0–27.5° µ = 2.71 mm−1 T = 93 K Block, red 0.20 × 0.08 × 0.05 mm F(000) = 1000.00 Dx = 1.671 Mg m−3 Mo Kα radiation, λ = 0.71075 Å Cell parameters from 5160 reflections θ = 3.0–27.5° µ = 2.71 mm−1 T = 93 K Block, red 0.20 × 0.08 × 0.05 mm 4518 independent reflections 4016 reflections with F2 > 2.0σ(F2) Rint = 0.028 θmax = 27.5°, θmin = 3.0° h = −13→13 k = −18→18 l = −17→17 4518 independent reflections 4016 reflections with F2 > 2.0σ(F2) Rint = 0.028 θmax = 27.5°, θmin = 3.0° h = −13→13 k = −18→18 l = −17→17 4518 independent reflections 4016 reflections with F2 > 2.0σ(F2) Rint = 0.028 θmax = 27.5°, θmin = 3.0° h = −13→13 k = −18→18 l = −17→17 Refinement Secondary atom site location: difference Fourier map Hydrogen site location: inferred from neighbouring sites H-atom parameters constrained w = 1/[σ2(Fo2) + (0.0039P)2 + 5.6867P] where P = (Fo2 + 2Fc2)/3 (Δ/σ)max < 0.001 Δρmax = 0.83 e Å−3 Δρmin = −0.80 e Å−3 Secondary atom site location: difference Fourier map Primary atom site location: structure-invariant direct methods sup-6 Acta Cryst. (2020). E76, 1139-1142 sup-6 supporting information p Refinement. Refinement was performed using all reflections. The weighted R-factor (wR) and goodness of fit (S) are based on F2. R-factor (gt) are based on F. The threshold expression of F2 > 2.0 sigma(F2) is used only for calculating R- factor (gt). Refinement. Refinement was performed using all reflections. The weighted R-factor (wR) and goodness of fit (S) are based on F2. R-factor (gt) are based on F. The threshold expression of F2 > 2.0 sigma(F2) is used only for calculating R- factor (gt). Fractional atomic coordinates and isotropic or equivalent isotropic displacement parameters (Å2) x y z Uiso*/Ueq Occ. Acta Cryst. (2020). E76, 1139-1142 Refinement (<1) Br1 0.39194 (6) 0.11344 (3) 0.73710 (4) 0.02535 (17) 0.807 (2) O2 −0.0653 (5) 0.0445 (4) 0.8859 (4) 0.0351 (15) 0.807 (2) C2 0.0332 (9) 0.0641 (9) 0.8536 (10) 0.037 (3) 0.807 (2) Br2 −0.0197 (5) 0.0661 (4) 0.8657 (4) 0.0403 (14) 0.193 (2) O3 0.4377 (18) 0.1006 (12) 0.7111 (13) 0.033 (4)* 0.193 (2) C3 0.339 (3) 0.092 (3) 0.746 (3) 0.076 (12)* 0.193 (2) Mn1 0.18158 (5) 0.08786 (4) 0.80533 (4) 0.02451 (14) O1 0.1797 (3) −0.10749 (19) 0.7376 (2) 0.0359 (6) N1 0.2796 (3) 0.0760 (2) 0.9421 (2) 0.0224 (6) N2 0.2005 (3) 0.22091 (19) 0.8440 (2) 0.0202 (6) N3 0.0937 (3) 0.1521 (2) 0.6822 (2) 0.0219 (6) C1 0.1807 (4) −0.0334 (3) 0.7637 (3) 0.0310 (8) C4 0.3125 (3) −0.0050 (3) 0.9917 (3) 0.0275 (8) H1 0.288826 −0.063547 0.960043 0.033* C5 0.3784 (4) −0.0070 (3) 1.0856 (3) 0.0298 (8) H2 0.400366 −0.065698 1.117441 0.036* C6 0.4125 (4) 0.0773 (3) 1.1335 (3) 0.0301 (8) H3 0.458098 0.077281 1.198549 0.036* C7 0.3789 (3) 0.1623 (3) 1.0847 (3) 0.0257 (7) H4 0.400583 0.221221 1.116233 0.031* C8 0.3132 (3) 0.1592 (2) 0.9894 (3) 0.0217 (7) C9 0.2711 (3) 0.2443 (2) 0.9303 (3) 0.0212 (7) C10 0.3006 (3) 0.3376 (2) 0.9548 (3) 0.0227 (7) H5 0.351261 0.352547 1.015754 0.027* C11 0.2548 (3) 0.4096 (2) 0.8886 (3) 0.0223 (7) C12 0.1818 (3) 0.3838 (2) 0.7994 (3) 0.0225 (7) H6 0.149023 0.431227 0.753384 0.027* C13 0.1572 (3) 0.2887 (2) 0.7779 (3) 0.0208 (7) C14 0.0918 (3) 0.2483 (2) 0.6854 (3) 0.0210 (7) C15 0.0335 (3) 0.3016 (3) 0.6061 (3) 0.0251 (7) H7 0.033795 0.368774 0.609582 0.030* C16 −0.0251 (3) 0.2560 (3) 0.5220 (3) 0.0274 (8) H8 −0.066553 0.291366 0.467603 0.033* C17 −0.0223 (3) 0.1587 (3) 0.5187 (3) 0.0277 (8) H9 −0.060962 0.125743 0.461531 0.033* C18 0.0376 (3) 0.1095 (3) 0.5994 (3) 0.0262 (7) H10 0.039210 0.042307 0.596314 0.031* C19 0.2863 (3) 0.5109 (2) 0.9128 (3) 0.0247 (7) C20 0.3268 (4) 0.5390 (3) 1.0100 (3) 0.0316 (8) H11 0.333688 0.493598 1.062254 0.038* Fractional atomic coordinates and isotropic or equivalent isotropic displacement parameters (Å Fractional atomic coordinates and isotropic or equivalent isotropic displacement parameters (Å2) Acta Cryst. (2020). E76, 1139-1142 sup-7 supporting information supporting information C21 0.3573 (4) 0.6332 (3) 1.0315 (3) 0.0385 (10) H12 0.385755 0.651422 1.098118 0.046* C22 0.3466 (4) 0.7000 (3) 0.9572 (4) 0.0379 (10) H13 0.367454 0.764157 0.972651 0.045* C23 0.3056 (4) 0.6739 (3) 0.8598 (3) 0.0376 (10) H14 0.297827 0.720240 0.808386 0.045* C24 0.2757 (4) 0.5797 (3) 0.8373 (3) 0.0299 (8) H15 0.247979 0.561819 0.770438 0.036* Atomic displacement parameters (Å2) U11 U22 U33 U12 U13 U23 Br1 0.0222 (3) 0.0293 (3) 0.0246 (3) 0.0054 (2) 0.0032 (2) 0.00736 (19) O2 0.038 (3) 0.028 (3) 0.041 (3) −0.0170 (18) 0.012 (2) 0.000 (2) C2 0.055 (7) 0.023 (3) 0.031 (4) 0.009 (5) −0.006 (5) −0.006 (3) Br2 0.060 (4) 0.0228 (17) 0.035 (2) 0.001 (3) −0.005 (3) −0.0077 (14) Mn1 0.0276 (3) 0.0170 (3) 0.0275 (3) 0.0001 (2) −0.0027 (2) 0.0030 (2) O1 0.0504 (18) 0.0244 (14) 0.0342 (15) 0.0084 (12) 0.0102 (13) 0.0043 (12) N1 0.0202 (14) 0.0197 (14) 0.0276 (15) 0.0010 (11) 0.0042 (12) 0.0048 (12) N2 0.0176 (13) 0.0179 (13) 0.0250 (15) 0.0001 (10) 0.0017 (11) 0.0029 (11) N3 0.0191 (14) 0.0215 (14) 0.0249 (15) 0.0006 (11) 0.0016 (12) 0.0015 (12) C1 0.0266 (19) 0.037 (2) 0.029 (2) −0.0021 (16) 0.0024 (15) 0.0106 (17) C4 0.0248 (18) 0.0231 (17) 0.035 (2) 0.0036 (14) 0.0064 (15) 0.0077 (15) C5 0.0276 (19) 0.0283 (19) 0.034 (2) 0.0070 (15) 0.0064 (16) 0.0115 (16) C6 0.0286 (19) 0.039 (2) 0.0235 (18) 0.0076 (16) 0.0040 (15) 0.0082 (16) C7 0.0239 (17) 0.0283 (18) 0.0252 (18) 0.0022 (14) 0.0041 (14) 0.0017 (14) C8 0.0182 (16) 0.0240 (17) 0.0236 (17) 0.0012 (13) 0.0059 (13) 0.0031 (14) C9 0.0177 (16) 0.0214 (16) 0.0249 (17) 0.0006 (13) 0.0042 (13) 0.0030 (13) C10 0.0192 (16) 0.0247 (17) 0.0244 (18) 0.0003 (13) 0.0028 (13) −0.0012 (14) C11 0.0180 (16) 0.0202 (16) 0.0297 (18) 0.0012 (12) 0.0064 (14) 0.0008 (14) C12 0.0208 (16) 0.0196 (16) 0.0270 (18) 0.0014 (13) 0.0028 (13) 0.0027 (14) C13 0.0172 (15) 0.0224 (17) 0.0232 (17) 0.0012 (12) 0.0036 (13) 0.0041 (13) C14 0.0173 (15) 0.0209 (16) 0.0251 (17) 0.0000 (12) 0.0037 (13) 0.0018 (13) C15 0.0221 (17) 0.0243 (17) 0.0289 (19) 0.0016 (14) 0.0023 (14) 0.0057 (14) C16 0.0225 (17) 0.0333 (19) 0.0261 (19) 0.0011 (15) 0.0016 (14) 0.0059 (15) C17 0.0227 (18) 0.036 (2) 0.0241 (18) −0.0009 (15) 0.0013 (14) −0.0016 (15) C18 0.0224 (17) 0.0247 (17) 0.0311 (19) −0.0016 (14) 0.0020 (14) −0.0005 (15) C19 0.0181 (16) 0.0208 (17) 0.036 (2) −0.0005 (13) 0.0065 (14) −0.0015 (14) C20 0.0290 (19) 0.0240 (18) 0.042 (2) 0.0000 (15) 0.0027 (17) −0.0041 (16) C21 0.031 (2) 0.031 (2) 0.053 (3) −0.0024 (16) 0.0019 (19) −0.0145 (19) C22 0.030 (2) 0.0206 (18) 0.065 (3) −0.0047 (15) 0.014 (2) −0.0097 (19) C23 0.038 (2) 0.0211 (18) 0.057 (3) −0.0022 (16) 0.018 (2) 0.0041 (18) C24 0.0288 (19) 0.0221 (17) 0.040 (2) 0.0013 (14) 0.0081 (16) 0.0006 (16) Atomic displacement parameters (Å2) supporting information supporting information Br1—Mn1 2.5170 (11) C10—H5 0.9500 O2—Br2 0.654 (5) C11—C12 1.398 (5) O2—C2 1.201 (11) C11—C19 1.496 (5) C2—Br2 0.597 (8) C12—C13 1.392 (5) C2—Mn1 1.790 (9) C12—H6 0.9500 O3—C3 1.194 (18) C13—C14 1.468 (5) Mn1—C1 1.803 (4) C14—C15 1.390 (5) Mn1—N2 1.954 (3) C15—C16 1.384 (5) Mn1—N1 2.012 (3) C15—H7 0.9500 Mn1—N3 2.019 (3) C16—C17 1.376 (5) O1—C1 1.103 (5) C16—H8 0.9500 N1—C4 1.350 (4) C17—C18 1.380 (5) N1—C8 1.364 (4) C17—H9 0.9500 N2—C9 1.346 (4) C18—H10 0.9500 N2—C13 1.351 (4) C19—C20 1.390 (5) N3—C18 1.343 (4) C19—C24 1.402 (5) N3—C14 1.360 (4) C20—C21 1.390 (5) C4—C5 1.371 (5) C20—H11 0.9500 C4—H1 0.9500 C21—C22 1.371 (6) C5—C6 1.381 (6) C21—H12 0.9500 C5—H2 0.9500 C22—C23 1.385 (6) C6—C7 1.395 (5) C22—H13 0.9500 C6—H3 0.9500 C23—C24 1.393 (5) C7—C8 1.387 (5) C23—H14 0.9500 C7—H4 0.9500 C24—H15 0.9500 C8—C9 1.481 (5) O3—Br1—C3 131 (4) N2—C9—C8 111.5 (3) Br2—O2—C2 15.7 (10) C10—C9—C8 126.8 (3) Br2—C2—O2 17.2 (12) C9—C10—C11 119.2 (3) O2—C2—Mn1 177.5 (10) C9—C10—H5 120.4 C2—Br2—O2 147 (2) C11—C10—H5 120.4 Br1—O3—C3 24 (2) C12—C11—C10 118.2 (3) Br1—C3—O3 24.3 (19) C12—C11—C19 121.5 (3) C2—Mn1—C1 87.9 (4) C10—C11—C19 120.3 (3) C2—Mn1—N2 98.6 (4) C13—C12—C11 120.0 (3) C1—Mn1—N2 173.57 (15) C13—C12—H6 120.0 C2—Mn1—N1 91.3 (4) C11—C12—H6 120.0 C1—Mn1—N1 100.98 (14) N2—C13—C12 120.4 (3) N2—Mn1—N1 79.05 (12) N2—C13—C14 112.0 (3) C2—Mn1—N3 93.0 (4) C12—C13—C14 127.5 (3) C1—Mn1—N3 100.66 (15) N3—C14—C15 121.5 (3) N2—Mn1—N3 79.05 (12) N3—C14—C13 114.1 (3) N1—Mn1—N3 158.08 (12) C15—C14—C13 124.4 (3) C2—Mn1—Br1 177.4 (4) C16—C15—C14 119.6 (3) C1—Mn1—Br1 89.68 (12) C16—C15—H7 120.2 N2—Mn1—Br1 83.89 (8) C14—C15—H7 120.2 N1—Mn1—Br1 88.42 (8) C17—C16—C15 118.8 (3) Acta Cryst. (2020). supporting information E76, 1139-1142 sup-9 supporting information pp g 88.26 (8) C17—C16—H8 120.6 117.4 (3) C15—C16—H8 120.6 126.8 (3) C16—C17—C18 119.1 (3) 115.8 (2) C16—C17—H9 120.4 120.4 (3) C18—C17—H9 120.4 119.7 (2) N3—C18—C17 123.1 (3) 119.3 (2) N3—C18—H10 118.4 117.9 (3) C17—C18—H10 118.4 126.7 (2) C20—C19—C24 118.5 (3) 115.5 (2) C20—C19—C11 121.0 (3) 179.5 (4) C24—C19—C11 120.5 (3) 123.2 (4) C21—C20—C19 120.5 (4) 118.4 C21—C20—H11 119.8 118.4 C19—C20—H11 119.8 119.4 (3) C22—C21—C20 120.6 (4) 120.3 C22—C21—H12 119.7 120.3 C20—C21—H12 119.7 118.9 (3) C21—C22—C23 120.1 (4) 120.5 C21—C22—H13 120.0 120.5 C23—C22—H13 120.0 118.8 (3) C22—C23—C24 119.8 (4) 120.6 C22—C23—H14 120.1 120.6 C24—C23—H14 120.1 122.3 (3) C23—C24—C19 120.5 (4) 113.7 (3) C23—C24—H15 119.8 124.0 (3) C19—C24—H15 119.8 121.7 (3) −0.7 (5) C11—C12—C13—N2 1.8 (5) −179.2 (3) C11—C12—C13—C14 −174.8 (3) 0.7 (6) C18—N3—C14—C15 0.3 (5) 0.0 (5) Mn1—N3—C14—C15 −179.4 (3) −0.5 (5) C18—N3—C14—C13 −179.0 (3) 0.1 (5) Mn1—N3—C14—C13 1.3 (4) 178.8 (3) N2—C13—C14—N3 −3.4 (4) −178.9 (3) C12—C13—C14—N3 173.5 (3) −0.2 (4) N2—C13—C14—C15 177.4 (3) 0.5 (5) C12—C13—C14—C15 −5.7 (6) 179.4 (3) N3—C14—C15—C16 0.5 (5) 0.7 (5) C13—C14—C15—C16 179.7 (3) 171.8 (2) C14—C15—C16—C17 −1.0 (5) −177.5 (3) C15—C16—C17—C18 0.7 (5) −6.4 (4) C14—N3—C18—C17 −0.6 (5) 4.1 (4) Mn1—N3—C18—C17 179.1 (3) −174.9 (3) C16—C17—C18—N3 0.1 (6) −174.0 (3) C12—C11—C19—C20 162.1 (3) 7.0 (5) C10—C11—C19—C20 −19.3 (5) 0.6 (5) C12—C11—C19—C24 −18.0 (5) pp g N3—Mn1—Br1 88.26 (8) C17—C16—H8 120.6 C4—N1—C8 117.4 (3) C15—C16—H8 120.6 C4—N1—Mn1 126.8 (3) C16—C17—C18 119.1 (3) C8—N1—Mn1 115.8 (2) C16—C17—H9 120.4 C9—N2—C13 120.4 (3) C18—C17—H9 120.4 C9—N2—Mn1 119.7 (2) N3—C18—C17 123.1 (3) C13—N2—Mn1 119.3 (2) N3—C18—H10 118.4 C18—N3—C14 117.9 (3) C17—C18—H10 118.4 C18—N3—Mn1 126.7 (2) C20—C19—C24 118.5 (3) C14—N3—Mn1 115.5 (2) C20—C19—C11 121.0 (3) O1—C1—Mn1 179.5 (4) C24—C19—C11 120.5 (3) N1—C4—C5 123.2 (4) C21—C20—C19 120.5 (4) N1—C4—H1 118.4 C21—C20—H11 119.8 C5—C4—H1 118.4 C19—C20—H11 119.8 C4—C5—C6 119.4 (3) C22—C21—C20 120.6 (4) C4—C5—H2 120.3 C22—C21—H12 119.7 C6—C5—H2 120.3 C20—C21—H12 119.7 C5—C6—C7 118.9 (3) C21—C22—C23 120.1 (4) C5—C6—H3 120.5 C21—C22—H13 120.0 C7—C6—H3 120.5 C23—C22—H13 120.0 C8—C7—C6 118.8 (3) C22—C23—C24 119.8 (4) C8—C7—H4 120.6 C22—C23—H14 120.1 C6—C7—H4 120.6 C24—C23—H14 120.1 N1—C8—C7 122.3 (3) C23—C24—C19 120.5 (4) N1—C8—C9 113.7 (3) C23—C24—H15 119.8 C7—C8—C9 124.0 (3) C19—C24—H15 119.8 N2—C9—C10 121.7 (3) C8—N1—C4—C5 −0.7 (5) C11—C12—C13—N2 1.8 (5) Mn1—N1—C4—C5 −179.2 (3) C11—C12—C13—C14 −174.8 (3) N1—C4—C5—C6 0.7 (6) C18—N3—C14—C15 0.3 (5) C4—C5—C6—C7 0.0 (5) Mn1—N3—C14—C15 −179.4 (3) C5—C6—C7—C8 −0.5 (5) C18—N3—C14—C13 −179.0 (3) C4—N1—C8—C7 0.1 (5) Mn1—N3—C14—C13 1.3 (4) Mn1—N1—C8—C7 178.8 (3) N2—C13—C14—N3 −3.4 (4) C4—N1—C8—C9 −178.9 (3) C12—C13—C14—N3 173.5 (3) Mn1—N1—C8—C9 −0.2 (4) N2—C13—C14—C15 177.4 (3) C6—C7—C8—N1 0.5 (5) C12—C13—C14—C15 −5.7 (6) C6—C7—C8—C9 179.4 (3) N3—C14—C15—C16 0.5 (5) C13—N2—C9—C10 0.7 (5) C13—C14—C15—C16 179.7 (3) Mn1—N2—C9—C10 171.8 (2) C14—C15—C16—C17 −1.0 (5) C13—N2—C9—C8 −177.5 (3) C15—C16—C17—C18 0.7 (5) Mn1—N2—C9—C8 −6.4 (4) C14—N3—C18—C17 −0.6 (5) N1—C8—C9—N2 4.1 (4) Mn1—N3—C18—C17 179.1 (3) C7—C8—C9—N2 −174.9 (3) C16—C17—C18—N3 0.1 (6) N1—C8—C9—C10 −174.0 (3) C12—C11—C19—C20 162.1 (3) C7—C8—C9—C10 7.0 (5) C10—C11—C19—C20 −19.3 (5) N2—C9—C10—C11 0.6 (5) C12—C11—C19—C24 −18.0 (5) sup-10 Acta Cryst. Symmetry codes: (i) −x+1, −y, −z+2; (ii) x, −y+1/2, z+1/2; (iii) −x, y+1/2, −z+3/2; (iv) x, −y+1/2, z−1/2. Acta Cryst. (2020). E76, 1139-1142 supporting information (2020). E76, 1139-1142 sup-10 supporting information C8—C9—C10—C11 178.5 (3) C10—C11—C19—C24 160.6 (3) C9—C10—C11—C12 −0.7 (5) C24—C19—C20—C21 −0.7 (6) C9—C10—C11—C19 −179.4 (3) C11—C19—C20—C21 179.2 (3) C10—C11—C12—C13 −0.5 (5) C19—C20—C21—C22 0.7 (6) C19—C11—C12—C13 178.2 (3) C20—C21—C22—C23 −0.2 (6) C9—N2—C13—C12 −1.9 (5) C21—C22—C23—C24 −0.3 (6) Mn1—N2—C13—C12 −173.0 (2) C22—C23—C24—C19 0.3 (6) C9—N2—C13—C14 175.2 (3) C20—C19—C24—C23 0.2 (5) Mn1—N2—C13—C14 4.1 (4) C11—C19—C24—C23 −179.7 (3) Hydrogen-bond geometry (Å, º) D—H···A D—H H···A D···A D—H···A C5—H2···Br1i 0.95 2.84 3.528 (4) 130 C7—H4···Br1ii 0.95 2.86 3.771 (4) 162 C12—H6···Br2iii 0.95 2.75 3.688 (7) 171 C12—H6···O2iii 0.95 2.55 3.491 (7) 173 C15—H7···Br2iii 0.95 2.81 3.759 (7) 175 C15—H7···O2iii 0.95 2.50 3.447 (7) 172 C16—H8···Br2iv 0.95 2.52 3.286 (7) 138 C16—H8···O2iv 0.95 2.57 3.363 (7) 141 C20—H11···Br1ii 0.95 2.81 3.743 (4) 168 C20—H11···O3ii 0.95 2.55 3.446 (18) 158 C24—H15···Br2iii 0.95 2.84 3.611 (7) 139 Symmetry codes: (i) −x+1, −y, −z+2; (ii) x, −y+1/2, z+1/2; (iii) −x, y+1/2, −z+3/2; (iv) x, −y+1/2, z−1/2. C8—C9—C10—C11 178.5 (3) C10—C11—C19—C24 160.6 (3) C9—C10—C11—C12 −0.7 (5) C24—C19—C20—C21 −0.7 (6) C9—C10—C11—C19 −179.4 (3) C11—C19—C20—C21 179.2 (3) C10—C11—C12—C13 −0.5 (5) C19—C20—C21—C22 0.7 (6) C19—C11—C12—C13 178.2 (3) C20—C21—C22—C23 −0.2 (6) C9—N2—C13—C12 −1.9 (5) C21—C22—C23—C24 −0.3 (6) Mn1—N2—C13—C12 −173.0 (2) C22—C23—C24—C19 0.3 (6) C9—N2—C13—C14 175.2 (3) C20—C19—C24—C23 0.2 (5) Mn1—N2—C13—C14 4.1 (4) C11—C19—C24—C23 −179.7 (3) Hydrogen-bond geometry (Å, º) Hydrogen-bond geometry (Å, º) D—H···A D—H H···A D···A D—H···A C5—H2···Br1i 0.95 2.84 3.528 (4) 130 C7—H4···Br1ii 0.95 2.86 3.771 (4) 162 C12—H6···Br2iii 0.95 2.75 3.688 (7) 171 C12—H6···O2iii 0.95 2.55 3.491 (7) 173 C15—H7···Br2iii 0.95 2.81 3.759 (7) 175 C15—H7···O2iii 0.95 2.50 3.447 (7) 172 C16—H8···Br2iv 0.95 2.52 3.286 (7) 138 C16—H8···O2iv 0.95 2.57 3.363 (7) 141 C20—H11···Br1ii 0.95 2.81 3.743 (4) 168 C20—H11···O3ii 0.95 2.55 3.446 (18) 158 C24—H15···Br2iii 0.95 2.84 3.611 (7) 139 sup-11 Acta Cryst. (2020). E76, 1139-1142 sup-11
https://openalex.org/W4312964994
https://revistas.ucm.es/index.php/ANHA/article/download/83058/4564456561480
es
Memoria oral del pueblo Mapuche en la reforma agraria y la dictadura. Aplicación del archivo en el proyecto Dungun, instalación sonora interactiva
Anales de historia del arte
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FORO Anales de Historia del Arte ISSN: 0214-6452 https://dx.doi.org/10.5209/anha.83058 Memoria oral del pueblo Mapuche en la reforma agraria y la dictadura. Aplicación del archivo en el proyecto Dungun, instalación sonora interactiva Luis Urquieta Robles1 Recibido: 1 de febrero de 2022 / Aceptado: 6 de junio de 2022 Resumen. El presente escrito se enmarca dentro de una pasantía realizada durante el año 2021 en el Museo de la Memoria y los Derechos Humanos, en Santiago de Chile. La finalidad de dicho proceso fue la utilización del extenso archivo del que dispone esta institución, tanto escrito como audiovisual, para trabajar y reflexionar en torno a un complejo proceso de la historia reciente de este país, las comunidades Mapuche durante la reforma agraria y la posterior dictadura perpetrada por Augusto Pinochet. Dicha etapa se encuentra soterrada debido a los graves crímenes de lesa humanidad que sufrió todo Chile durante esa fatídica época, relegando a un segundo plano las violaciones contra el pueblo Mapuche. El trabajo investigativo en este Museo culminó con la creación de una propuesta de una instalación artística titulada Dungun, un proyecto interactivo sonoro que se nutre de toda la información recopilada durante la pasantía, generando una propuesta estética sonora en torno a la memoria, el archivo y el acto de escuchar. Palabras clave: museo; archivo; Mapuche; memoria; instalación; arte sonoro. [en] Oral Memory of the Mapuche People during the Agrarian Reform and the Dictatorship. Application of the Archive in the Dungun Project, Interactive Sound Installation Abstract. This paper is part of an internship carried out during the year 2021 at the Museum of Memory and Human Rights in Santiago, Chile. The purpose of this process was the use of the extensive archive that this institution has, both written and audiovisual, to work and reflect on a complex process of the recent history of this country, the Mapuche communities during the agrarian reform and the subsequent dictatorship perpetrated by Augusto Pinochet. This stage is buried due to the serious crimes against humanity suffered by all Chile during that fateful time, relegating to the background the violations against the Mapuche people. The research work in this museum culminated with the creation of an artistic installation proposal entitled Dungun, an interactive sound project that draws on all the information collected during the internship, generating an aesthetic sound proposal around memory, the archive and the act of listening. Keywords: museum; archive; Mapuche; memory; installation; sound art. Sumario: 1. Introducción. 2. Las comunidades Mapuche y la reforma agraria. 3. Dictadura en la Araucanía. 4. La historia oral y el archivo en la preservación de la memoria. 5. Proyecto arte sonoro Dungun. 6. Referencias. 7. Instalación. 8. Conclusiones. 9. Conflicto de intereses. 10. Referencias bibliográficas. 1 Universidad Politécnica de Valencia (UPV). E-mail: lurquieta.robles@gmail.com An. his. arte 32 (2022): 77-97 TERCERAS-AnalesHdA32.indd 77 77 4/10/22 16:26 Urquieta Robles, L. An. his. arte 32 (2022): 77-97 78 Cómo citar: Urquieta Robles, Luis (2022). Memoria oral del pueblo Mapuche en la reforma agraria y la dictadura. Aplicación del archivo en el proyecto Dungun, instalación sonora interactiva, en Anales de Historia del Arte nº 32 (2022), 77-97. 1. Introducción El inicio de la memoria oral, abordada desde el punto de vista de la institucionalidad historiográfica, de archivo o documental, está vinculada de manera irrestricta a los procesos sociales de nuestra historia más reciente. Fue en 1948 cuando el concepto de historia oral comenzó a ser utilizado. Allan Nevins, profesor de la Universidad de Columbia, funda la Oral History Research Office en la misma institución2. En el caso del Cono Sur, también tenemos importantes ejemplos de cómo se usó la historia oral para preservar las memorias de personas vinculadas a colectividades que lograron sobrevivir, como familiares de detenidos desaparecidos o torturados políticos en Argentina, a raíz de la cruenta dictadura de Videla de 1976. Lo mismo ocurre en el caso chileno de 1973 con Pinochet. Sobre este último, el Museo de la Memoria y los Derechos Humanos (MMDH) ha realizado una enorme labor de archivar, documentar y crear memoria en torno a los crímenes de lesa humanidad ocurridos bajo la dictadura cívico-militar de Augusto Pinochet, disponiendo de una extensa documentación de casos. Para este artículo, nos centraremos en aquellas zonas rurales donde se vio afectada la población mapuche3. Como veremos más adelante, este vasto archivo de memoria oral mapuche será aplicado en un caso concreto expositivo de una obra de arte sonoro. Para ello, ahondaremos en los antecedentes históricos sobre el pueblo Mapuche, la vida y las condiciones de las comunidades, previas y durante el golpe cívico militar. Después de ello, indagaremos en torno al archivo oral mapuche que posee el Museo y su importancia tanto en lo concerniente a la memoria como a sus aspectos técnicos, y cómo estas cuestiones se pueden abordar en una obra de arte, a través de un caso en concreto. 2. Las comunidades Mapuche y la reforma agraria Hasta los primeros atisbos de la reforma agraria en Chile durante el año 19624, las comunidades Mapuche sólo disponían de pequeñas mercedes de tierra, que les fue2 3 4 Si bien los inicios de la historia oral, como tal, se enfocaron en personalidades ilustres, como Grover Cleveland, presidente de los Estados Unidos, con el tiempo, el enfoque usado en la memoria oral fue dando un giro hacia la historia social, poniendo el énfasis en personas comunes, que, al dar sus testimonios, permiten mantener la memoria de determinados hechos de colectividades más invisibilizadas. Este cambio estuvo influenciado en gran medida por lo realizado previamente por estudios sociológicos en la Universidad de Chicago, los cuales abordaban temáticas vinculadas a las capas marginales de la sociedad. En este informe usaremos la palabra «Mapuche» en singular para referirnos a la comunidad en su conjunto, pues, consideramos que, al tratarse de una palabra que significa «gente de la tierra», ya tiene de manera implícita la pluralidad. Por otra parte, estimamos que debemos respetar el modo en que el mismo pueblo Mapuche se llama a sí mismo, sobre todo si se trata de un acto de resistencia a partir de su lengua, tema principal de este artículo. Nos abocaremos únicamente al proceso de la reforma agraria en adelante, sin profundizar en el conflicto previo entre el Estado chileno y el pueblo Mapuche, durante la llamada «Pacificación de la Araucanía». TERCERAS-AnalesHdA32.indd 78 4/10/22 16:26 Urquieta Robles, L. An. his. arte 32 (2022): 77-97 79 ron entregadas en compensación a todo su terreno perdido durante la «Pacificación de la Araucanía», reduciendo su territorio de manera drástica, no sólo en tamaño sino también en calidad, ya que las tierras entregadas estaban en lugares cordilleranos o muy próximos a la costa, haciendo muy difícil poder llevar a cabo una buena labor en lo agrícola. Tras la derrota durante la Ocupación de la Araucanía, «cinco millones de hectáreas entre el Malleco y Valdivia son rematadas»5, obligando al Mapuche a desplazarse a otras zonas. Muy pocos pudieron quedarse en sus terrenos ancestrales, debiendo convivir, en muchos casos, con colonos tanto chilenos como extranjeros. «En este periodo sólo retuvieron unas 510 mil hectáreas, es decir, el 6,3% de su territorio original»6. Esta estrategia fue muy beneficiosa para el gobierno, ya que pudo hacerse con una extensa cantidad de hectáreas, las cuales comenzaron a ser destinadas a las plantaciones agrícolas. La situación territorial de las comunidades Mapuche no varió positivamente hasta 1962, año en que se produjo un hecho que, más allá de lo tangible en términos de recuperación de territorios, se convirtió en un momento simbólico que propició el empoderamiento de dichas comunidades. Durante ese año, se dio la primera reforma agraria, impulsada por el gobierno de Alessandri en respuesta al poco aprovechamiento y abandono de la tierra, tanto en los fundos como en grandes latifundios, además de la creciente demanda de alimentos debido a la crisis alimentaria suscitada en ese entonces. Tras su implementación, el resultado de esta medida fue tan exiguo que fue conocida como la «ley macetero», debido a las paupérrimas recuperaciones de territorio para con las comunidades campesinas, alcanzando la cifra de 491 nuevos propietarios agrícolas a nivel nacional7. De éstas, tan solo una pequeña fracción fue destinada al pueblo Mapuche. Como señalamos anteriormente, la Ley Nº 15.020, más allá de cambios administrativos –como la creación de la Corporación de la Reforma Agraria (CORA)8– no generó un cambio sustancial en la entrega de terrenos al campesinado, pero sirvió para posicionar esta demanda en todo el territorio por parte de las comunidades Mapuche, que vieron aquí una gran oportunidad de exigir de manera más formal las tierras arrebatadas tras la guerra ocurrida a mediados del siglo XIX. Con el cambio de presidencia del país y el desplazamiento del eje político de este gobierno, pasando de un mandato de derecha altamente conservador a una postura de centro izquierda, Eduardo Frei Montalva impulsó tempranamente la nueva Ley de Reforma Agraria, despachándola al congreso en 1965, siendo aprobada en 19679. Previamente a la aprobación de la nueva reforma agraria, se lograron expropiar 1.209.975 hectáreas10 a través de la antigua ley, cifra que, al compararla con la administración anterior, habla de la poca voluntad política por parte del gobierno de 5 6 7 8 9 10 Bengoa, J. (1985). Historia del Pueblo Mapuche (siglos XIX y XX). Santiago de Chile: LOM, 336. Araya, J. (1999). Conflicto mapuche: Algunas razones y derechos en este nuevo enfrentamiento. Derechos Humanos y trabajadores. Sobre el derecho de los ex prisioneros políticos a ser indemnizados por el Estado. Santiago de Chile: LOM, 12. Comisión Verdad Histórica y Nuevo Trato con los Pueblos Indígenas. (2008). Informe de la Comisión Verdad Histórica y Nuevo Trato con los Pueblos Indígenas. Santiago de Chile: Gobierno de Chile, 403. La Corporación de Reforma Agraria, nació en 1962 como la empresa estatal encargada de llevar a cabo lo desarrollado por la ley del mismo nombre, efectuando las subdivisiones de tierras que se iban aprobando. Correa, M., Molina, R., y Yáñez, N. (2005). La Reforma Agraria y las tierras mapuche: Chile 1962 – 1975. Santiago de Chile: LOM, 79. Ibid., 80. TERCERAS-AnalesHdA32.indd 79 4/10/22 16:26 Urquieta Robles, L. An. his. arte 32 (2022): 77-97 80 Alessandri por favorecer al campesinado, dada la baja extensión de terreno entregado durante su mandato. Con la promulgación de la Ley de Reforma Agraria Nº 16.640, se amplían las causas para que un predio pueda ser expropiado, adoptando una postura más proactiva para con el campesinado, entregándole capacitación, promoviendo el desarrollo económico de la comunidad y suministrando infraestructura mínima para que la familia beneficiada pueda desempeñar la normal explotación de su terreno. En esta nueva ley, se incluye el «problema indígena» dentro de la discusión, dado que, en la ley anterior, se hablaba únicamente del campesinado. Lamentablemente, todo esto se realizó de manera tangencial, ya que no afrontaba la situación desde la etnicidad de las comunidades, haciendo imposible abordar estas demandas desde lo histórico. Pese a ello, las reivindicaciones mapuches por sus tierras siguieron su curso, obteniendo buenos resultados en aquellas comunidades que se organizaron en torno a Cooperativas y Comités Campesinos, incrementando la posibilidad de acceder a créditos en infraestructura, asesoría técnica, aumento de becas de estudios, etc. Aquel impulso iniciado con la primera reforma agraria ya se hacía notar con mayor fuerza para la segunda mitad de la década de los sesenta. La acción directa cobra relevancia en el quehacer indígena, sin dejar de lado la vía legal, siendo esta última la que daba pie a las tomas de predios y las posteriores corridas de cerco. El número de tomas fue creciendo a medida que las comunidades fueron viendo cómo otras agrupaciones sí estaban teniendo resultados, procediendo a expropiar predios, cada vez con mayor frecuencia. Ejemplo de ello fue lo ocurrido en la comuna de Lumaco, donde la Confederación Indígena Campesina de diversas zonas de la comuna se tomó el fundo Reñico, el cual perteneció a Raimán Pichulmán y había sido usurpado. Para finales de 1969, las tomas de terreno se extendieron en la provincia de Malleco, llegando hasta Ercilla, en el centro de la provincia, dando muestra de que el proceso parecía no tener marcha atrás. Fue en este mismo año que comienzan a suceder las corridas de cerco11, acto que consistía básicamente en restablecer los límites legales existentes hasta antes de lo que había sido usurpado de los Títulos de Merced12. Para ello, se movían los cercos perimetrales hasta el punto señalado anteriormente. Estas medidas insurreccionales se debían a las discrepancias existentes entre, por un lado, las leyes de corte reformista y, por otro lado, las necesidades urgentes y la mirada revolucionaria de muchos sectores del campesinado mapuche, sumadas a la larga e infructuosa historia de peticiones y reclamos que no terminaban en algo concreto. Además hicieron que, para finales de 1970, las tierras recuperadas llegaran a las casi 100.000 hectáreas. La llegada al poder por parte de la Unidad Popular, liderada por Salvador Allende, allanó el camino para que las tomas y corridas de cerco fueran en aumento, sobre todo considerando que, dentro del gobierno de Allende, se contemplaba un programa que se hacía cargo específicamente de la problemática territorial mapuche. Aquí, se hablaba de defender la integridad de las comunidades indígenas asegurando tierras suficientes, apoyo técnico y crediticio para su normal funcionamiento. 11 12 Las corridas de cerco fueron propiciadas en su gran mayoría por el Movimiento Campesino Revolucionario (MCR), agrupación de corta data, que aglutinó personas provenientes del campesinado mapuche y del Movimiento de Izquierda Revolucionario (MIR). Comisión Verdad Histórica y Nuevo Trato con los Pueblos Indígenas. (2008) op.cit., 406. TERCERAS-AnalesHdA32.indd 80 4/10/22 16:26 Urquieta Robles, L. An. his. arte 32 (2022): 77-97 81 Si bien hubo un notable apoyo por parte del gobierno de Allende respecto a las tomas y corridas de cerco, éste mantenía su postura de realizar esta recuperación de territorio por medio de la vía institucional. Para ello, trasladó el Ministerio de Agricultura desde Santiago a la ciudad de Temuco, capital de la provincia de Cautín, constituyendo la «Comisión de Restitución de Tierras Mapuche Usurpadas»13 con la finalidad de poder trabajar en terreno y otorgar una pronta salida a aquellas comunidades donde el nivel de conflictividad era mayor. Para poder concretar los traspasos de tierra a los pueblos originarios, Allende promulga, el 15 de septiembre de 1972, la Ley Nº 17.729, conocida como Ley de Indígenas, la cual tenía la particularidad de restituir las tierras mapuches que fueron perdidas durante el proceso de la Ocupación de la Araucanía. Además, se estipulaba que no se podrían «enajenar, gravar ni dar en arrendamiento o aparcería los goces que posean en la reserva, ni los derechos que les correspondan en la comunidad, excepto en favor de otro u otros miembros de la misma»14. Esta ley, sin duda fue un avance cualitativo y sustancial, no solo en el proceso de recuperación de tierras ancestrales, ya que por primera vez existían medios legales efectivos, sino también en el otorgamiento de dignidad a las comunidades: al ser reconocidas como tales y brindarles diversas ayudas, más allá de su condición de campesinos, eran entregadas por el hecho de ser mapuche. Lamentablemente, este hito no duró mucho. El golpe cívico-militar de Pinochet, derribó inmediatamente estos avances para con las comunidades Mapuche. Es más, en muchos relatos se habla que el golpe de Estado de 1973 no se originó ni en Valparaíso ni en Santiago, y tampoco un 11 de septiembre, sino en la Araucanía y durante el mes de agosto de ese año. Esto se explica porque, debido a la Ley de Control de Armas, se realizó una fuerte fiscalización por parte de las fuerzas armadas y de aire en los caminos rurales de la zona y en particular contra los principales Centros de Reforma Agraria, llevándose a cabo allanamientos y detenciones incluso con resultado de torturas y muerte15. Entre el 4 de noviembre de 1970 y el 11 de septiembre de 1973 se expropiaron en la Araucanía 574 fundos, con una superficie de 636.288,3 hectáreas. Los predios expropiados a favor de comunidades mapuche o con participación mapuche fueron 138, con una superficie total de 132.115,78 hectáreas físicas16. 3. Dictadura en la Araucanía El golpe cívico-militar impulsado por Pinochet tuvo un particular ensañamiento en la actual Región de la Araucanía debido a todo lo ocurrido en relación a la recuperación de tierras por parte de las comunidades Mapuche. Este clima reivindicatorio atrajo, 13 14 15 16 Navarrete, J. (2020). Movimiento Campesino Revolucionario. Lucha mapuche, política de clase y «proyecto socialista» durante el gobierno de la Unidad Popular (Cautín, 1970-1971). En R. Austin (Eds.), La vía al socialismo chileno 50 años después (pp. 495-519). Buenos Aires: Clacso. Chile. (1972). Ley 17.729. Propiedad intelectual. Obtenido de https://www.bcn.cl/leychile/navegar?idNorma=29250 [Consulta: 18 de octubre de 2021]. Correa, M., Molina, R., y Yáñez, N. (2005) op. cit., 244. Comisión Verdad Histórica y Nuevo Trato con los Pueblos Indígenas. (2008) op. cit., 403. TERCERAS-AnalesHdA32.indd 81 4/10/22 16:26 Urquieta Robles, L. An. his. arte 32 (2022): 77-97 82 durante la Unidad Popular, la llegada de «organizaciones políticas como el Partido Socialista, el Partido Comunista, el MAPU-OC (Movimiento de Acción Popular Unitaria, Obrero Campesino), y políticas-militares como el MIR y el PCR a través de sus sub-orgánicas MCR y Netuaiñ Mapu»17. Las personas agrupadas en confederaciones, cooperativas y comités campesinos fueron las primeras en sufrir persecución, tortura y muerte. Todo lo construido a partir de la primera reforma agraria se esfumó. La dictadura de Pinochet acabó con las libertades colectivas eliminando de raíz todo medio de organización, como partidos políticos, entidades públicas indígenas, consejos comunales, organizaciones sociales, cooperativas y comités. Otra arista no menos importante fueron las consecuencias territoriales generadas a partir del golpe de estado. La junta de gobierno encabezada por Pinochet inició una fuerte contrarreforma agraria desmantelando todo lo avanzado en políticas públicas de otorgamiento de tierras a las comunidades indígenas, restituyendo dichos terrenos a sus antiguos dueños. Dicha medida, además, les despojó de sus bienes materiales –herramientas, tractores, etc.– obligando a una gran cantidad de familias a salir de su comunidad sin bienes, en busca de oportunidades laborales; muchas migraron a Temuco o ciudades aledañas a sus comunidades, mientras otras se debieron instalar en Santiago, e incluso huir a Argentina cruzando la cordillera. En términos legislativos, la dictadura de Pinochet en 1978, por medio del Decreto Ley Nº 2.405, disolvió la Corporación de la Reforma Agraria sepultando con esto la reforma iniciada en 1962. En reemplazo de la CORA se constituyó la Oficina de Normalización Agraria (ODENA), la cual tendría la misión de finiquitar toda situación pendiente de la extinta CORA, teniendo la potestad completa de su predecesora18. En 1979 promulgó el Decreto de Ley 2.568 liquidando las comunidades Mapuche19. Hasta esa fecha, la Ley Indígena se mantuvo pero, al eliminar por completo el Instituto de Desarrollo Indígena, la Ley 17.729 no podía ser utilizada. Desde el punto de vista organizativo, se constituyeron entidades, como el Comité Ejecutivo Agrario (CEA), que tenía la finalidad de restituir las tierras a los latifundistas que habían sido expropiados. Con los años, estas tierras arrebatadas a los Mapuche terminaron transformándose en monocultivos de pinos o eucaliptos. La CONAF20 había rematado una gran extensión de hectáreas, las cuales fueron compradas a precios ínfimos por empresas forestales. Dicha aparición se ve favorecida con la aplicación del Decreto Ley Nº 701, de 1974, de fomento forestal, el que estableció́ una serie de incentivos a las plantaciones forestales, entre ellas que el Estado, bonificaba en un 75 % los costos netos de forestación, por un periodo de 10 años, incluidos los gastos de manejo correspondiente en que incurran las personas naturales o jurídicas de cualquier 17 18 19 20 Farías, C. (2012). Pueblo mapuche y dictadura: desde el terror hacia la nueva esperanza. Elementos configurativos para el entendimiento de la represión efectuada por el Régimen Militar hacia el pueblo mapuche. (Informe final de práctica profesional). Santiago de Chile: Museo de la Memoria y los Derechos Humanos. Chile. (1978). Ley 2.405. Propiedad intelectual. Obtenido de https://www.bcn.cl/leychile/navegar?idNorma=6911 [Consulta: 3 de noviembre de 2021]. Comisión Verdad Histórica y Nuevo Trato con los Pueblos Indígenas. (2008) op.cit., 415. La Corporación Nacional Forestal es una institución del Estado creada en 1970 bajo el gobierno de Eduardo Frei Montalva. Esta institución contempla la realización de diversas tareas tales como: administrar las áreas silvestres del país, combatir incendios forestales y administrar las políticas del Estado en torno a lo forestal. TERCERAS-AnalesHdA32.indd 82 4/10/22 16:26 Urquieta Robles, L. An. his. arte 32 (2022): 77-97 83 naturaleza, además de declarar inexpropiables los predios afectos a dicho Decreto, y se declaró la libertad de comercio para todos los productos del bosque. Recién transcurridos 6 meses de vigencia de la norma sufre la primera modificación, y el 25 de marzo de 1975, se amplía a 20 años el plazo para el pago de bonificaciones forestales, es decir las condiciones se hacen aún más beneficiosas para el negocio forestal21. Volviendo a lo referente a las violaciones de derechos humanos en las comunidades Mapuche, estas fueron horrendas, de un ensañamiento brutal, tanto por parte de las fuerzas armadas y los carabineros, como de vecinos no mapuches de las distintas comunidades. La facilidad con la que los militares localizaron a campesinos mapuches vinculados a organizaciones o colaboradores con la reforma agraria se debió al apoyo, tanto logístico como de recursos humanos para cazar, una a una, a personalidades mapuches que tuvieron cierta vinculación en las recuperaciones de terrenos. Los testimonios de los sobrevivientes a la tortura de estos deleznables hechos fueron vitales para la conformación de un archivo oral respecto a sus vivencias durante la dictadura, situación sobre la que ahondaremos en el siguiente apartado. 4. La historia oral y el archivo en la preservación de la memoria La historia oral, como señalamos al inicio, nació de la mano de Nevins con su trabajo sobre el presidente Grover Cleveland, una investigación en torno a la vida y obra de esta personalidad de la sociedad estadounidense. Con los años, este modo de hacer historia fue variando, extendiéndose a otras latitudes, acuñando otras miradas vinculadas con la historia social y sus modos de entender esta parte de las ciencias sociales, buscando abrir espacios a aquellos grupos menos favorecidos en diversos procesos que, por resultar perdedores en un hecho, no son considerados como efectivamente lo merecen en los libros oficiales y registros de historia. Estos antecedentes los encontramos en hitos como el trabajo de Ronald Fraser en España, rescatando testimonios de personas vinculadas al bando republicano, perdedores de la Guerra Civil española; la labor realizada por Oscar Lewis en México, investigando sobre la vida de la gente pobre de la capital durante finales de la década del cincuenta; o los History Workshops de Raphael Samuel en Reino Unido22, los cuales buscaban construir un nuevo paradigma en torno a estudios de historia popular en los años sesenta. La historia oral, tal como se conoce ahora, tiene por objetivo rescatar aquellas voces que continúan con vida, que pese a ser excluidas de la historia oficial y ser consideradas partes del medio cotidiano, deben ser estimadas por el hecho de ser testigos o protagonistas de un hecho histórico. De esa manera, es posible constituir un proceso de construcción de fuentes que antes no eran tomadas en cuenta por la historiografía, para así establecer una memoria colectiva a partir de la memoria individual de cada entrevistado. Permite además el desarrollo de proyectos tanto investigativos 21 22 Correa, M., y Mella, E. (2009). El territorio mapuche de Malleco: Las razones del Illkun. Santiago de Chile: ODHPI, 122. Yusta, M. (2002). Historia oral, historia vivida. El uso de las fuentes orales en la investigación histórica. Pandora: revue d’etudes hispaniques, (2). Obtenido de https://dialnet.unirioja.es/servlet/articulo?codigo=3160107 [Consulta: 25 de octubre de 2021]. TERCERAS-AnalesHdA32.indd 83 4/10/22 16:26 Urquieta Robles, L. An. his. arte 32 (2022): 77-97 84 como prácticos, de manera interdisciplinar, aunando diversos enfoques y realizando cruces entre variadas especialidades, otorgando mayor profundidad a un proyecto determinado. A través de ella, se pueden recuperar testimonios de hechos históricos, trabajando de forma paralela con la oficialidad, por medio de personajes invisibilizados por la misma. De este modo se aportan nuevos antecedentes a un mismo hecho, a partir de las diversas miradas existentes. En el caso de Chile, la historia oral tiene una relación directa con el golpe cívico-militar perpetrado por Pinochet que se extendió entre los años 1973 y 1990. Fue durante esta represión y ocultamiento de la información por parte de los medios de comunicación oficiales cuando diversas organizaciones que se encontraban resistiendo en el territorio chileno, apoyados por la iglesia y otras instituciones extranjeras de defensa de los derechos humanos, procedieron a la toma de testimonios, no con un afán (inicialmente) de archivar, sino de defender, proteger y denunciar al mundo las atrocidades cometidas por las fuerzas armadas y de orden23. En ese sentido, el MMDH ha realizado una invaluable labor en términos de archivar, preservar y visibilizar testimonios, tanto de familiares de detenidos desaparecidos (DD.DD.), ejecutadas y ejecutados políticos, como de víctimas de tortura durante la dictadura de Pinochet, transformando dichos testimonios en patrimonio intangible para las futuras generaciones, y lo más importante: aprender del pasado para que desde el presente seamos conscientes de la defensa de los derechos humanos y así no cometer los mismos errores a futuro. Sobre crímenes de lesa humanidad ocurridos en la Araucanía durante la Dictadura, el Museo cuenta con una serie de archivos audiovisuales y documentos escritos alusivos a este fatídico periodo. Dentro de lo destacado, encontramos el trabajo realizado por la Corporación de Promoción y Defensa de los Derechos del Pueblo (CODEPU)24, quienes deciden iniciar un proyecto ante «la necesidad de documentar la represión vivida por los pueblos indígenas durante el periodo de dictadura militar comprendido entre el 11 de septiembre de 1973 y el 10 de marzo de 1990»25. CODEPU denotó esta especificidad al referirse a lo ocurrido en la Araucanía debido a lo complejo de la situación político-social previa al golpe en dicha zona, pero también por lo anómalo del proceso de detenciones y torturas vividas en estos territorios rurales, los cuales impactaron profundamente a la comunidad. Otro punto no menor dentro de las motivaciones de la CODEPU al trabajar con las comunidades Mapuche víctimas de tortura fue que la Comisión Nacional sobre Prisión Política y Tortura (CNPPT), institución encargada en el año 2004 de llevar a cabo el proceso de registro y toma de testimonio a víctimas de tortura, no contaba con los mecanismos para recibir y gestionar los antecedentes de los pueblos originarios vejados en este tipo de crímenes. Fue así como en 2005 CODEPU se dispuso a continuar la labor de registrar los testimonios de dichas víctimas, las cuales no habían compadecido 23 24 25 Museo de la Memoria y los Derechos Humanos. (2013). Archivo Oral Museo de la Memoria y los Derechos Humanos. Proyecto Piloto «Maestranza de San Bernardo» [Ponencia]. V Encuentro Latinoamericano de Historia Oral, San Salvador. Obtenido de http://www.cedocmuseodelamemoria.cl/wpcontent/uploads/2013/05/ MM_Chile_SSalvador.pdf [Consulta: 28 de octubre de 2021]. CODEPU nació en la ciudad de Santiago en noviembre de 1980 con la finalidad de dar soporte y asistencia a las víctimas y a familiares de las víctimas de violaciones de los derechos humanos. Colipi, G. (Ed.). (2011). Mirar hacia atrás. Memoria oral del pueblo mapuche. Una expresión sobre la represión y tortura en la Comuna de Tirúa, Chile (1973-marzo 1990). Santiago de Chile: Caballo de mar, 3. TERCERAS-AnalesHdA32.indd 84 4/10/22 16:26 Urquieta Robles, L. An. his. arte 32 (2022): 77-97 85 frente a la CNPPT, quedando fuera de la Comisión Valech26 de 2004 y la Comisión Rettig27 de 1991, imposibilitando cualquier tipo de reparación por parte del Estado. «Se logró calificar 4.073 personas, que aportaron antecedentes probados de tortura y prisión política, entre ellos a personas del pueblo Mapuche»28. Las comunidades concurrentes a este llamamiento por parte de la CODEPU y la Fundación Salvador Allende prestaron declaración de manera colectiva, siendo esto desestimado posteriormente por las organizaciones convocantes ya que, para continuar con lo realizado por la comisión Valech, debían regirse bajo los mismos preceptos. Fue así como nació la propuesta de memoria oral mapuche, llevada a cabo por CODEPU y titulada «Memoria Mapuche de las Violaciones Graves de Derechos Humanos: Historia oral, documental, fotográfica del pueblo Mapuche de la VII y IX Región durante la Dictadura Militar (1973-1990)»29. A partir de este proyecto de testimonios individualizados, CODEPU propuso la realización de entrevistas a las 46 comunidades que ya habían participado del proceso anterior, creando un archivo pormenorizado de 624 entrevistas, tanto a diversos lonkos30, como a mujeres y hombres de estas colectividades, con la finalidad de rescatar estas memorias y sus experiencias respecto a las violaciones de DDHH sufridas, y crear un respaldo de dichas memorias para, en una segunda etapa del proyecto, poder crear la Casa Museo de la Memoria Mapuche. Para lograr esta recopilación de memorias, se recurrió a la realización de una matriz de entrevistas. En palabras de Eugenia Meyer, «una entrevista puede definirse brevemente como la conversación entre dos o más personas, con una finalidad concreta, que por otra parte está encaminada a obtener cierta información»31. En este caso, la finalidad de dichas entrevistas era poder reflexionar en torno a las comunidades Mapuche durante el periodo comprendido entre 1973 y 1990, sensibilizar respecto a la importancia de preservar estas memorias y reconstruir el pasado individual de cada entrevistado. Para esto, se recurrió a una serie de preguntas ya definidas, tales como nombre, edad, grupo familiar, etc. Las siguientes preguntas guardaban relación con la vida de las y los entrevistados previa al golpe de estado y con el modo en que se desenvolvían con su comunidad y con la gente winka32 del sector; también con lo ocurrido el mismo martes 11 de septiembre de 1973 en su entorno cotidiano y su familia más cercana, con el modo en que actuaron las fuerzas armadas y de orden en su comunidad, si fueron víctimas directas o si tuvieron algún familiar que sufriera represión, las consecuencias físicas y psicológicas de dichos actos, el tiempo en el que se produjeron las vejaciones, el lugar al que fueron trasladados, si hubo civiles 26 27 28 29 30 31 32 La Comisión Nacional Sobre Prisión Política y Tortura fue un organismo chileno dirigido por el sacerdote, Monseñor Sergio Valech, con la finalidad de identificar a las personas víctimas de prisión y tortura llevadas a cabo por agentes del Estado en el periodo comprendido entre 1973 y 1990. La Comisión Nacional de Verdad y Reconciliación fue un organismo chileno creado por el presidente Patricio Aylwin que estuvo presidida por Raúl Rettig entre los años 1990 y 1991. La finalidad de esta comisión era la de esclarecer la verdad respecto a los crímenes de lesa humanidad cometidos por la dictadura de Pinochet entre 1973 y 1990. Colipi, G. (Ed.). (2011) op.cit., 15. Ibid., 17. Lonko, Longko (cabeza) o Cacique, corresponde al líder de una comunidad Mapuche. Meyer, E. (1971). La historia oral. Origen, metodología, desarrollo y perspectivas. Historia Mexicana, volumen 21 (2). Obtenido de https://www.jstor.org/stable/25134913 [Consulta: 8 de septiembre de 2021]. Winka, Wingka o Huinca, es un término mapudungun, que inicialmente se usaba para referirse a los españoles conquistadores. Con el tiempo, comenzó a usarse para referirse a cualquier persona no mapuche. TERCERAS-AnalesHdA32.indd 85 4/10/22 16:26 Urquieta Robles, L. An. his. arte 32 (2022): 77-97 86 involucrados de manera directa o indirecta en estos hechos, si el Estado se hizo cargo de estos crímenes al volver la democracia, etc. Dadas las particularidades que tuvo la represión en esta zona, las entrevistas se realizaron de manera semi estructurada, para darle al entrevistado la libertad de contar lo que estimara necesario, respetando su privacidad e intimidad. El proyecto de CODEPU Mirar hacia atrás cuenta con un total de treinta entrevistas registradas en formato de video, donde es posible conocer en extenso las vidas de las personas entrevistadas. Cada una de estas memorias se encuentra disponible en el MMDH, convirtiéndose en un documento audiovisual fundamental para conocer de primera fuente las vejaciones cometidas en dictadura contra el pueblo Mapuche, así como reconocer la labor realizada por este pueblo en la recuperación de la democracia. Esta importancia se acentúa por medio del uso de la memoria oral, como motor testimonial de un pueblo, que, a través de generaciones, ha construido su cultura, traspasando conocimientos por este medio. La oralidad es vital para el pueblo Mapuche, sin la palabra sería imposible preservar la lengua y, sin ella, desaparecería el pensamiento propio de toda una comunidad. Cada entrevistado proporcionó su historia de vida, desde aspectos vinculados tanto a su infancia y las carencias materiales existentes en esa etapa de su vida, como a su adolescencia, donde muchos de ellos se incorporaron a la escuela, insertándose en un lugar donde convivían periódicamente con niños y niñas winka, con todo lo que esto significaba en aquella época, siendo en muchos casos, víctimas de humillaciones, tanto por sus compañeros de curso como por algunos profesores. Durante el golpe de Estado, muchos de los entrevistados eran ya adultos, con familia constituida y padres ya mayores. En esta etapa se siente con mayor énfasis la dureza de lo vivido durante la dictadura. Es aquí donde la voz se torna a momentos más frágil y el ritmo del relato cambia, haciendo de las pausas algo cada vez más frecuentes: La escucha atenta permite detectar –a partir de tonos, gestos, silencios, rupturaso quiebres de discurso– sentidos actuales, vinculados al propio relato, al paso del tiempo, a las valoraciones que de ellos se haga y al contexto cultural y epocal en que se recuperan. Se trata de fragmentos del pasado (...) incorporados o silenciados, siempre reelaborados en función de factores ideológicos, generacionales, culturales o históricos que al ser narrados sostienen la expresión de las voces y las actitudes33. La forma en que se relatan estos episodios dice mucho del modo en que padecieron esta etapa, lo extremo de estas experiencias que, en muchos casos, mantuvieron en silencio por distintos motivos. No fue hasta la constitución de la Comisión Rettig que «se atrevieron a contar algo de lo ocurrido»34. La importancia de los actos de relatar lo sucedido es abordable, a nuestro parecer, desde dos importantes aristas: la primera de ellas viene dada por el componente psicológico, en dónde muchas de las personas que prestaron testimonio no habían comentado nunca algunos hechos debido a la vergüenza y al dolor que supone revi33 34 Diamant, A. (2012). La historia oral. Buenos Aires: BMN, 15. Bengoa, J. (2002). Historia de un conflicto. El Estado y los mapuches en el siglo XX. Santiago de Chile: Planeta, 154. TERCERAS-AnalesHdA32.indd 86 4/10/22 16:26 Urquieta Robles, L. An. his. arte 32 (2022): 77-97 87 vir estas situaciones, o al no querer que sus hijos se enteraran de esta parte de la vida personal de quien está relatando. El peso de cargar con estas memorias finalmente se libera a partir del acto de hablar y contar lo ocurrido, propiciando el primer paso para lograr la sanación personal ante estos dolorosos acontecimientos. La segunda arista guarda relación con el profundo vínculo existente entre el acto de hablar y el pueblo Mapuche, quienes, por una cuestión cultural y estructural intrínseca, le otorgan al habla un valor que es difícil cuantificar para el mundo occidental. El habla da forma y soporte al mundo conocido por ellas y ellos, es la encargada de entregar identidad a través de cómo conciben el mundo, donde cada palabra guarda un profundo significado que se vincula con su entorno natural. Más aún si la lengua madre del pueblo Mapuche, el mapudungun, se encuentra bajo seria amenaza de desaparecer. Según la UNESCO, se han extinguido alrededor de 200 idiomas, de un universo de 6.000 durante las últimas tres generaciones35. De ahí la importancia de preservar la cosmovisión y, por ende, la lengua mapuche: Cuando se pierde una lengua, todos nosotros perdemos el conocimiento que contienen las palabras y la gramática de esa lengua, un conocimiento que nunca podrá recuperarse si la lengua no ha sido estudiada o grabada. No toso estos conocimientos tiene un beneficio práctico inmediato, por supuesto, pero todos son vitales para enseñarnos diferentes formas de pensar en la vida, de guiar nuestra existencia cotidiana en el planeta Tierra36. 5. Proyecto arte sonoro Dungun A partir de toda la información recopilada en el MMDH en torno al archivo y las memorias mapuches en dictadura, nos proponemos realizar un proyecto archivístico-instalativo sonoro, que por un lado contenga parte de estas memorias en formato audiovisual y, por otra parte, contemple la realización de una instalación sonora que ahonde en torno al mapudungun, como lengua en resistencia por la pervivencia de esta cultura ancestral. Dungun (nombre del proyecto propuesto) es una palabra de origen mapuche que en su forma verbal significa hablar. Se trata de una lengua de carácter espiritualista: en todas las ceremonias mapuches el habla es la estructura fundamental. El mapudungun se desarrolló inicialmente como lenguaje oral, mnemónico y no escrito. No fue hasta 1982 que se creó el primer grafemario mapuche con el objetivo de mantener el idioma como símbolo de la identidad y cultura colectiva37. La pervivencia del mapudungun se ha visto amenazada en varias ocasiones: la primera vez fue con la llegada de los españoles en 154138 y la posterior ocupación de la Araucanía en 1860 por parte del Estado Chileno, lo que significó la mayor pérdida 35 36 37 38 Iglesias, L. (2009). Lenguas en peligro. El correo de la UNESCO, (2). Obtenido de https://unesdoc.unesco.org/ ark:/48223/pf0000186521_spa/PDF/186521spa.pdf.multi [Consulta: 21 de septiembre de 2021]. Everett, D. (2012). Lenguage: The Cultural Tool. Londres: Pantheon Books, 303. Traducción del autor. Álvarez, P., Forno, A., y Risco, E. (2015). Propuestas de grafemarios para la lengua mapuche: desde los fonemas a las representaciones político-identitarias. Alpha, (40). Obtenido de https://www.scielo.cl/scielo.php?script=sci_arttext&pid=S0718-22012015000100009 [Consulta: 3 de septiembre de 2021]. Retamal, J., (1980). Descubrimiento y conquista de Chile. Santiago de Chile: Salesiana, 14. TERCERAS-AnalesHdA32.indd 87 4/10/22 16:26 Urquieta Robles, L. An. his. arte 32 (2022): 77-97 88 de territorio por parte del pueblo Mpuche, siendo concentrados en pequeñas reservas en el sur del país, reduciendo su territorio para finales del siglo XIX a poco más de quinientas mil hectáreas39. Actualmente el pueblo Mapuche se encuentra diseminado en varias regiones del país; los resultados de la Ocupación de la Araucanía, la pérdida de sus tierras y el empobrecimiento que ha azotado a la región histórica de los Mapuche, obligó a muchas familias a buscar nuevos horizontes para así lograr mejorar su calidad de vida. Según el INE, en 2017 existen alrededor 1.700.000 mapuches en Chile40, de los cuales cerca de 615.000 están afincados en la Región Metropolitana. De ese total nacional, cerca del 65% no habla ni entiende su lengua. 6. Referencias El proyecto Dungun se nutre de diversos artistas para su conformación teórica y técnica; comenzando con Sebastián Calfuqueo, artista chileno de origen mapuche que ha realizado obra en torno a sus raíces y las relaciones biopolíticas existentes entre el mundo occidentalizado y el mundo mapuche. Cada una de sus intervenciones tiene como eje la constante tensión existente entre la cultura mapuche y la sociedad chilena, ya sea cuestionando el tema territorial mapuche, la creciente problemática del agua, la lengua, la identidad y la sexualidad. Dentro de la extensa lista de obras con las que cuenta Calfuqueo, nacido en 1991, todas poseen como hilo conductor la materialidad y la técnica, que en su gran mayoría son instalaciones, trabajos en cerámica, performance o video. De ellas podemos destacar Palabras a las aguas de 2021, obra que busca reflexionar en torno a la importancia de las aguas para la vida, no solo humana, sino para el ecosistema completo41, más aún tratándose de Chile, país que lleva años atravesando por serios problemas hídricos tanto a nivel ambiental como socioeconómico. Esta instalación está constituida por 70 metros de tela azul, 12 piezas de cerámica esmaltada azul con textos en color blanco, 3 luces de color azul y 2 canales de audio. La tela que zigzaguea a través de la sala lleva una inscripción en letras blancas que dice «un río que no corre es solo un reflejo», haciendo alusión a la privatización de los ríos y napas subterráneas de nuestro territorio. Sobre la tela se posicionan las 12 piezas de cerámica azul, cada una de las cuales contiene una inscripción que hace referencia a este elemento, pero abordado desde los distintos intereses que cruzan la obra de Calfuqueo: el agua desde lo elemental para el pueblo mapuche, como bien público y de acceso universal, como creadora de vida y como elemento fluido de cuerpo líquido. Los últimos elementos a destacar de esta instalación son los dos audios: se trata de dos poemas que suenan al mismo tiempo y hablan de la potencia y fluidez del no binarismo que representan las aguas, así como de la trascendencia que tienen las aguas para la vida en el planeta. 39 40 41 Almonacid, F. (2009). El problema de la propiedad de la tierra en el sur de Chile (1850-1930). Historia (Santiago), 1 (42). Obtenido de https://www.scielo.cl/scielo.php?script=sci_arttext&pid=S0717-71942009000100001 [Consulta: 3 de septiembre de 2021] Instituto Nacional de Estadísticas. (s.f). Obtenido de http://resultados.censo2017.cl/ [Consulta: 21 de septiembre de 2021] Calfuqueo, S. (2022). Palabras a las aguas. Obtenido de https://sebastiancalfuqueo.com/2022/04/09/palabrasa-las-aguas-2021/ [Consulta: 17 de junio de 2022] TERCERAS-AnalesHdA32.indd 88 4/10/22 16:26 Urquieta Robles, L. An. his. arte 32 (2022): 77-97 89 Continuando con las referencias existentes en Dungun, encontramos el proyecto Argentina suena del año 201642, dirigido por el investigador y docente de la Universidad Nacional de Tres de Febrero, Raúl Minsburg, quien convocó a una serie de artistas sonoros de distintas zonas de Argentina para realizar grabaciones de campo de sus respectivos pueblos o ciudades y, de esa manera, componer una obra. En palabras de Minsburg, «no se intentó realizar un mapa sonoro, (…) sino que se convocó a compositores que vivieran en zonas lo más alejadas posibles entre sí»43. El resultado del proyecto se tradujo en 14 piezas, las cuales buscan plasmar la identidad sonora de sus respectivas regiones a través de los sonidos más característicos. Dentro de estas propuestas, muy particulares y distintas unas de otras, quisiéramos detenernos en la realizada por Federico Barabino, artista sonoro y docente, que realizó la pieza titulada Jujuy 441, Merlo Buenos Aires. Esta pieza se desmarca de las demás por la presencia protagónica de la voz como relato, encontrando testimonios de dos personas que comparaban los sonidos de sus respectivos barrios en el pasado y en el presente. Se hace referencia a antiguos clubes de baile, los cuales colmaban el barrio de música, o la tranquilidad que ofrecía cierto lugar al no contar con el flujo aéreo que sí posee ahora. Todo esto acompañado de quiebres sonoros, ruidos, errores, bucles de otras grabaciones, potenciando y enriqueciendo la pieza final. Sin duda, el ajedrez electroacústico de John Cage Reunion44 es vital en la creación de Dungun. Cage, junto a Marcel Duchamp, realizó la primera exhibición en 1968 en el Ryerson Theatre de Toronto, Canadá. Reunion corresponde a un ajedrez electrónico que generaba sonidos que iban variando de acuerdo con la posición de las piezas en el tablero y los fotosensores ubicados en cada una de las 64 casillas del tablero. La infinidad de movimientos y combinaciones que permite el ajedrez le otorgaba a esta pieza una riqueza de posibilidades sonoras insospechadas. Otra referencia fundamental, esta vez desde lo técnico-estético, es el proyecto del año 2006 de los alemanes Hauert & Reichmuth, titulado Instant City45. Esta obra consiste en una mesa interactiva que combina diversos elementos, tales como: instrumento musical digital, juego de mesa, montaje experimental psicológico, etc. Se trata de una mesa sonora a la que uno o más jugadores pueden añadir y superponer bloques geométricos semitransparentes para así realizar distintas composiciones de sonidos en base a la ubicación y disposición de los bloques puestos en el tablero. Se trata de una composición sonora realizada en un micro tablero situacionista, el cual va cobrando vida y emitiendo sonidos a medida que la ciudad va aumentando su volumen. Las posibilidades son innumerables ya que el sonido resultante es consecuencia del número de bloques utilizados y su disposición en el espacio de la mesa, ya que incluso se pueden poner unos sobre otros, modificando de esta manera el tono o ritmo de la composición. 42 43 44 45 Minsburg, R. (2016). Identidad y arte sonoro: el proyecto Suena Argentina. Listen Over , 12 (1), 44–52. https:// doi.org/10.14393/OUV18-v12n1a2016-3 Ibid. 51. Idis. (2022). Ajedrez electroacústico Duchamp vs Cage. Obtenido de https://proyectoidis.org/ajedrez-electroacustico-duchamp-vs-cage/ [Consulta: 15 de junio de 2022] Atelier Hauert Reichmunth. (2022). Instant city. Obtenido de http://www.hauert-reichmuth.ch/en/projekte/instant-city/ [Consulta: 15 de junio de 2022] TERCERAS-AnalesHdA32.indd 89 4/10/22 16:26 90 Urquieta Robles, L. An. his. arte 32 (2022): 77-97 7. Instalación Dungun es una instalación interactiva constituida por dos elementos centrales. El primero de ellos consiste en un ajedrez sonoro en el que se podrá realizar una (o muchas) partida(s) conformada por dos equipos, las piezas blancas y las negras; estas a su vez están vinculadas a dos audios distintos: las blancas están asociadas a un fragmento del poema épico de Alonso de Ercilla La Araucana (primer registro escrito que habla de la resistencia mapuche ante los españoles en 1570) en castellano, mientras que las piezas negras están vinculadas al mismo fragmento del poema, pero recitado en mapudungun. Al iniciar la partida, los audios simultáneos se encontrarán con el mismo nivel de volumen, pero, a medida que un equipo vaya eliminando piezas de su oponente, estos reducirán el volumen en función del número de piezas que han sido tomadas. El sonido predominante de la instalación se escuchará en relación al equipo que tenga más piezas en el tablero, creando la metáfora de la utilización del territorio, determinado por el mismo tablero de ajedrez. Figura 1. Modelado 3D de instalación. El tablero es una metáfora del territorio que habita el pueblo mapuche, donde buscan mantener vivas sus tradiciones a través de su lengua. Son un colectivo casi sin presencia a nivel mediático, que salvo por situaciones ligadas a la contingencia logran algo de visibilidad. A fecha de hoy esta visibilidad ha aumentado gracias a la Convención Constitucional y el rol que ha jugado particularmente Elisa Loncon, activista mapuche, al presidir dicho órgano. Aún así, es importante mantener la discusión sobre la necesidad de revitalizar el mapudungun desde lo cotidiano a través del empoderamiento local. La inclusión del poema de La Araucana es crucial, ya que es la primera crónica que registra el conflicto librado entre el pueblo Mapuche y los conquistadores es- TERCERAS-AnalesHdA32.indd 90 4/10/22 16:26 Urquieta Robles, L. An. his. arte 32 (2022): 77-97 91 pañoles. Es el punto de inicio de una larga historia de lucha por la pervivencia de un pueblo y su tierra, por ende, de su lengua, la cual es un territorio cognitivo libre desde donde se puede resistir. Esta lucha es extrapolable a cada comunidad Mapuche, resida en el sur de Chile, en la capital o en algún cerro de la ciudad de Valparaíso. Digo que norte sur corre la tierra, y báñala de oeste la marina; a la banda del este va una sierra que el mismo rumbo mil leguas camina: en medio es donde el punto de la guerra por uso y ejercicio más se afina: Venus y Amón aquí no alcanzan parte, sólo domina el iracundo Marte. Pikum willi püle winufküley mapu konwe antü püle muñetuniey lhafkenh, puwel püle müley pire winkul mapu feichi rüpü ka inaniyey waragka legua; ragintu mew müley kiñe küme lelffüm, zuampeyel, mümülpeyüm, weychapeyüm: Venus egu Amón gelay faw ta ñi müleam, fofoillkum Marte fey mütem güneniyey46 El objetivo de este ajedrez es visibilizar por medio de la voz una problemática sostenida a través de décadas: la constante disminución del mapudungun a lo largo del territorio chileno debido a factores políticos, sociales y culturales. El ajedrez es una buena metáfora de este problema, ya que este milenario juego ahonda en torno a la disputa de territorio entre dos grupos, de cómo se desenvuelven estos en un territorio, y de cómo conviven en un mismo espacio. En este caso, la disputa de territorio no es solo física, es también simbólica, puesto que a medida que los mapuches afincados en distintas comunidades van perdiendo espacio para desarrollar sus actividades, su lengua también va perdiendo protagonismo en el diario vivir, por lo que es importante, como país, prestar atención a la participación y cabida de nuestras lenguas originarias en más sitios, como por ejemplo algunos centros de asistencia médica primarios, que disponen de señalética en mapudungun. De esta manera, se potencia el desarrollo del mapudungun dándole difusión e integrándose además como parte de la herencia cultural que posee Chile. 46 Schwemberg, H., y Azocar, A. (2006). La Araucana – Ta Awkan mapu Mew. De Alonso de Ercella y Zuñiga. Versión Castellano-Mapuzungun. Santiago de Chile: Ministerio de Educación. Gobierno de Chile, 23. TERCERAS-AnalesHdA32.indd 91 4/10/22 16:26 92 Urquieta Robles, L. An. his. arte 32 (2022): 77-97 Figura 2. Modelado 3D de instalación El juego del ajedrez tiene reglas predeterminadas, como los turnos, los distintos tipos de piezas y los movimientos que hace cada una de ellas, las diferentes jugadas o posiciones existentes en una partida, también, las múltiples estrategias que se pueden utilizar contra el oponente. Por otra parte, al tratarse de una instalación artística, estas reglas pueden adaptarse a la conveniencia del proyecto. En este caso, también se puede usar de manera libre, sin la necesidad de seguir las reglas clásicas de una partida de ajedrez, y usarse como una mesa sonora en donde se puede cotejar cada poema de manera separada, o en conjunto realizando un sinfín de variaciones en torno al volumen y cómo estas dos lenguas pueden coexistir en un mismo espacio. Esta modalidad está pensada principalmente para niñas y niños que, sin tener nociones de cómo jugar ajedrez, pueden igualmente interactuar con la instalación y ahondar en torno al mapudungun. La posibilidad de escuchar el poema épico de Ercilla en ambos idiomas de manera simultánea, nos ofrece la oportunidad de adentrarnos en un idioma que para el chileno medio es identificable, pero puede resultar a la vez, ininteligible. Poder contrastar y comparar ambas frases puede convertirse en la puerta de entrada para aquellos que se quieran iniciar en el conocimiento y aprendizaje del mapudungun. TERCERAS-AnalesHdA32.indd 92 4/10/22 16:26 Urquieta Robles, L. An. his. arte 32 (2022): 77-97 93 Figura 2. Modelado 3D de instalación El segundo elemento importante de esta instalación es una serie de pantallas donde la memoria oral se hace presente. Aquí también se distinguen dos grupos de audiovisuales. El primer grupo consiste en dos pantallas, cada una contiene una de las entrevistas hecha por CODEPU y archivada por el MMDH, donde un miembro de la comunidad relata lo sucedido durante su infancia, adolescencia, el golpe de estado y las consecuencias que este hecho tuvo para su comunidad. Las otras dos pantallas corresponden a los testimonios de dos personalidades del mundo mapuche que hablarán sobre la importancia de la lengua en la cosmovisión mapuche, de lo vital que es la preservación del mapudungun como puente de memoria y transmisión de conocimientos. Dichos testimonios serán realizados de manera personal, utilizando la pauta que CODEPU efectuó al momento de llevar a cabo sus entrevistas y que dejó registrada en su libro. El proyecto Dungun remite a las memorias de un pueblo que se ha mantenido en resistencia desde que se tienen registros escritos del mismo. Comenzando con la primera aproximación que tuvo con el ejército conquistador español; en el combate de las tropas de Cornelio Saavedra a lo largo de la ocupación de la Araucanía; durante la dictadura de Pinochet y las violaciones de los Derechos Humanos; o en el actual conflicto forestal en las tierras ancestrales de esta comunidad, las actuales provincias de Malleco y Cautín, donde grupos empresariales tanto extranjeros como nacionales –la maderera japonesa Volterra, Forestal Mininco del grupo Matte, Forestal Arauco del grupo Angelini, o Forestal Bosques Cautín47–, se han enriquecido de manera ilícita, a la luz de la documentación entregada, a costa de las tierras que han pertenecido de manera milenaria al pueblo Mapuche. Esa misma resistencia se traslada al campo de las memorias ya que, como señala Elizabeth Jelin, «encontramos una situación de luchas por las representaciones del 47 Mapuexpress. (2016). Recuperado de https://www.mapuexpress.org/2016/11/21/araucania-listado-de-empresas-consideradas-transgresoras-de-derechos-humanos-y-la-naturaleza/ [Consulta: 24 de septiembre de 2021]. TERCERAS-AnalesHdA32.indd 93 4/10/22 16:26 Urquieta Robles, L. An. his. arte 32 (2022): 77-97 94 pasado, centradas en la lucha por el poder, por la legitimidad y el reconocimiento»48, otorgándole un nombre a un hecho en base de lo que se recuerda. En ese sentido, es fundamental mantener y reforzar dichas memorias en la disputa social, para que la hegemonía no siga invisibilizando la resistencia de este pueblo. 8. Conclusiones Desde el fin de la ocupación de la Araucanía, el pueblo Mapuche sufrió una notable disminución de territorio que se tradujo en movilizaciones forzadas, empobrecimiento y estigmatización, lo que ha conllevado la pérdida gradual pero constante de su lengua. Esta situación comenzó a cambiar después de la Ley de Reforma Agraria de 1962, de 1967 y la Ley Indígena de 1972, logrando un significativo avance en la recuperación de las tierras que habían sido arrebatadas un siglo atrás. Lamentablemente, todo este proceso de reconocimiento, empoderamiento y trabajo colectivo entre las distintas comunidades Mapuche se vio truncado con el golpe de Estado perpetrado por Pinochet, desbaratando en muy poco tiempo, con ayuda de civiles de la zona, lo construido por las diversas agrupaciones sociales, campesinas e indígenas, y las instituciones gubernamentales de la Unidad Popular, volviendo todo, por medio de persecución, prisión, torturas y desapariciones, a un punto con las mismas características que tenía Chile previamente a la reforma de 1962. Con el retorno a la democracia, instituciones como CODEPU llevaron a cabo una invaluable labor, al realizar el registro audiovisual de comuneros mapuches en las VIII y IX regiones sobre hechos acontecidos en la comuna de Tirúa, que dieron testimonio respecto a la represión vivida durante los años de dictadura y las consecuencias que ésta tuvo a nivel personal y colectivo. Dicho registro se encuentra actualmente en el MMDH así como en otros sitios afines. Documentos como estos son vitales, ya que constituyen parte de nuestro patrimonio, son el reflejo de una actividad política y social de una época determinada en una localidad específica; son memoria, permitiendo configurar nuestro presente y futuro a partir de un hecho pasado; también son conocimiento, dada la enorme y valiosa cantidad de información adjunta en cada testimonio, que se puede además contrastar con otras fuentes historiográficas. Así mismo, este tipo de documentos proporciona pautas técnicas en torno a cómo llevar a cabo, desde lo audiovisual, una entrevista de estas características, convirtiéndose en un manual para aquellos realizadores que se están adentrando en el tema documental. Desde hace ya varias décadas, se ha creado una suerte de vínculo entre arte, memoria y archivo, asociado principalmente al Holocausto y horrores posteriores que son importantes recordar. En el Cono Sur, dicho proceso ha estado marcado, al igual que la historia oral, por la profunda huella dejada por las dictaduras de estos países, a la que Chile no es ajeno. Fue casi una pulsión de la coyuntura aunar estas disciplinas, una búsqueda de verdad y justicia por parte de creadores y creadoras cercanas a los Derechos Humanos, con la convicción de que, por medio de sus obras, se pudiese vencer el olvido. En el caso de Dungun, proyecto expositivo surgido a raíz de la investigación en el MMDH, la memoria se aborda desde aquella que se quiso atribuir en el poema 48 Jelin, E. (2002). Los trabajos de la memoria. Buenos Aires: Siglo XXI, 36. TERCERAS-AnalesHdA32.indd 94 4/10/22 16:26 Urquieta Robles, L. An. his. arte 32 (2022): 77-97 95 de Ercilla, asignándole categorías épicas al pueblo Mapuche, así como desde los testimonios del trabajo de CODEPU proyectados y los relatos que hablan sobre el acto de hablar. Se trata de un proyecto, que, desde el punto de vista de la memoria, es una pieza «en tanto que archivo», tanto por el acto de trabajar con un documento literario para integrarlo a una obra de arte interactiva, como por usar un archivo como parte fundamental de la obra. «Esta recuperación de la memoria (“recordar como una actividad vital humana define nuestros vínculos con el pasado, y las vías por las que recordamos nos define en el presente”) rehabilita los necesarios diálogos pasado-presente y sincronía-diacronía»49 y, ya que el pasado se encuentra en constante disputa, nos permite hacernos cargo de él trabajando con y para las memorias. Dichas memorias, añadidas a una instalación interactiva, no sólo se abordan desde lo lúdico, sino que también son abordables desde la trascendencia que tienen para entender de manera cabal la experiencia humana. En ese sentido, la labor del MMDH es crucial, no solo por proteger, archivar y visibilizar la historia de un periodo determinado de Chile, sino también por brindar espacios culturales para la creación y reflexión en torno a su archivo, con vistas a un futuro en donde el atropello de la vida y la aniquilación de culturas ancestrales no vuelva a ocurrir. Desde lo sonoro, es pertinente mencionar la importancia que tiene trabajar con voces mapuches, ya que tanto esta como muchas otras lenguas prehispánicas, se encuentran relegadas a un segundo plano debido al proceso de colonización50. En este sentido, la dictadura cívico-militar de Pinochet se encargó de soterrar cualquier rasgo que fragmentara la unidad de Estado nación que el mismo régimen quería impulsar, y en ese sentido el mapudungun era disruptivo. El mapudungun, en el momento de ser hablado, se proyecta como un acto de resistencia, ya que dicha lengua es identitaria de un pueblo que pervive pese a los embates recibidos durante siglos. Hablar no solo es comunicar y transmitir un mensaje a otra persona, es también hacer presente un territorio, material y simbólico51, más aún tratándose de una lengua donde las onomatopeyas son vitales y la configuración de las palabras está en directa relación con el sonido de sus bosques, ríos, volcanes y aves. Por este motivo, hablar es resistir; cada vez que se habla mapudungun, se recuerda un territorio asediado, un bosque que lucha por no ser talado, un río secado o prisionero de una empresa papelera. De esta manera, realizar proyectos de arte que aborden la problemática mapuche desde lo sonoro es resignificar el habla y extender sus alcances a otras áreas del conocimiento, transformando Dungun en un dispositivo que pretende reflejar la lucha que este pueblo ha dado, desde que se tienen registros de su existencia, gracias a las crónicas realizadas por Alonso de Ercilla. 49 50 51 Guasch, A. (2005). Los lugares de la memoria. El arte de archivar y recordar. Matèria: Revista d’ art, 5. Obtenido de https://annamariaguasch.com/es/Publicaciones/Los_lugares_de_la_memoria:_el_arte_de_archivar_y_ de_recordar [Consulta: 11 de septiembre de 2021]. García, E. (2016). Imágenes y sonidos del Wall Mapu. El proyecto de descolonización del universo visual y sonoro del Pueblo Mapuche. Empiria. Revista De metodología De Ciencias Sociales, (35), 125–151. https://doi. org/10.5944/empiria.35.2016.17171 Soto-Silva, I., Millán, F., Silva-Zurita, J., & Núñez Pertucé, M. (2021). La Mímesis Musical en la construcción de un discurso de identidad mapuche en la música popular del sur de Chile: una aproximación preliminar a partir de tres casos en la Región de Los Lagos. Panambí. Revista De Investigaciones Artísticas, (12). https://doi. org/10.22370/panambi.2021.12.2486 TERCERAS-AnalesHdA32.indd 95 4/10/22 16:26 96 Urquieta Robles, L. An. his. arte 32 (2022): 77-97 9. Conflicto de intereses Ninguno. 10. Referencias bibliográficas Almonacid, F. (2009). El problema de la propiedad de la tierra en el sur de Chile (18501930). Historia (Santiago), 1 (42). Obtenido de https://www.scielo.cl/scielo.php?script=sci_arttext&pid=S0717-71942009000100001 [Consulta: 3 de septiembre de 2021] Álvarez, P., Forno, A., Risco, E. (2015). Propuestas de grafemarios para la lengua mapuche: desde los fonemas a las representaciones político-identitarias. Alpha, (40). Obtenido de https://www.scielo.cl/scielo.php?script=sci_arttext&pid=S0718-22012015000100009 [Consulta: 3 de septiembre de 2021] Araya, J. (1999). Conflicto mapuche: Algunas razones y derechos en este nuevo enfrentamiento. Derechos Humanos y trabajadores. Sobre el derecho de los ex prisioneros políticos a ser indemnizados por el Estado. Santiago de Chile: LOM, 12. Atelier Hauert Reichmunth. (2022). Instant city. Obtenido de http://www.hauert-reichmuth. ch/en/projekte/instant-city/ [Consulta: 15 de junio de 2022] Bengoa, J. (1985). Historia del Pueblo Mapuche (siglos XIX y XX). Santiago de Chile: LOM, 336. Bengoa, J. (2002). Historia de un conflicto. El Estado y los mapuches en el siglo XX. Santiago de Chile: Planeta, 154. Calfuqueo, S. (2022). Palabras a las aguas. Obtenido de https://sebastiancalfuqueo. com/2022/04/09/palabras-a-las-aguas-2021/ [Consulta: 17 de junio de 2022] Chile. (1972). Ley 17.729. Propiedad intelectual. Obtenido de fttps://www.bcn.cl/leychile/ navegar?idNorma=29250 [Consulta: 3 de noviembre de 2021] Chile. (1978). Ley 2.405. Propiedad intelectual. Obtenido de https://www.bcn.cl/leychile/ navegar?idNorma=6911 [Consulta: 3 de noviembre de 2021] Colipi, G. (Ed.). (2011). Mirar hacia atrás. Memoria oral del pueblo mapuche. Una expresión sobre la represión y tortura en la Comuna de Tirúa, Chile (1973-marzo 1990). Santiago de Chile: Caballo de mar, 3. Comisión Verdad Histórica y Nuevo Trato con los Pueblos Indígenas. (2008). Informe de la Comisión Verdad Histórica y Nuevo Trato con los Pueblos Indígenas. Santiago de Chile: Gobierno de Chile, 403. Correa, M., y Mella, E. (2009). El territorio mapuche de Malleco: Las razones del Illkun. Santiago de Chile: ODHPI, 122. Correa, M., Molina, R., y Yáñez, N. (2005). La Reforma Agraria y las tierras mapuche: Chile 1962 – 1975. Santiago de Chile: LOM, 79. Diamant, A. (2012). La historia oral. Buenos Aires: BMN, 15. Everett, D. (2012). Lenguage: The Cultural Tool. Londres: Pantheon Books, 303. Farías, C. (2012). Pueblo mapuche y dictadura: desde el terror hacia la nueva esperanza. Elementos configurativos para el entendimiento de la represión efectuada por el Régimen Militar hacia el pueblo mapuche. (Informe final de práctica profesional). Museo de la Memoria y los Derechos Humanos, Santiago de Chile. García, E. (2016). Imágenes y sonidos del Wall Mapu. El proyecto de descolonización del universo visual y sonoro del Pueblo Mapuche. Empiria. Revista De metodología De Ciencias Sociales, (35), 125–151. https://doi.org/10.5944/empiria.35.2016.17171 TERCERAS-AnalesHdA32.indd 96 4/10/22 16:26 Urquieta Robles, L. An. his. arte 32 (2022): 77-97 97 Guasch, A. (2005). Los lugares de la memoria. El arte de archivar y recordar. Matèria: Revista d’ art, 5. Obtenido de https://annamariaguasch.com/es/Publicaciones/Los_lugares_de_ la_memoria:_el_arte_de_archivar_y_de_recordar [Consulta: 11 de septiembre de 2021] Idis. (2022). Ajedrez electroacústico Duchamp vs cage. Obtenido de https://proyectoidis.org/ ajedrez-electroacustico-duchamp-vs-cage/ [Consulta: 15 de junio de 2022] Iglesias, L. (2009). Lenguas en peligro. El correo de la UNESCO, (2). Obtenido de https:// unesdoc.unesco.org/ark:/48223/pf0000186521_spa/PDF/186521spa.pdf.multi [Consulta: 21 de septiembre de 2021] Instituto Nacional de Estadísticas. (s.f). Obtenido de http://resultados.censo2017.cl/ [Consulta: 21 de septiembre de 2021] Jelin, E. (2002). Los trabajos de la memoria. Buenos Aires: Siglo XXI, 36. Mapuexpress. (2016). Recuperado de https://www.mapuexpress.org/2016/11/21/araucania-listado-de-empresas-consideradas-transgresoras-de-derechos-humanos-y-la-naturaleza/ [Consulta: 24 de septiembre de 2021] Meyer, E. (1971). La historia oral. Origen, metodología, desarrollo y perspectivas. Historia Mexicana, volumen 21 (2). Obtenido de https://www.jstor.org/stable/25134913 [Consulta: 8 de septiembre de 2021] Minsburg, R. (2016). Identidad y arte sonoro: el proyecto «Suena Argentina». ListenOver , 12 (1), 44–52. https://doi.org/10.14393/OUV18-v12n1a2016-3 Museo de la Memoria y los Derechos Humanos. (2013). Archivo Oral Museo de la Memoria y los Derechos Humanos. Proyecto Piloto “Maestranza de San Bernardo” [Ponencia]. V Encuentro Latinoamericano de Historia Oral, San Salvador. Obtenido de http://www.cedocmuseodelamemoria.cl/wpcontent/uploads/2013/05/MM_Chile_SSalvador.pdf [Consulta: 28 de octubre de 2021] Navarrete, J. (2020). Movimiento Campesino Revolucionario. Lucha mapuche, política de clase y “proyecto socialista” durante el gobierno de la Unidad Popular (Cautín, 19701971). En R. Austin (Eds.), La vía al socialismo chileno 50 años después (pp. 495-519). Buenos Aires: Clacso. Retamal, J., (1980). Descubrimiento y conquista de Chile. Santiago de Chile: Salesiana, 14. Schwemberg, H., y Azocar, A. (2006). La Araucana – Ta Awkan mapu Mew. De Alonso de Ercella y Zuñiga. Versión Castellano-Mapuzungun. Santiago de Chile: Ministerio de Educación. Gobierno de Chile, 23. Soto-Silva, I., Millán, F., Silva-Zurita, J., & Núñez Pertucé, M. (2021). La Mímesis Musical en la construcción de un discurso de identidad mapuche en la música popular del sur de Chile: una aproximación preliminar a partir de tres casos en la Región de Los Lagos. Panambí. Revista De Investigaciones Artísticas, (12). https://doi.org/10.22370/panambi.2021.12.2486 Yusta, M. (2002). Historia oral, historia vivida. El uso de las fuentes orales en la investigación histórica. Pandora: revue d’etudes hispaniques, (2). Obtenido de https://dialnet. unirioja.es/servlet/articulo?codigo=3160107 [Consulta: 25 de octubre de 2021] TERCERAS-AnalesHdA32.indd 97 4/10/22 16:26
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https://repositorioinstitucional.ceu.es/bitstream/10637/14376/1/News_Pou_CCAC_2022.pdf
English
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News coverage of the Church dealing with the pandemic: Spanish and Italian newspapers
Church, communication and culture
2,022
cc-by
11,678
Church, Communication and Culture Church, Communication and Culture ISSN: (Print) (Online) Journal homepage: https://www.tandfonline.com/loi/rchu20 ISSN: (Print) (Online) Journal homepage: https://www.tandfonline.com/loi/rchu20 Full Terms & Conditions of access and use can be found at https://www.tandfonline.com/action/journalInformation?journalCode=rchu20 ABSTRACT This paper focuses on media representation of the Catholic Church during the first months of the coronavirus pandemic in Italy and Spain. The purpose is to know when and how the Catholic Church starred in the news; how its actions were pre- sented, and using which frames. The work analyses the coverage of the Church in Corriere della Sera and El Paıs between January and May 2020 and connects the deductive process (news frames by Semetko and Valkenburg) with the inductive process (setting common places or topoi) in order to get a better understanding of the news-framing. Findings show that both newspapers gave prevalence to news about the charitable work of the Catholic Church during the pandemic instead of about conflict with the political authorities because of restrictive measures. When it comes to the relationship with civil institutions, a positive image of the Church was offered, one that portrayed her as ready to col- laborate and apply the preventive measures recommended by the health authorities. The positive perception is more frequent than the negative one when there are dualities in the topoi that are found in the interpretation of the facts. KEYWORDS Covid-19; pandemic; content analysis; framing; topoi María-José Pou-Amérigo María-José Pou-Amérigo To cite this article: María-José Pou-Amérigo (2022) News coverage of the Church dealing with the pandemic: Spanish and Italian newspapers, Church, Communication and Culture, 7:1, 154-174, DOI: 10.1080/23753234.2022.2040373 To link to this article: https://doi.org/10.1080/23753234.2022.2040373 © 2022 The Author(s). Published by Informa UK Limited, trading as Taylor & Francis Group. Published online: 10 May 2022. Submit your article to this journal Article views: 393 View related articles View Crossmark data © 2022 The Author(s). Published by Informa UK Limited, trading as Taylor & Francis Group. Published online: 10 May 2022. Submit your article to this journal Article views: 393 View related articles View Crossmark data Full Terms & Conditions of access and use can be found at https://www.tandfonline.com/action/journalInformation?journalCode=rchu20 Full Terms & Conditions of access and use can be found at https://www.tandfonline.com/action/journalInformation?journalCode=rchu20 CHURCH, COMMUNICATION AND CULTURE 2022, VOL. 7, NO. 1, 154–174 https://doi.org/10.1080/23753234.2022.2040373 News coverage of the Church dealing with the pandemic: Spanish and Italian newspapers Marıa-Jose Pou-Amerigo Journalism, Universidad CEU Cardenal Herrera, Valencia, Spain Marıa-Jose Pou-Amerigo Marıa-Jose Pou-Amerigo Journalism, Universidad CEU Cardenal Herrera, Valencia, Spain Journalism, Universidad CEU Cardenal Herrera, Valencia, Spain ARTICLE HISTORY Received 30 June 2021 Revised 11 November 2021 Accepted 22 November 2021 KEYWORDS Covid-19; pandemic; content analysis; framing; topoi ARTICLE HISTORY Received 30 June 2021 Revised 11 November 2021 Accepted 22 November 2021 ARTICLE HISTORY Received 30 June 2021 Revised 11 November 2021 Accepted 22 November 2021 CONTACT Marıa-Jose Pou-Amerigo mpou@uchceu.es Journalism, Universidad CEU Cardenal Herrera, Valencia, Spain  2022 The Author(s). Published by Informa UK Limited, trading as Taylor & Francis Group. This is an Open Access article distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/ licenses/by/4.0/), which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited. ARTICLE HISTORY Received 30 June 2021 Revised 11 November 2021 Accepted 22 November 2021 Journalism, Universidad CEU Cardenal Herrera, , p  2022 The Author(s). Published by Informa UK Limited, trading as Taylor & Francis Group. 1. Introduction The most representative image of the hardest lockdown in Europe in 2020 was absence: streets, squares, or historical monuments, that so often used to be full of neighbors and visitors, appeared devoid of life and movement, like a static scene captured by a photo- graphic camera. Among all of them, Saint Peter’s Square, a point of reference for all Catholic people and a universal tourist destination, was the best example of the excep- tional measures taken in those days. There, one of the iconic images summarized the effects of the pandemic on social life. It was April 26, and the Pope was preparing to impart, alone, the Urbi et Orbi blessing. This square, designed to embrace everyone, was completely empty. mpou@uchceu.es CHURCH, COMMUNICATION AND CULTURE 155 The image travelled around the world with adjectives about the exceptional scene (‘historical’, ‘extraordinary’ or ‘unusual’). In the same way, the development of religious activity without the physical presence of the people transferred the meeting of com- munities to the digital network. This was a challenge for the Catholic Church and news for a large part of public opinion. The life of the Church did not stop, but the approach required the mediation of the media. This paper focuses on this mediation role and analyzes the story about the work of the Catholic Church during the first months of the coronavirus pandemic what was published by the Italian press and the Spanish press. Our objective is to know when and how the Catholic Church starred in the news; how its actions were presented, and through which frames it was seen. This paper analyzes Corriere della Sera and El Paıs, the best-selling daily newspapers in Italy and Spain. The aim is to know what image the media offered about the Church during a global crisis. The interest in the question is related, on the one hand, to the efforts made by religious entities, especially the Catholic Church, to present their message to society without distortions and, on the other, to the knowledge of the keys that produce these distortions. 2. The Church and the pandemic in Italy and Spain In Italy, the lockdown began in Lombardy, but on March 9th, Prime Minister Conte extended it to the whole country; in Spain, on the 11th, President Sanchez announced it for March 14th. In this country, the measures did not include the closure of places of worship but made it difficult, in fact, to participate in religious ceremonies and activities, especially due to the impossibility of traveling to the spaces dedicated to worship. In Spain, the Royal Decree 463/2020, of March 14th (BOE 2020) said in article 11 Attendance at places of worship and civil and religious ceremonies, including funerals, is conditioned on the adoption of organizational measures consisting of avoiding crowds of people, depending on the size and characteristics of the places, in such a way that guarantee attendees the possibility of respecting the distance between them of at least one meter. In Italy, however, although places of worship were not closed in the Decree of the President of the Consiglio of March 8th (Gazetta Uficciale della Repubblica Italiana 2020) religious ceremonies, including funerals were suspended by Article 1: i) the opening of places of worship is conditional on the adoption of organizational measures that avoid gatherings of people, considering the size and characteristics of the places, and such as to guarantee visitors the possibility of respecting the distance between them of at least one meter as per attachment 1 letter d). Civil and religious ceremonies, including funeral ones, are suspended. As in the case of other individual freedoms, restrictions generated protests and reproaches. The Council of Europe (2020, 6) had to remind governments of the requirement of proportionality in these initiatives: Effective enjoyment of all these rights and freedoms guaranteed by Articles 8, 9, 10 and 11 of the Convention is a benchmark of modern democratic societies. Restrictions on them are only permissible if they are established by law and proportionate to the legitimate aim pursued, including the protection of health. The significant restrictions to usual social activities, including access to public places of worship, public gatherings and wedding and funeral ceremonies, may inevitably lead to arguable complaints under the above provisions. It is for the authorities to ensure that any such restriction, whether it is based on a derogation, is clearly established by law, in compliance with relevant constitutional guarantees and proportionate to the aim it pursues. 2. The Church and the pandemic in Italy and Spain In December 2019, the World Health Organization (WHO) admitted the existence of a new coronavirus, SARS-CoV-2, and the disease associated with it, Covid-19, initially located in Wuhan (China). From that moment, the EU began to take measures to respond to this threat. Thus, on January 17th, the Health Security Committee met to address the new coronavirus for the first time under the indications of the European Center for Disease Prevention and Control. The risk was considered low or very low (European Commission 2020, 2). A few days later, on January 24th, the first case was known in Europe, in France. At that time the disease was still linked to China or to people who had just returned from there, however, on January 28th, the first ‘native’ case was known in Europe; it was a German person who had not traveled to Wuhan. In Italy, the first cases were known on January 30th. They were two Chinese tourists who had been in Milan on January 23rd and, later, traveled to Rome; the first non- imported case was found in the hospital in Codogno (Lombardy) on February 21st. In Spain, the first known patient was a German tourist on the island of La Gomera, on January 31st, but the first local case was known on February 26th. In Europe, the first deaths occurred in France on February 15th and in Italy on February 22nd. In Spain, the first death occurred on February 13th, but its cause was only known later. From that moment, cases multiplied. In Italy, Lombardy became the ‘ground zero’ of the pandemic, and one of the most affected regions in Europe. Spain would also lead with the worst statistics during the first wave, looking itself in the Italian mirror to gain an idea from the advance of the pandemic in that country how things might go on the Iberian Peninsula. In both countries, the authorities began to take measures as soon as the risks were known and, above all, when the first diagnosed cases appeared. Just a few days sepa- rated the declaration of a state of alarm in both countries: on March 8th, in Italy; on M.-J. POU-AMÉRIGO 156 March 14th, in Spain. The pandemic in Italy seemed to advance on a timeline approxi- mately a week ahead of Spain’s. In addition, both governments approved severe measures of lockdown almost at the same time. 2. The Church and the pandemic in Italy and Spain The religious authorities also showed their disappointment, especially when, despite the fact that in some cases where no rules were being broken, the forces the law inter- vened to end ceremonies and to fine those responsible. The debate was transferred to the media, although the respective Episcopal Conferences had given instructions, from the beginning, to obey the sanitary rules. Thus, the Executive Commission of the Spanish Episcopal Conference (CEE 2020) published a note on March 13th, in order to make ‘ … a call to follow the instructions of those responsible for health to avoid the accelerated progression of the disease’ while its president, Cardinal Omella, invited people to follow the masses live-streamed on radio and television. The Italian Episcopal Conference (CEI 2020), in a note published on March 8th, regretted the hard CHURCH, COMMUNICATION AND CULTURE 157 measures adopted, but he accepted them for the common good: ‘The acceptance of the decree is mediated only by the will to do, even in this juncture, one’s own part to con- tribute to the protection of public health’. Italy and Spain share values, history, and religious tradition, although Italy’s close- ness with Vatican City has always made it unique. However, essentially the two are comparable, since both are countries with a Catholic majority (around 80% baptized), but with a much lower rate of daily religious practice. What has been called a ‘Catholic imprinting’ (Verzichelli 2007) remains in this majority, and it spreads religious values in social life, although some public officials reproach the religious authorities for this. These values have remained stable even during the pandemic, a period in which religi- osity has not suffered (Reeskens et al. 2021, 159), because paradoxically, ‘in a certain sense it is precisely at the moment of greatest difficulty that the need for support in spiritual terms becomes stronger, in the hope of finding comfort in one’s faith and beliefs’ (Coppola et al. 2021). Despite Italy and Spain having a similar profile in terms of religious values and the influence and the role of the Catholic Church in public life, the social debate about the difficult balance between religious freedom and health care differed in the two coun- tries during the first months of the pandemic. 2. The Church and the pandemic in Italy and Spain In Italy, some voices recalled the cap- acity of Catholic Church to decide the best measures in liturgical celebrations to prevent contagion, but in Spain this issue was hardly raised in public opinion – there were hardly any complaints about the restrictions on religious activity, and these came only from people critical of power. During the pandemic, misinformation grew; in fact, the development of the disease was plagued with fake news (Perez-Dasilva, Meso-Ayerdi, and Mendiguren-Galdospın 2020). In this sense, it is necessary to know if the media also offered a misleading view of the Church’s work during the pandemic. It is relevant to the extent that it helps us to understand the role of the press in building the public image of the Catholic Church. In a context of fake news and disinformation such as the one experienced, it is necessary to analyze the process of the construction of reality. In this way, it will be possible to delve into, on the one hand, the decision guidelines and productive routines of the media, and on the other, the implications that the process of construction of an ‘alternative reality’ in the media may have for social organizations, especially the Catholic Church. 3. Methodology and the field of the study In this context, the comparative analysis of Italy and Spain seems especially timely. Both countries share religious tradition (both are Catholic, with a similar percentage of believers and religious practice; and with the presence of the Catholic Church in public life along with other religious communities) and both experienced a simultaneous development of Covid-19 and in the same geographical area of the south of Europe, on the Mediterranean. However, the voice of the Church in Italy is received differently than it is in Spain, hence the relevance of the comparison. If the objective is to know M.-J. POU-AMÉRIGO 158 whether the media discourse varies between the Italian and Spanish press, and what influence the frames and topoi have on it, the comparison can help. Therefore, the research objectives are: 1. Quantify the presence of the Catholic Church in the Italian and Spanish quality press; uantify the presence of the Catholic Church in the Italian and Spanish quality press; 1. Quantify the presence of the Catholic Church in the Italian and Spanish quality press; 2. Identify which frames and topoi are in the news about the activities of the Church during the pandemic; 2. Identify which frames and topoi are in the news about the activities of the Church during the pandemic; 3. Compare the journalistic treatment of the Church and religious activity in the Spanish and Italian press. 3. Compare the journalistic treatment of the Church and religious activity in the Spanish and Italian press. We believe there are common features in the Spanish and Italian press regarding the image of the Catholic Church during the pandemic. In this context, the social system had acquired an important role, so some groups appear as ‘close collaborators of the citizenry’ (Argi~nano and Bilbao 2020, 19). Thus, given the social role of the Church, it is probable that its presence in the press during the pandemic was defined by its tasks of spiritual and charitable assistance, and not by its political role in conflict with the authorities, as might have been expected after restrictive measures on the part of the government. Conflict and deviation-negativity (Van Dijk 1990) are very recurrent news-values in journalistic information (Contreras 2006) and, although we wouldn’t expect it in a tra- gedy such as the coronavirus pandemic, disappointment with political measures was on the table. 3. Methodology and the field of the study In addition, conflict is behind much of religious news because often jour- nalists are more interested in news when it is connected to scandals, conflicts, and negativity (Radde-Antweiler and Zeiler 2020, 4), without forgetting that the pandemic ‘has increased polarization in an already polarized media context’ (Iranzo-Cabrera and Lopez-Garcıa 2021, 264). Often conflict is the key to journalistic interpretation of information about the Church because it is presented as a political entity (Pou-Amerigo, 2007), however, the same does not happen when the story is about the Church assisting the disadvantaged. We established various research questions: Q1: How many texts about the Catholic Church in the Italian and Spanish press were published in the selected titles during the pandemic? published in the selected titles during the pandemic? Q2: What actions are described in them? Q3: In what percentage do actions of a spiritual or assistance nature predominate? Q4: In what percentage is the Church a factor of conflict? Q5: Are there differences between the Italian and Spanish press? Thus, hypothesis 1 is regarding the frames of reference through which the role of the Church is interpreted when the story is a global tragedy: H1. The Italian and Spanish press gave prevalence to the charitable work of the Catholic Church during the pandemic instead of to the conflict with the political authorities over restrictive measures. CHURCH, COMMUNICATION AND CULTURE 159 Likewise, the paper seeks to establish the keys to the journalistic construction of the image of the Church in the context of the pandemic: What natural topoi are activated in the news about the activity of the Church? Q6. What natural topoi are activated in the news about the activity of the Chur Q7. What cultural topoi are activated in the news about the activity of the Church? Q8. Which are positive actions on the part of the Church, according to the press? Q9. Which are negative actions on the part of the Church, according to the press? Q10. What are the differences between the Italian and Spanish press? Q10. What are the differences between the Italian and Spanish press? Hypotheses 2 and 3 regard to the evaluation criteria that the media use around the Church: H2. The Italian and Spanish press valued the spiritual and welfare work of the Church. H2. The Italian and Spanish press valued the spiritual and welfare work of the Church. H3. 3. Methodology and the field of the study POU-AMÉRIGO 160 hand, in the printed edition, the limitation of space and the absence of additional con- tent from the web allow the editorial criteria to be assessed more clearly. The selection was made based on the presence of the terms ‘catholic’, ‘church’, ‘priest’, ‘bishop’ or ‘pope’, together with ‘covid’, ‘coronavirus’ or ‘virus’. The search focused on the informative and interpretive genres (news, report, interview and chronicle) since in opin- ion texts the author’s interpretive framework is explicit and is not imposed on the reader, as it is a personal point of view. However, the informative texts are offering an account of verified facts, not a questionable opinion. Thus, the unit of analysis was each unitary informative text. Three coders – two researchers and a Ph.D. student in Communication – worked on the coding, as well as two researchers from outside the team who acted as external inter-judges to validate the results. The selection of the topoi was developed from a ran- dom reading of two pieces every week from each paper throughout the period. The process was inductive. Subsequently, the presence of the topoi was verified in the entire sample, so that each coder analyzed two thirds of the total sample to ensure that each third was analyzed by two different coders. Two types of data were obtained from the units of analysis: identifying data and analytical data. The identification data is necessary to identify each piece; they are date, headline and page or URL. The analysis data refers to the content and narrative options of the author. They are, therefore, the frame: the kind of collaboration with the author- ities; the type of activity and the common places or natural and cultural ‘topoi’. The ‘news frames’ are part of the journalistic story because they serve as a concep- tual framework (Sadaba and Virgili 2007, 210) from which the journalist analyzes, gives meaning and, finally, narrates what happened. It is the place, therefore, where the jour- nalist’s interpretation and the ‘framing strategy’ of the media are found (Lopez- Rabadan 2010). It is a way of approaching the mediation process through the journalis- tic story, with which the media becomes an ‘essential actor in the process of production and construction of public discourse, but far from the only one’ (Valera Ordaz 2016, 15). That work involves a selection process, which means accepting and rejecting. 3. Methodology and the field of the study The Italian and Spanish press criticized the Church’s opposition to government measures, reintroducing it as an entity with a political role. H3. The Italian and Spanish press criticized the Church’s opposition to government measures, reintroducing it as an entity with a political role. We chose the two most representative newspapers of public opinion in each of the countries, with a significant penetration among the population. In Italy, we chose Corriere della Sera, the most read newspaper in 2020 according to the ADS (Accertamenti Diffusione Stampa) (Prima online 2020). In Spain, we chose El Paıs, the general information newspaper with the greatest circulation (EGM 2020), only sur- passed by the sports newspaper Marca. Both constitute the quality press (Casasus and Roig 1981, 30) and they have what Imbert and Vidal Beneyto (1986, 26) call the ‘performative power’ of a newspaper that is a dominant reference, ‘power to institu- tionalize what it says, to give a reality check to everything it publishes and, conse- quently, to symbolically nullify what it omits’. The period included in the analysis was four months from the date of the first known case in each country, 30 January 2020, for Italy’s Corriere della Sera and 31 January 2020, for Spain’s El Paıs. The purpose was to obtain a wide period that included the main starting moments and first wave of the coronavirus pandemic in both countries. In the research, we chose journalistic stories in which focus was placed on references to the Catholic Church directly linked to the coronavirus, discarding others about the Church, but not connected to the virus, such as the elections in the Spanish Episcopal Conference or the death of Ernesto Cardenal. Thus, we search terms such as ‘catholic’, and ‘virus’, in the virtual archives of each of the newspapers (El Paıs, https://elpais.com/diario/, Corriere della Sera, http://archi- vio.corriere.it/Archivio/interface/landing.html). We didn’t include news with circum- stantial mentions. The sample was selected through the virtual newspaper libraries of the newspapers that collect the editions published on paper. It is, therefore, the printed edition that is referenced, although online instruments have been used for its compilation. We con- sider the printed reference relevant given the peculiarities of production routines in the digital environment, that is, the fact that the implementation of SEO techniques and the constant flow of new information conditions the selection of content. On the other M.-J. 3. Methodology and the field of the study  We will refer to the human interest when the text reflects the suffering of people, members of the Church or not, who need comfort or assistance from the Church, as well as when the texts provoke emotions: anger, tenderness, sadness. p g  The economic consequences are present due the effect of the pandemic on the economic situation of the Church.  The economic consequences are present due the effect of the pandemic on the economic situation of the Church.  Finally, we will consider the moral approach when judging behavior. Conflict about the Church often arises linked to civil authorities and political leaders. In this case, the imposition of restrictive measures with a direct impact on Catholic commun- ities was fertile ground for controversy. Failure to comply with the norm, the intervention of the police in churches or religious ceremonies, fines to some clerics or Catholic people for breaking lockdown, the internal division between different bishops or Catholic groups about the rules of the Episcopal Conference or the requirement of stopping the bans on celebrations were cause for confrontation, controversy or disagreement. However, next to this negativity, the opposite behavior was the framework of responsibility. We found this framework in the news that showed the Church comply- ing with health regulations to avoid contagion, even going beyond those established by the authorities; the network of coordination in parishes and dioceses to do prevention pedagogy; the alteration of the liturgical calendar and the proposal of alternative dates for the main festivities such as Easter; and prayers, blessings and liturgies without peo- ple being present – an exceptionally unusual circumstance. The third frame is that of human interest. It includes the charitable behaviors of priests or religious people: food distribution, help over the telephone; the presence and prayer of priests in cemeteries. 3. Methodology and the field of the study As Entman (1993, 54) says, ‘most frames are defined by what they omit as well as include, and the omissions of potential problem definitions, explanations, evaluations, and rec- ommendations may be as critical as the inclusions in guiding the audience’. The first step in analyzing the building of the image of the Church during the pandemic is to draw a map with what they include as preferred issue and what they put in the background. In this work we connect the deductive process (Ardevol-Abreu 2015; Ballesteros Herencia 2015, 52) from the five news frames established by Semetko and Valkenburg (2000) as generic frames (conflict, attribution of responsibility, human interest, eco- nomic consequences, and morality), with the inductive process, to discover the com- mon places or topoi present in the analyzed news. They will be our specific frames. So, we can organize into categories the events narrated by the media, according to the focus of the journalists. The objective is to be able to quantify the presence of gen- eric frames about the Church in the pandemic: conflict (due to opposition to restric- tions), human interest (due to assistance activity), attribution of responsibility (due to the implementation of prevention measures), morality (due to the judgment of the CHURCH, COMMUNICATION AND CULTURE 161 actions) and the economic consequences (due to the impact of the pandemic on the finances of the Church).  We will refer to conflict whenever there is a confrontation or disagreement (phys- ical, verbal or ideological) between the authorities (including the police) and the members of the Church.  We will refer to conflict whenever there is a confrontation or disagreement (phys- ical, verbal or ideological) between the authorities (including the police) and the members of the Church.  We will talk about attribution of responsibility, when the Church is pointed out as being responsible for the development of the pandemic or the possible contagion due to its action or omission, as well as when it is related to possible solutions.  We will talk about attribution of responsibility, when the Church is pointed out as being responsible for the development of the pandemic or the possible contagion due to its action or omission, as well as when it is related to possible solutions. 3. Methodology and the field of the study It also includes giving words of encouragement, teach- ing and testimony urging people to accept the lockdown situation and to prepare for the return of normality with greater sensitivity towards people who suffer; concern for those who are alone or suffering, for those hospitalized and for the families of those who die and cannot accompany them; tributes to health personnel; prayer to ask for the end of the pandemic; dispensation from Lenten abstinence, and understanding for those who have difficulties or fear after lockdown in fulfilling their religious obliga- tions; missionary activity in a pandemic situation; care even for unbelievers in need of comfort. The fourth frame is the economic consequences, very common in the information on the pandemic. It is difficult to link this one to the Church except regarding finances or the economic impact of the suppression of some religious celebrations such as Holy Week in emblematic places such as Seville or Rome. 162 M.-J. POU-AMÉRIGO 162 M.-J. POU-AMÉRIGO 162 The last frame is linked to morality and therefore to the moral assessment of behav- iors both by the media and by selected sources. The media are often critical of the peo- ple in question, especially of some bishops with very personal positions on the complicated issues. We have searched in the units of analysis for the media keys to present each event. Because of this, we have focused on the main elements in the news: headlines, summaries and highlights. We have applied the content analysis (Krippendorff 2012) because it helps us to show the hidden words behind the obvious, in other words the subliminal content. This is the way of trying to get an idea of the journalists’ point of view (Pi~nuel 2002). This is the connection between saying and thinking, the link between expression and understanding as Van Dijk (1993, 147) explains: ‘the structures and meanings [ … ] reflect the structures and contents of the specific mental model of that individual reporter about [a] specific event’. In any case, whether the decision is conscious or involuntary, the result is part of the message that the reader receives. This is our object- ive. We do not seek to inquire about the motivations of the media but to show the con- structed image to the reader. 3. Methodology and the field of the study In the analyzed texts, the events narrated explain the contribution of the Church during the pandemic, both in a positive and negative sense. The list of positive behav- iors is made up of the following:  compliance with sanitary regulations  establishment of guidelines to avoid contagion  making available different ways of following the liturgy  charitable activities  spiritual or emotional accompaniment  to stay when others do not, such as at funerals  recommendations and testimonies for living during the lockdown  to stay when others do not, such as at funerals  recommendations and testimonies for living during the lockdown There are also negative behaviors and some can even be ambiguous. Among the negatives:  non-compliance with the rules  opposition to the rules of the government or recommendations of the Vatican or the Episcopal Conference  to deny the virus  peculiar and extravagant behavior, according to the media  to deny the virus  peculiar and extravagant behavior, according to the media We have considered a behavior ambiguous when, depending to the observer, it can be described as positive or negative. For example, the claim of religious freedom because of the intervention of the police in a legal mass, according to the rules. For people who accept the rule, it is legitimate; for people who consider any assembly dan- gerous, even if it is legal, it will be interpreted as an unnecessary provocation. In any case, the doubt disappears when we read how the media has presented the fact. Therefore, is important to focus on headlines and prominent elements of the way the information is presented. CHURCH, COMMUNICATION AND CULTURE 163 We have analyzed two kinds of issues: the relationship between the Church and the government, on the one hand, and the Church’s activity during the pandemic, on the other. The relationship with the authorities is one of the most relevant elements because the restrictive measures implied, de facto, an intervention in the life of the Church. Truly, the context imposed restrictions in all social fields and this is why it was interesting to know the attitude of ‘collaboration’, ‘non-collaboration’, or ‘other’ (cases non explicit in the text) on the part of the Church. 3. Methodology and the field of the study We have differentiated the activity of the Church during the first months of the pandemic into: ‘volunteer and social assistance tasks’; ‘Liturgy and sacraments’; ‘concern for herself’ and ‘others’, when not specified. To differentiate a positive or negative action, we have analyzed the topoi. The topoi or common places are ideas shared by the community, naturally or acquired, that ‘allow to establish values and hierarchies’, according to Perelman and Olbrechts-Tyteca (1994, 146). Their analysis has been developed by the research group ‘Family and Media’ (Gonzalez Gaitano 2015) and addresses the presence of common places related to social virtues (Perez Martınez 2016). Some works about it are published by Norberto Gonzalez Gaitano (2015), Giovanni Tridente (2009) or Piotr Studnicki (2016). Their presence in the news is essential (Gonzalez Gaitano 2010, 76) and contributes to developing the frames with a constellation of issues based on the different events and the way of interpreting them. Barthes (2009, 237) called them the ‘reserve of themes’, that are not questioned because of their extension and assumption by the community. That is why they are effective. We can find two kind of topoi, natural and cultural (Gonzalez Gaitano 2015). The topoi of the first group are natural to the human being, they are anthropological; the topoi of the second group are cultural products, the result of ideologies. Since the topoi are presupposi- tions for the journalist’s narration, and that they share with the reader to the point of ‘guiding the judgment’ (Studnicki 2016), it is possible to find more than one topos in the same text. The topoi are related to frames and indicate the predominant frame. In relation to the action of the Church during the pandemic, we find the following common places. We have formulated them as statements of latent content in the news. The natural topoi are:  suffering and vulnerable people are the priority  priests know how to accept death and spiritual pain  the Church consoles and accompanies in the tragedy  the pandemic is an occasion for improving our spirituality  the Church complies with prevention rules And the cultural topoi:  religion is tradition  religion is fanaticism  religion has an immutable ancestral liturgy; our astonishment at the mediatization of religious rites is natural 164 M.-J. POU-AMÉRIGO M.-J. 3. Methodology and the field of the study POU-AMÉRIGO  religious stubbornness leads to violation of law and conflict  there are two kinds of clergy: the collaborator and the intolerant  the Church is only concerned with its finances  the Church considers the pandemic a divine punishment  religion leads to peculiar attitudes 4.1. Generic frames The sample is made up a total of 62 pieces in El Paıs and 73 in Corriere della Sera (see Table 1). The two most relevant frames in the information published on the actions of the Church during the first wave of covid-19 are the ‘attribution of responsibilities’ and ‘human interest’. In El Paıs the ‘attribution of responsibilities’ predominates (33.8%) over ‘human interest’ (30.6%) while in Corriere della Sera it is the opposite, ‘human interest’ predominates notably (61.6%) over ‘attribution of responsibilities (26%). ‘Conflict’ is the third frame, with a much lower percentage, in both newspapers (19.3%, El Paıs, and 9.5%, Corriere della Sera) and it is the same with morality. The ‘economic consequences’ are only relevant in El Paıs (4.8%). About the attitude of the Church to the authorities, the highest percentage is for the field ‘others’ that do not specify the degree of collaboration (see Table 2). This indicates that it is not the priority for the media. In addition, both in El Paıs (30.6%) and in Corriere della Sera (17.8%), the number of texts that show the willingness of the Church to collaborate is higher, almost double, compared to those which do not (17, 7% and 8.2, respectively). Regarding the actions of the Church, the difference between El Paıs and Corriere della Sera is striking. Thus, for El Paıs, the main activities of the Church are the liturgy, especially the Eucharist – sometimes ‘clandestine’, or funerals (22.5%). In contrast, assistance to the underprivileged and spiritual care remain in the background (4.8%). Corriere della Sera is very interested in publishing news about initiatives other than the Mass, such as blessings in cemeteries, showing relics or sick calls. It has a greater num- ber of pieces on liturgy than El Paıs (26%), however, the number of pieces about Table 1. News frames of the Church by newspapers. F /N El P  Table 1. News frames of the Church by newspapers. Frames/Newspapers El Paıs Corriere della Sera Attribution of responsibility 33.8% (21) 26% (19) Human interest 30.6% (19) 61.6% (45) Conflict 19.3% (12) 9.5% (7) Morality 11.2% (7) 2.7% (2) Economic consequences 4.8% (3) 0 Total 62 73 Source: Author’s compilation. Table 2. Attitude of the Church to the authorities. Newspapers/Attitudes Collaboration No collaboration Other El Paıs 30.6% (19) 17.7% (11) 51.6% (32) Corriere della Sera 17.8% (13) 8.2% (6) 73.9% (54) Source: Author’s compilation. Table 2. 4.1. Generic frames Attitude of the Church to the authorities. CHURCH, COMMUNICATION AND CULTURE 165 charitable assistance is also higher (10.9%). The only higher percentage in El Paıs is about the lack of activity (14.5%). It is possibly one of the biggest differences between the two newspapers. Corriere della Sera focuses on the hard work of priests and their sacrifice in caring for the patients and families. The evidence is that Corriere della Sera includes a lot of news about sick bishops, priests and nuns, if they are victims of Covid-19, but El Paıs does not. Furthermore, Corriere della Sera uses the interview genre to give religious people a voice, an initiative that is lacking in El Paıs (see Table 3). About the frame ‘attribution of responsibility’, both newspapers show how the Church has implemented measures to prevent contagion and, in fewer cases, point out when it has not been done (see Table 4). Half of the pieces refer to how the Church has taken care of prevention, hygienic measures and compliance with Health regula- tions. In El Paıs, 42.8% do so, compared to 9.5% that address the opposite issue. In Corriere della Sera, 58.8% have to do with following regulations, and there is no piece that reports on the opposite phenomenon. Both newspapers emphasize the celebration of rites in solitude or the closing of iconic churches, such as the Duomo of Milan, which offers an image of exceptionality. This percentage corresponds to the focus on liturgical actions by El Paıs compared to the focus on assistance by Corriere della Sera. Thus, in El Paıs, 23.8% of pieces show surprise about rites never having been celebrated in such a way before (alone or with- out the sign of peace). Corriere della Sera also echoes this but with a lesser percentage (11.7%). El Paıs, for example, jokes about the disappointment of the parishioners who in 2020 were not allowed to kiss the Christ of Medinaceli on Good Friday according to custom. The newspaper said: ‘It is not clear if the spell loses strength when the kiss is lacking’ (Quesada 2020). Most of the cases are from Holy Week celebrations, especially in the Vatican, with the image of the Pope in a completely empty St. Peter’s Square. 4.1. Generic frames The frame ‘conflict’ includes resistance to the measures implemented by govern- ments or ecclesiastical authorities and their consequences as fines or sanctions. There are incidents of police intervention and, sometimes, the journalist is at the scene and evaluates the ‘illegal’ situation. Its funny but there is an article in which a journalist describes an ‘illegal’ Mass and explains the rules – and the conclusion is that the rules are not being violated, according to his own story. When we say ‘sanctions’, we refer to fines or something like this, because, if the police had to intervene, the behavior prob- ably will be punished. In El Paıs, non-compliance and sanctions are more frequent (50%) than in Corriere della Sera (42%), although it differentiates as to whether there was only a complaint or also a fine. In Corriere della Sera, on the other hand, more emphasis is placed on the different positions taken up regarding the norms imposed by the civil authorities, or the different sensitivities in relation to the freedom of the Church to self-regulate its activity (57.1%). Corriere della Sera interviews bishops reluctant to avoid the celebration of the Eucharist; something that, however, we do not find in El Paıs (16.6%). In the latter, on the other hand, one finds more importance given to the political use of the argument of religious freedom by some groups or parties (25%) (see Table 5). This difference in the main conflict between the Church and the respective govern- ments must be sought on the one hand, in the interest of the media, but also in the reality of the country itself. In fact, El Paıs emphasizes the criticism of some Spanish bishops for the recommendations of the Episcopal Conference but includes a descrip- tion of the Italian situation and the role of the Pope in the relationship between the Church and government. El Paıs, in addition, insists on presenting an image of the Church as divided. It presents the bishop of Alcala, Mons. Reig, as a counterpoint to the president of the council of bishops, Cardinal Omella. We can see the same in Corriere della Sera in a piece that recounts the ins and outs of the tensions between the Church and the Government: ‘A telephone call to Casa Santa Marta. Thus the premier has disarmed the CEI [Italian Bishops’ Conference]’ (Franco 2020). 4.1. Generic frames Both newspapers include anecdotical news; El Paıs writes about a Detroit priest who sprinkles holy water for blessings with a toy water pistol to keep his distance, and in Corriere della Sera, about another American priest who celebrates mass while the parishioners remain in the car and called it a ‘drive-in mass’ (Maniaci 2020). Table 3. Main activities of the Church. Newspapers/Activities Assistance Liturgy Not activity Other El Paıs 4.8% (3) 22.5% (14) 14.5% (9) 58% (36) Corriere della Sera 10.9% (8) 26% (19) 4.1% (3) 58.9% (43) Source: Author’s compilation Table 4. Frame ‘attribution of responsibility’. Frame/Newspapers El Paıs Corriere della Sera The Church implements measures to prevent contagion 42.8% (9) 58.8% (10) No preventive measures are taken 9.5% (2) 0 Celebrations behind closed doors an unusual image of the Church 23.8% (5) 11.7% (2) Pedagogy of prevention in parishes and dioceses 9.5% (2) 0 Later celebration of Easter 4.7% (1) 0 Alternatives to liturgical activity via electronic means 4.7% (1) 23.5% (4) Peculiar and extravagant behaviors. in the opinion of the media 4.7% (1) 5.8% (1) Source: Author’s compilation 166 M.-J. POU-AMÉRIGO 166 In addition, both newspapers talk about sharing the celebration of the liturgy through electronic media, television, and the Internet. Corriere della Sera devotes more attention to it (23.5% compared to 4.7% in El Paıs) not only because of the novelty but especially because of Corriere della Sera’s interest in priests. The Italian paper dedicated more pieces to the tasks of priests and among them the necessary adaptation of litur- gical activity through new media. Regarding celebrating Easter later, the impact was particularly noted in Seville, where there are the confraternities, groups of people devoted to the image of Christ or the Virgin. These became the protagonists of the news (El Paıs 4.7%). In fact, one of the colorful bits of news collected is about the sale of a hand sanitizer with a ‘brother’s scent’. Finally, we have included news about the task of spreading the message about prevention of contagion among the parishioners only in El Paıs (9.5%). Furthermore, as has been said, peculiar behaviors are mentioned but they are not very significant (4.7% in El Paıs and 5.8% in Corriere della Sera). In other texts we can find these keys but not predominantly, so they are not included as prevalent frames in the quantification. 4.1. Generic frames Finally, conspiracy theories and denialism are not present in the Corriere della Sera. They are in El Paıs CHURCH, COMMUNICATION AND CULTURE 167 Table 5. Frame ‘conflict’. Frame/Newspapers El Paıs Corriere della Sera non-compliance with the rules without sanction 25% (3) 0 Sanctions 25% (3) 42.8% (3) opposition to guidelines from both the government and the Vatican or the Episcopal Conference 16.6% (2) 57.1% (4) Denialism 0 0 conspiracy theories appealing to the religious factor 8.3% (1) 0 Political use of religious freedom 25% (3) 0 Source: Author’s compilation. Source: Author’s compilation. Table 6. Frame ‘human interest’. Frame/Newspapers El Paıs Corriere della Sera assistance activities 26.3% (5) 17.7% (8) spiritual or emotional accompaniment 10.5% (2) 31.1% (14) Being present when others cannot be (funerals) 15.7% (3) 28.8% (13) Alerts, recommendations and testimonials to help people withstand lockdown 36.8% (7) 2.2% (1) Family or social traditions 10.5% (2) 0 Victims (bishops, priests and religious) 0 20% (9) Source: Author’s compilation. but only as a prejudice (8.3%), that is, attributing to the Church the explanation of the pandemic as a ‘divine punishment’ when it is not true. The frame of ‘human interest’ focuses on the activity of the Church in assisting the underprivileged with the distribution of food or direct aid; soup kitchens or the work of Caritas, but also includes the spiritual comfort of patients or their families as well as emotional comfort for any person addressing the Church. El Paıs devotes more atten- tion to assistance to vulnerable, 26%, compared to 17.7% in Corriere della Sera, but the latter focuses much more (31.1%) on the issue of spiritual and emotional accompani- ment, especially on the part of parish priests and bishops, whereas in El Paıs this is pre- sent in a tiny minority of pieces (10.5%) (see Table 6). In the same way, Corriere della Sera, as has been said, dedicates a lot of space to showing the work of priests and bishops who feel close to their community, especially when it comes to funerals or blessings in the cemetery in the absence of loved ones (28.8% in Corriere della Sera, compared to 15.7% in El Paıs). In this field, references to death and the disposition of coffins in cemetery chapels or in Churches until they can be buried have been included. 4.1. Generic frames The state of alarm, which establishes that you can only leave home for essential activities such as going to work or shopping, does not prohibit the liturgy of the Eucharist, but conditions its celebration to "avoid crowds" since it is possible to "guarantee a minimum distance of one meter" between assistant and assistant." (Carranco 2020) Only two pieces appear in Corriere della Sera, one about the preconceived idea that the pandemic is divine punishment and another that criticizes a priest for imposing preventive measures. The text criticizes an ‘excessive zeal’. In summary, in El Paıs, the frame ‘attribution of responsibility’ predominates above all. The focus is on the acceptance by the Church of sanitary rules, and its collaboration in the prevention of contagion. The second frame is related to the Church’s activity in caring for the vulnerable and providing spiritual assistance, and the presence of priests at funerals. The conflict frame is related to the division of opinions regarding the gov- ernmental measures and the resistance to the restrictions of some voices within the Church, even outside the Church, but using the argument of religious freedom to demand a change in the measures. Morality is present, in the appeals to topoi that asso- ciate religion with superstition, charlatanism, denialism or conflict. Finally, the news does not delve into the economic consequences except linked to the suppression of Holy Week liturgies, the closing of the churches or the absence of pilgrims on the Camino de Santiago. g In Corriere della Sera the most relevant frame is that of ‘human interest’ because it gives more prominence to the interview genre, and thus gives information about spe- cific people, previously unknown, who are interviewed either because of their experi- ence or because of their involvement in the support and care of the community. In this sense, it is common to find the voice of bishops or priests concerned about the faithful of their diocese or parish; engaged in assistance tasks or promoting charity, mutual aid and greater sensitivity with others in difficult times. In El Paıs, these words come more frequently from the important messages of the Pope, and connect with the frame of ‘conflict’ and the division between different sectors of the Church, which is much more prevalent in El Paıs than in Corriere della Sera. 4.1. Generic frames This testimony also joins that of bishops, missionaries, volunteers, Catholic people and even some patients who have found their faith strengthened after their hard experience. El Paıs has incorpo- rated it with more dedication (36.8%) but the figure in Corriere della Sera (2.2%) can be misleading. The reason is that this figure is separated from the one that corresponds to the labors of priests. In that sense, it is striking that only Corriere della Sera docu- mented the victims of the virus among bishops, priests and religious. For Corriere della Sera, including them as protagonists, sources or voices of social relevance seems more natural than for the Spanish newspaper. Finally, a reference to traditions in El Paıs (10.5%) has been included, because some people spoke of consoling religious rites as part of a family tradition that is no longer used, but that is missed. Corriere della Sera doesn’t mention it. 68 M.-J. POU-AMÉRIGO 168 Regarding the frame ‘Economic consequences’ in El Paıs there are only three pieces that refer to the economic impact of the suppression of Holy Week, to the marketing of a hand sanitizer with the smell of incense (in both cases it is not an activity of the Church itself, but of some Catholic people) and another on the impact of the corona- virus on the finances of the Churche and its repercussion on the economy of the par- ishes. In Corriere della Sera there are no uses of this frame. Finally, the frame ‘morality’ is present mainly in opinion texts where moral evalua- tions of behaviors can be made. In El Paıs it appears in a veiled way in a couple of informative pieces that focused on a hidden breach of the norm (the headline of one of them is ‘Attending mass through the back door’), however, they show in the text that no norm prohibits what is being described: The priest, Miguel Angel Nieto, explains in a statement published on social networks that the agents invited them to leave the Church or, otherwise, they would be sanctioned. "We were only five parishioners and the priest, so the safety distances were more than assured," he says. 4.2. Topoi or common places The frames are complemented with the analysis of topoi or common places that delve into the latent content of the information. Thus, for example, the attribution of respon- sibility, with the topoi, has a different dimension. It is not a question of responsibility as an attribution of cause-effect, but in a deeper sense, of assumption of basic princi- ples concerning human nature and interaction with others in society. In fact, among the topoi present in the news, we have found ‘the Church complies with prevention measures’ or ‘the Church consoles and accompanies in the tragedy’ as an example of ‘attribution of responsibility’ and at the same time of ‘human interest’. With this other important frame present in this paper, something similar happens: ‘human interest’ finds its roots in those common places: ‘the suffering and vulnerable are the priority’. ‘Morality’ also connects with the judgment which is the basis of admiration and grati- tude towards priests, ‘the priest knows how to remain close in the death’, or the convic- tion that this time will generate a spiritual renewal, ‘the pandemic is an occasion of greater spirituality’. On the other hand, the ‘ideological’ or cultural topoi include prejudices or the point of view from which the Church is judged, which emanates from dominant thoughts such as: ‘the Church is tradition’, ‘the Church is fanaticism’, ‘religion has unchanging traditional rites, so it is natural to be surprised by changes such as going online’, ‘the Church considers the pandemic a divine punishment’ or ‘religion fosters peculiar and strange attitudes’. In this field appear the topoi linked to economic consequences such as ‘the Church is concerned about its finances’; or conflict, such as ‘stubbornness leads to violation of law and conflict’ or ‘there are two types of clerics: collaborators and rebels’. The natural topoi at the basis of judgments on religious activity are shown in Table 7. The cultural topoi at the basis of judgments on religious activity are shown in Table 8. 4.1. Generic frames Finally, it is striking that the Corriere della Sera offers more variety of Catholic voices but also fewer moral judgments about the Church even when some bishops or the CEI CHURCH, COMMUNICATION AND CULTURE 169 demand a more careful treatment by the authorities regarding the religious life of the community. Source: Author’s compilation. 4.2. Topoi or common places the Church being concerned about itself (‘the Church is concerned about its finances’), we find that the Church is focused on those who suffer (‘the suffering and vulnerable are the priority ‘and’ the Church consoles and accompanies in the midst of tragedy’). In this sense, the results are always favorable to the Church: the references to compli- ance with prevention are more numerous than references to violations (12.9% com- pared to 4.8%, in El Paıs and 5.4% compared to 4%, in Corriere della Sera). In the same way, the Church is seen as more focused on others, especially the suffering and vulner- able, than on itself (9.6% compared to 1.6% in El Paıs; 19% compared to 0 in Corriere della Sera). The more unusual topoi in both newspapers are those linked to a negative view of the Church and the role of religion in society. In El Paıs there predominates a Manichean view of Catholics, especially the clergy (16.1%, compared to 6.8% in Corriere della Sera) and a constant conviction about religious fanaticism (11.2% com- pared to 0 in the Italian newspaper). In Corriere della Sera, in contrast, the work of priests and bishops stands out; they are seen as dedicated to the needy, rather than engaged in litigation. Finally, both newspapers share a somewhat stagnant vision of the Church; that’s why technological developments are repeated as surprising news (14.5% in El Paıs and 19% in Corriere della Sera). The interest of the press in this is logical but focus on the novelty and not so much on the adaptability of the Church itself is striking. 4.2. Topoi or common places In some cases, the topoi can be paired as opposites, thus, in contrast with compli- ance (‘the Church complies with prevention’), non-compliance appears (‘stubbornness leads to violation of law and conflict’); in contrast to the frame of the sacrificial and self-surrendering priest (‘the priest knows how to accompany people who are dying’), there is the frame that the clergy is divided by different opinions about the measures against Covid (‘there are two types of clergy: collaborators and rebels’) or in contrast to Table 7. Topoi or common places: natural topoi. Natural topoi/newspapers El Paıs Corriere suffering and vulnerable people are the priority 9.6% (6) 19% (14) the priest knows how to accompany people who are dying 6.4% (4) 21.9% (16) the Church consoles and accompanies in the midst of tragedy 3.2% (2) 13.6% (10) the pandemic is an occasion for greater spirituality 8% (5) 8.2% (6) the Church complies with prevention 12.9% (8) 5.4% (4) Source: Author’s compilation. 170 M.-J. POU-AMÉRIGO 170 M.-J. POU-AMÉRIGO Table 8. Topoi or common places: cultural topoi. Cultural topoi/Newspapers El Paıs Corriere della Sera Religion is tradition 4.8% (3) 0 Religion is fanaticism 11.2% (7) 0 Religion has unchanging traditional rites, so it is natural to be surprised by changes such as going online 14.5% (9) 19% (14) Stubbornness brings about violation of rules and conflict 4.8% (3) 4% (3) There are two types of clerics: collaborative and rebellious 16.1% (10) 6.8% (5) The Church is concerned about its finances 1.6% (1) 0 The Church considers the pandemic a divine punishment 1.6% (1) 1.3% (1) Religion fosters peculiar and strange attitudes 4.8% (3) 0 Source: Author’s compilation. the Church being concerned about itself (‘the Church is concerned about its finances’), we find that the Church is focused on those who suffer (‘the suffering and vulnerable are the priority ‘and’ the Church consoles and accompanies in the midst of tragedy’). In this sense, the results are always favorable to the Church: the references to compli- ance with prevention are more numerous than references to violations (12.9% com- pared to 4.8%, in El Paıs and 5.4% compared to 4%, in Corriere della Sera). In the same way, the Church is seen as more focused on others, especially the suffering and vulner- able, than on itself (9.6% compared to 1.6% in El Paıs; 19% compared to 0 in Corriere della Sera). 5. Conclusions The Catholic Church was subject of news interest during the first months of the Covid-19 pandemic with a similar volume of information in Spain and Italy. However, Corriere della Sera gave voice to Catholic protagonists more frequently than El Paıs, above all, to bishops, priests and volunteers. In this sense, it showed the hard and necessary work of priests, especially at death-beds and at lonely funerals, as well as through the proximity of bishops and parish priests to their parishioners. Therefore, the first hypothesis is confirmed, regarding the frames of reference through which the role of the Church is interpreted when the story is a global tragedy: The quality press in Italy and Spain gave precedence to the charitable work of the Catholic Church during the pandemic, as opposed to the conflict with the political authorities over restrictive measures. Moreover, when it comes to the relationship with civil institutions, a positive image of the Church is offered, ready to collaborate and CHURCH, COMMUNICATION AND CULTURE 171 apply the preventive measures recommended by the health authorities. Discrepancies regarding the application of the norms on liturgical activity appear as a factor of internal division, especially in El Paıs, but conflict is not the predominant framework. El Paıs insists on showing the Church’s disappointment with the measures, while Corriere della Sera shows the imaginative effort to implement novel ways to broadcast the celebrations of the liturgy through the Internet. In both cases, the surprise at this ability to adapt to the online context is constant. In the same way, the idea of excep- tionality accompanies the account of ceremonies and rites in the absence of the community. 5. Conclusions The topoi or common places present in the information are, on the one hand, of a natural or anthropological nature: ‘suffering and vulnerable people are the priority’, ‘the priest knows how to accompany people who are dying’, ‘the Church consoles and accompanies in the midst of tragedy’, ‘the pandemic is an occasion for greater spiritual- ity’, ‘the Church complies with prevention measures’; and on the other hand, cultural or ideological: ‘religion is tradition’, ‘religion is fanaticism’, ‘religion has unchanging traditional rites, so it is natural to be surprised by changes such as going online’, ‘stubbornness leads to violation of law and conflict’, ‘there are two types of clergy: col- laborative and rebellious’, ‘the Church is concerned about its finances’, ‘the Church considers the pandemic a divine punishment’, ‘religion fosters peculiar and strange attitudes’. Hypotheses 2 and 3 are confirmed, since in the media analyzed both in Italy and in Spain, the spiritual and welfare work of the Church is valued (H2) and its oppos- ition to government measures is criticized (H3). However, the two are not compar- able since the positive assessment predominates in the story when there are dualities in the topoi found in the interpretation of the facts. Thus, the Church appears more concerned about others (‘the suffering and vulnerable are the priority’ and ‘the Church consoles and accompanies in the midsttragedy’) than for itself (‘the Church is concerned about its finances’). Likewise, it makes more effort in preven- tion, caring for people and the common good (‘the Church complies with preven- tion’) than in confrontation and controversy (‘stubbornness leads to violation of law and conflict’). The negative view of the Church and the role of religion in society can also be paired with opposite frames: in contrast to the frame of self-sacrificial priests (‘the priest knows how to accompany people who are dying’), we read that the the clergy is divided by the prevention measures (‘there are two types of clergy: collaborative and rebellious’). We see this duality in both newspapers. In El Paıs Manichean prejudice prevails over Catholic sense, especially with regard to the clergy. In Corriere della Sera, on the other hand, the work of priests and bishops stands out: they are seen as dedi- cated to the needy, rather than engaged in litigation. Disclosure statement No potential conflict of interest was reported by the author(s). 5. Conclusions We can therefore appreciate that the construction of the image of the Church in the media largely depends on the weight of prejudices or socially accepted topoi on the situation that the media is willing to accept. As seen in the global crisis of Covid-19, the media that give voice to the protagonists enrich the story with points of view that could not have been foreseen. However, those that ignore the news that does not cor- respond with the pre-established patterns obscure an important part of reality. 172 M.-J. POU-AMÉRIGO 172 M.-J. POU-AMÉRIGO Notes on contributor Marıa-Jose Pou-Amerigo has a PhD in Communication and Journalism from the Complutense University of Madrid (1999); a Bachelor’s degree in Communications and Journalism from the Polytechnic University of Valencia-CEU San Pablo (1992); a Bachelor’s in Humanities from UOC of Barcelona (2009), and a Master’s in Teaching Spanish as a Foreign Language from UNED (2012). 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References “Christian Democratic Parliamentarians: From a Century of Multifaceted Recruitment to the Convergence within "Larger Family"?” In Democratic Representation in Europe: Diversity, Change, and Convergence edited by Maurizio Cotta and Heinrich Best, 253–283. Oxford: Oxford University Press.
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Palmitic acid inhibits vascular smooth muscle cell switch to synthetic phenotype via upregulation of miR-22 expression
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ABSTRACT Synthetic phenotype switch of vascular smooth muscle cells (VSMCs) has been shown to play key roles in vascular diseases. Mounting evidence has shown that fatty acid metabolism is highly associated with vascular diseases. However, how fatty acids regulate VSMC phenotype is poorly understood. Hence, the effects of palmitic acid (PA) on VSMC phenotype were determined in this study. The effect of the PA on VSMCs was measured by live/dead and EdU assays, as well as flow cytometry. Migration ability of VSMCs was evaluated using transwell assay. The underlying targets of miR-22 were predicted using bioinformatics online tools, and confirmed by luciferase reporter assay. The RNA and protein expression of certain gene was detected by qRT- PCR or western blot. PA inhibited VSMC switch to synthetic phenotype, as manifested by inhibiting VSMC proliferation, migration, and synthesis. PA upregulated miR-22 in VSMCs, and miR-22 mimics exerted similar effects as PA treatment, inhibiting VSMC switch to synthetic phenotype. Inhibition of miR-22 using miR-22 inhibitor blocked the impacts of PA on VSMC phenotype modulation, suggesting that PA modulated VSMC phenotype through upregulation of miR-22 expression. We found that ecotropic virus integration site 1 protein homolog (EVI1) was the target of miR-22 in regulation of VSMC phenotype. Overexpression of miR-22 or/and PA treatment attenuated the inhibition of EVI1 on switch of VSMCs. These findings suggested that PA inhibits VSMC switch to synthetic phenotype through upregulation of miR-22 thereby inhibiting EVI1, and correcting the dysregulation of miR-22/EVI1 or PA metabolism is a potential treatment to vascular diseases. AGING 2022, Vol. 14, No. 19 Palmitic acid inhibits vascular smooth muscle cell switch to synthetic phenotype via upregulation of miR-22 expression Correspondence to: Congxia Wang; email: wcxcirculation@163.com, https://orcid.org/0000-0002-3423-4644 Keywords: VSMC, palmitic acid, synthetic phenotype, miR-22 Received: August 17, 2022 Accepted: October 3, 2022 Published: October 12, 2022 Copyright: © 2022 Hu 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. www.aging-us.com www.aging-us.com Yanchao Hu1, Yajie Fan1, Chunyan Zhang1, Congxia Wang1,& 1Department of Cardiovascular Medicine, The Second Affiliated Hospital of Xi'an Jiaotong University, Shaanxi, Xi'an 710004, China 1Department of Cardiovascular Medicine, The Second Affiliated Hospital of Xi'an Jiaotong University, Shaanx Xi'an 710004, China Correspondence to: Congxia Wang; email: wcxcirculation@163.com, https://orcid.org/0000-0002-3423-4644 Keywords: VSMC, palmitic acid, synthetic phenotype, miR-22 Received: August 17, 2022 Accepted: October 3, 2022 Published: October 12, 2022 Correspondence to: Congxia Wang; email: wcxcirculation@163.com, https://orcid.org/0000-0002-3423-4644 Keywords: VSMC, palmitic acid, synthetic phenotype, miR-22 Received: August 17, 2022 Accepted: October 3, 2022 Published: October 12, 2022 INTRODUCTION VSMC phenotypic switch regulation is important in vascular diseases. Vascular function is largely dependent on vascular smooth muscle cells (VSMCs). Different from the skeletal muscle cells or cardiomyocytes, VSMCs remain possessing remarkable phenotypic plasticity in response to multiple stimuli [1]. VSMCs switch from a contractile state to a dedifferentiated, synthetic phenotype, playing crucial roles in several vascular diseases [2–4]. The synthetic phenotype induces migration to the intima and enhances proliferation and extracellular matrix protein synthesis, thereby resulting in an impaired contractility of VSMC [5]. Therefore, exploration of the underlying mechanisms involved in There are multiple environmental stimuli have been identified as factors which lead to VSMC phenotype switch, such as growth factors, reactive oxidative species (ROS), and mechanical injury [6, 7]. Recent studies have shown that metabolites were also involved in regulation of VSMC phenotype [8]. For example, lactate, a product of glucose metabolism, was found to promote the synthetic phenotype of VSMCs, which links glucose metabolism to VSMC phenotypic switch [8]. Mounting evidence has shown that fatty acid metabolism is abnormal in vascular diseases, which AGING 8046 www.aging-us.com fluorescence was read using a microplate reader (FLUOstar® Omega). plays an important role in the development of atherosclerosis and other vascular diseases [9, 10]. These advances suggest that fatty acids metabolism may play a role in regulation of VSMC phenotype. However, how fatty acids regulate VSMC phenotype is poorly understood. As the most common saturated fatty acid found in organism, palmitic acid (PA) serves as an energy source or component of partially biochemicals and cellular structures. The circulating level of PA is increased in metabolic disorders and correlated with the adverse outcomes of cardiovascular diseases [11–13]. Here, we aimed to examine the impacts and underlying mechanism of PA on VSMC phenotype. fluorescence was read using a microplate reader (FLUOstar® Omega). EdU assay Proliferation of VSMCs was analyzed using the Click- it EdU kit (C10086, Invitrogen, USA). Briefly, cells were seeded on the slides at a density of 1.0 × 103 cells in 12-well plate each well. After treatment, cells were incubated with 50 μmol/L EdU solution at 37°C for 2 h. Then, cells were washed with cooled PBS for twice and fix 4% PFA at 4°C for 15 min. Following this, 100 μl Apollo reaction cocktail was added into cells followed by nucleus staining with Hoechst 33342 according to the manufacturer’s protocol. The fluorescence signal was then analyzed under a fluorescence microscope. EdU incorporation (%) = EdU positive cells/(EdU-positive cells + Hoechst- positive cells) ×100%. VSMC phenotype switch has been widely studied in transcriptional and epigenetic levels [14, 15]. We were very interested in the growing evidence supporting a critical role for miRNAs in regulating VSMC differentiation and phenotypic switch [16, 17]. A series of miRNAs have been reported as regulators of VSMC phenotype, including miR-21 [18], miR-22 [16], miR- 23b [19], miR-100 [20], miR-124 [16], miR-133 [21], miR-143/145 [22], miR-146a [23], miR-195 [24], miR- 221/222 [25] and miR-424 [26]. Here, we found that PA inhibits VSMC switch to synthetic phenotype via upregulation of miR-22. These results suggested that PA plays a role in regulation of VSMC phenotype. Transwell assay Primary VSMCs were isolated from 8–10 weeks old male SD rat (weighed 170–250 g) thoracic aorta as reported previously [27]. Briefly, thoracic aortas were excised followed by phosphate buffered saline (PBS) washing for 3 times. After these, the aortic media layer was dissected, cut into pieces, and seeded onto a 6-well plate. Cells were maintained in DMEM supplemented with 10% fetal bovine serum (FBS), 1% penicillin and streptomycin at 37°C in a humidified incubator with 5% CO2 in atmosphere for 2 weeks. All animal procedures in this study were conducted in accordance with the National Institutes of Health Guidelines on the Use of Laboratory Animals, and were approved by the Xi’an Jiaotong University Second Affiliated Hospital. VSMCs were seeded in the upper chamber of transwell (12 µm) and placed in a 24-well plate at a density of 1.0 × 105 cells/well in 200 μl DMEM contained with 0.5% FBS. The lower chamber was filled up with 600 μl DMEM contained with 10% FBS. After incubation with for 24 h, medium was discarded and the lower chamber membrane was fixed with methanol at room temperature for 15 min. Subsequently, cells were stained with 0.1 crystal violet-methanol solution for 15 min at room temperature. Finally, the migrated cells were pictured and calculated under a light microscope. Apoptosis determination The apoptosis of VSMCs was detected using an Annexin V-FITC apoptosis detection kit (C1062, Beyotime, China). Briefly, cells were collected after treatment, washed with cooled PBS twice, resuspended with 1 mL AnnexinV-FITC, and maintained for 10 min at room temperature according to the kit’s protocol. Following this, cells were subjected to flow cytometry analysis. Cell transfection MiR-22 mimics (5′-AAGCUGCCAGUUGAAGAA CUGU-3′), miR-23b mimics (5-′AUCACAUUGCCA GGGAUUACCAC-3′), miR-125b mimics (5′- UCCCUGAGACCCUAACUUGUGA-3′), negative control mimics (NC mimics, #miR1N0000001-1-10), miR-22 inhibitors (5′-ACAGUUCUUCAA CUGGCAGCUU-3′), and NC inhibitors (#miR2N0000001-1-10) were synthesized by RIBOBIO Co., Ltd Chin (Guangzhou, China). Empty vector (pcDNA3.1) and EVI1 overexpression plasmid (pcDNA3.1-EVI1 OE) were purchased from GeneChem (Shanghai, China). miRNA mimics (100 nmol/L), inhibitors (200 nmol/L), or NC (5′- UUCUCCGAACGUGUCACGUTT-3′) (100 nmol/L) were transfected using Lipofectamine™ 3000 (Invitrogen) according to manufacturer’s instruction. After 60 h post-transfection, the transfected cells were harvested and utilized for further analyses. Dual-luciferase reporter assay VSMCs were treated with PA (0, 100, 200 or 400 μM) for 3 d. Live/Dead assay suggested that PA treatment decreased cell viability, and increased cell death in a dose-dependent manner in VSMCs (Figure 1A). Moreover, EdU assay suggested that PA treatment significantly decreased the EdU incorporation of VSMCs in a dose-dependent manner (Figure 1B and 1C). Further analysis indicated that PA treatment could significantly increase VSMCs apoptosis (Figure 1D and 1E). Western blot analysis presented that PA treatment markedly increase the Bax and cleaved- caspase-3 expression but decreased Bcl-2 expression (Figure 1F). These findings suggested that PA may inhibit the VSMC switch to synthetic phenotype. In addition, PA treatment (200 μM) suppressed the migration of VSMCs as detected by transwell assay (Figure 1G). Furthermore, PA treatment (200 μM) for 3 d increased protein levels for markers of the contractile phenotype, including α-SMA, calponin, and SMMHC, and decreased protein levels of the synthetic phenotype, including vimentin, collagen I, and osteopontin (OPN) (Figure 1H). These results reinforced the notion that PA inhibits the VSMC switch to synthetic phenotype. Wt and Mt ecotropic virus integration site 1 protein homolog (EVI1) 3′UTR sequence was acquired using PCRmethod, and then cloned into SpeI and HindIII sites of pMir-Report Luciferase vector (Applied Biosystems). The resulting construct was transfected (5ng) into 293T cells with 20 nM control mimics or miR-22 mimics using Lipofectamine-2000 (Invitrogen). After 24 h post- transfection, luciferase activity of cells was assessed using a Luciferase Assay System (Promega). Western blot For immunoblotting, proteins were isolated from cells using RIPA buffer. Total protein extracts (15–50 μg) were separated using sodium dodecyl sulfate polyacrylamide gel electrophoresis and transferred onto polyvinylidene fluoride membrane. Membranes were then probed with anti-bax (1:2000; #ab32503; Abcam), bcl-2 (1:2000; #ab196495; Abcam), cleaved-caspase- 3/caspase-3 (1:2000; #ab184787; Abcam), SM22α (1:2000; #ab14106; Abcam), calponin (1:500; #ab227661; Abcam), SMMHC (1:2000; #ab125884; Abcam), vimentin (1:2000; #ab92547; Abcam), collagen I (1:1000; #ab270993; Abcam), osteopontin (OPN; 1:1000; #ab63856; Abcam), LAMC1 (1:1000; #ab233389; Abcam), EVI1 (1:1000; #SAB2100723; Sigma), AKT3 (1:2000; #ab152157; Abcam), TP53INP1 (1:2000; #ab202026; Abcam), and β-actin (1:2000; #ab8226; Abcam) at room temperature for Live/dead cell assay RNAiso Plus reagent (Code No.: 9108, Takara) was used for the RNA isolation as the manufacturer recommended. The cDNA was synthesized using the isolated RNA (500 ng/sample) and amplification of certain genes was performed using a SYBR Green PCR kit (Takara) in a CFX200 (Bio-Rad) with the cycles of 95°C for 10 min and 40 cycles of 95°C for 5 s, 58°C for 30 s, and 72°C for 10 s. The mRNA level of each The 2-color fluorescence with the LIVE/DEAD Viability/Cytotoxicity kit (Molecular Probes) was used to quantify the living and dead cells in this study as directed by the manufacturer’s protocol. Briefly, cells were harvested after treatment, washed with PBS twice, and incubated with 300 μl of live/dead solution for half an hour at 37°C in the dark room. Then, the AGING 8047 www.aging-us.com 1.5 h. Then, membranes were immersed with the HRP- conjugated secondary antibody at room temperature for 1 h. Following this, the BM chemiluminescence blotting system (Thermo Scientific) was used for detection and protein bands were quantified using Image J software (NIH, USA). gene was normalized to housekeeping gene, namely, GAPDH or U6. The primer sequences are listed in Supplementary Table 1. Statistical analysis All data are presented as mean ± standard deviation, and comparisons were performed using one-way ANOVA or two-way ANOVA followed by an unpaired t-test, as appropriate. P < 0.05 was considered statistically significant. Availability of data and materials The datasets used and/or analyzed during the present study are available from the corresponding author upon reasonable request. PA increased miR-22 expression in VSMCs Figure 2A, 15 miRNAs were detected, and miRNAs, 3 miRNAs were increased and 1 miRNA were decreased in PA-tre compared with that in untreated VSM this, the top 3 increased miRNAs were ov VSMCs via using transfecting with ncreased miR-22 expression in VSMCs. (A) MiRNAs levels in VSMCs treated with PA. (B) The mRNA miR-125b in VSMCs after transfecting with their specific miRNA mimics. (C) The protein expression of tin, collagen I, and osteopontin in VSMCs treated with miR-22, miR-23b or miR-125b mimics. (D) Migration h miR-122 mimics determined by transwell assay. Scale bar=100 μm. (E, F) The cell proliferation of VSMCs cted by EdU assay. (G, H) Apoptosis of VSMCs treated with miR-22 mimics determined by flow cytometr x, bcl-2, caspase-3, and cleaved-caspase-3 in VSMCs treated with miR-22 mimics. n = 3. *P < 0.05, **P < 0.0 miRNA were decreased in PA-treated VSMCs compared with that in untreated VSMCs. Following this, the top 3 increased miRNAs were overexpressed in VSMCs via using transfecting with their specific miRNAs which are involved in alteration of VSMC phenotype switch were screened in PA-treated VSMCs. As shown in Figure 2A, 15 miRNAs were detected, and among these miRNAs, 3 miRNAs were increased and 1 which are involved in alteration of VSMC switch were screened in PA-treated VSMCs. in Figure 2A, 15 miRNAs were detected, and se miRNAs, 3 miRNAs were increased and 1 miRNA were decreased in PA-trea compared with that in untreated VSMC this, the top 3 increased miRNAs were ove VSMCs via using transfecting with t A increased miR-22 expression in VSMCs. (A) MiRNAs levels in VSMCs treated with PA. (B) The mRNA le d miR-125b in VSMCs after transfecting with their specific miRNA mimics. (C) The protein expression of SM entin, collagen I, and osteopontin in VSMCs treated with miR-22, miR-23b or miR-125b mimics. (D) Migration a with miR-122 mimics determined by transwell assay. Scale bar=100 μm. (E, F) The cell proliferation of VSMCs tr etected by EdU assay. (G, H) Apoptosis of VSMCs treated with miR-22 mimics determined by flow cytometry f bax, bcl-2, caspase-3, and cleaved-caspase-3 in VSMCs treated with miR-22 mimics. n = 3. *P < 0.05, **P < 0.01. Figure 2. PA increased miR-22 expression in VSMCs. (A) MiRNAs levels in VSMCs treated with PA. (B) The mRNA levels of miR-22, miR-23b, and miR-125b in VSMCs after transfecting with their specific miRNA mimics. PA increased miR-22 expression in VSMCs To test whether miRNA is involved in regulation of VSMC phenotype switch induced by PA, the report AGING 8048 www.aging-us.com A inhibited VSMC switch to synthetic phenotype. VSMC morphologies after PA treatments (0, 100, 200 o ead cell assay of PA-treated VSMCs. (B and C). EdU assay to detect the proliferation on VSMCs treated with PA. (D detect the apoptosis of PA-treated VSMCs. (F) Western blot determined the expression of apoptosis associated f PA d (200 M) VSMC S l b 100 (H) E i l l f SM22 l i SMMHC i Figure 1. PA inhibited VSMC switch to synthetic phenotype. VSMC morphologies after PA treatments (0, 100, 200 or 400 μM) for 3d. (A) Live/dead cell assay of PA-treated VSMCs. (B and C). EdU assay to detect the proliferation on VSMCs treated with PA. (D and E). Flow cytometry to detect the apoptosis of PA-treated VSMCs. (F) Western blot determined the expression of apoptosis associated markers. (G) Transwell assay of PA-treated (200 μM) VSMCs. Scale bar = 100 μm. (H) Expression levels of SM22α, calponin, SMMHC, vimentin, collagen I, and osteopontin in PA-treated (200 μM for 3 d) VSMCs. n = 3. *P < 0.05, **P < 0.01. 8049 AGING www.aging-us.com miRNAs which are involved in alteration of VSMC henotype switch were screened in PA-treated VSMCs. s shown in Figure 2A, 15 miRNAs were detected, and mong these miRNAs, 3 miRNAs were increased and 1 miRNA were decreased in PA-treated VSMCs compared with that in untreated VSMCs. Following this, the top 3 increased miRNAs were overexpressed in VSMCs via using transfecting with their specific gure 2. PA increased miR-22 expression in VSMCs. (A) MiRNAs levels in VSMCs treated with PA. (B) The mRNA levels of miR-22, iR-23b, and miR-125b in VSMCs after transfecting with their specific miRNA mimics. (C) The protein expression of SM22α, calponin, MMHC, vimentin, collagen I, and osteopontin in VSMCs treated with miR-22, miR-23b or miR-125b mimics. (D) Migration ability of VSMCs ansfecting with miR-122 mimics determined by transwell assay. Scale bar=100 μm. (E, F) The cell proliferation of VSMCs treated with miR- 2 i i d d b EdU (G H) A i f VSMC d i h iR 22 i i d i d b fl (I) Th i ch are involved in alteration of VSMC itch were screened in PA-treated VSMCs. PA treatment enhanced the effect of miR-22 on abolishing EVI1mediated VSMC phenotype switch To test whether EVI1 contributes to the effects of PA on VSMC phenotype switch, EVI1 and miR-22 were overexpressed in VMSCs followed by PA treatment. The qRT-PCR demonstrated that EVI1 overexpression did not change the levels of miR-22 in VSMCs, but PA treatment could enhance the upregulation of miR-22 (Figure 5A). Overexpression of miR-22 mimic significantly suppressed the EVI1 expression and PA treatment further enhanced this inhibition on the expression of EVI1 (Figure 5A). Moreover, PA treatment promoted cell proliferation as detected by EdU staining in VSMCs with EVI1 overexpression, while miR-22 mimic expression aborted this upregulation and PA treatment markedly enhanced this inhibitive effect mediated by miR-22 (Figure 5B). Flow cytometry analysis presented that overexpression of EVI1 had no obvious effect on the apoptosis of VSMCs, but miR-22 and PA treatment could enhance the apoptosis of EVI1 (Figure 5C). Similarly, EVI1 had no obviously effect on the expression of bax, bcl-2, caspase-3, and cleaved caspase-3, while miR-22 mimic and PA treatment could significantly promote the bax and cleaved-caspase-3 but decreased bcl-2 expression (Figure 5D). Transwell assays indicated that EVI1 significantly increased the migration of VSMCs, while miR-22 and PA treatment obviously attenuated this promotion to suppress the migration of VSMCs (Figure 5E and 5F). In addition, EVI1 also inhibited the contractile markers of SM22α, calponin, and SMMHC and promoted the synthetic markers of vimentin, collagen I, and OPN, while miR-22 and PA treatment attenuated these changes (Figure 5G). These results reinforced the notion that PA inhibits the VSMC switch to synthetic phenotype through regulation of miR-22/EVI1 axis. PA increased miR-22 expression in VSMCs (C) The protein expression of SM22α, calponin, SMMHC, vimentin, collagen I, and osteopontin in VSMCs treated with miR-22, miR-23b or miR-125b mimics. (D) Migration ability of VSMCs transfecting with miR-122 mimics determined by transwell assay. Scale bar=100 μm. (E, F) The cell proliferation of VSMCs treated with miR- 22 mimics detected by EdU assay. (G, H) Apoptosis of VSMCs treated with miR-22 mimics determined by flow cytometry. (I) The protein expression of bax, bcl-2, caspase-3, and cleaved-caspase-3 in VSMCs treated with miR-22 mimics. n = 3. *P < 0.05, **P < 0.01. AGING 8050 www.aging-us.com miRNA mimics, respectively (Figure 2B). Following this, the expression of synthetic and contractile markers was detected in VSMCs. As shown in Figure 2C, miR-22, miR-23b, and miR-125b mimics all increased the mRNA levels for SM22α, calponin, and SMMHC, and decreased mRNA levels of vimentin, collagen I, and OPN, suggesting that PA may inhibit the VSMC switch to synthetic phenotype via upregulation of these miRNAs. Specifically, miR-22 presented the most significant effect among these miRNAs. Thus, we had chosen miR-22 for the following investigation. Moreover, transwell assay suggested that increased expression of miR-22 mimics obviously inhibited the VSMCs migration (Figure 2D). Overexpression of miR-22 mimics also inhibited the proliferation of VSMCs (Figure 2E and 2F). In addition, flow cytometry suggested that overexpression of miR-22 mimic increased the apoptosis of VSMCs (Figure 2G and 2H). Correspondingly, the western blot analysis showed that miR-22 mimic increased the expression of Bax and clveaed-caspase-3, but decrease Bcl-2 expression (Figure 2I). These results suggested that PA may inhibit the VSMC switch to synthetic phenotype via upregulation of miR-22. candidates (Figure 4A), and 10 of them are associate with cell proliferation, migration, or apoptosis (Figure 4B). Among these candidates, overexpression of miR- 22 reduced EVI1 mRNA levels significantly in VSMCs (Figure 4B). Western blot array showed the miR-22 mimic could significantly decrease the EVI1 expression, but miR-22 inhibitor largely enhanced the EVI1 expression (Figure 4C and 4D), indicating EVI1 acted as a candidate target of miR-22. This result was further confirmed by dual-luciferase reporter assay and presented that miR-22 reduced luciferase activity for EVI1 wild-type 3′UTR constructs but had no effect on the mutated binding site (Figure 4E and 4F). In addition, PA treatment abolished the EVI1 protein expression, while miR-22 inhibitor attenuated these downregulation, thereby upregulating EVI1 expression (Figure 4G). These results suggested that EVI1 is a target of miR-22. miR-22 inhibitor abolished the effects of PA on VSMC phenotype switch To test whether miR-22 is involved in the PA’s effects on VSMC phenotype switch, miR-22 inhibitor was used to inhibit the PA-upregulated miR-22. As shown in Figure 3A, miR-22 inhibitor decreased the miR-22 levels in VSMCs. As a result, PA treatment (200 μM) significantly inhibited the cell viability but increased apoptosis in VSMCs, while overexpression of miR-22 inhibitor attenuated the impacts of PA on the proliferation and apoptosis of VSMCs (Figure 3B–3E). Correspondingly, the western blot analysis presented that overexpression of miR-22 inhibitor attenuated the effect of PA in increasing bax and cleaved-caspase-3 expression, and decreasing bcl-2 expression (Figure 3F). Transwell analysis showed that overexpression of PA treatment significantly decreased the migration of VSMCs, but miR-22 inhibitor obviously aborted this enhancement (Figure 3G). In addition, the western blot analyses showed that PA treatment obviously accumulated the expression of SM22α, calponin, and SMMHC, but decreased the expression of vimentin, collagen I, and OPN; while overexpression of miR-22 inhibitor attenuated the effect of PA in VSMCs (Figure 3H). These results reinforced the notion that PA inhibits the VSMC switch to synthetic phenotype. EVI1 was the target of miR-22 The potential target genes of miR-22 were predicted by miRDB, ENCOR1, and TargetScan. There were 50 AGING 8051 www.aging-us.com DISCUSSION atherosclerosis and other vascular diseases [9, 10]. However, how fatty acid regulates VSMC phenotype switch has not been studied. Here, we found that PA, the most common saturated fatty acid in circulation, us com 8052 ION vidence has shown that disorder in fatty acid plays a casual role in the development of atherosclerosis and other vascular disease However, how fatty acid regulates VSMC switch has not been studied. Here, we foun the most common saturated fatty acid in c R-22 inhibitor abolished the effects of PA on VSMC phenotype switch. (A) MiR-22 inhibitor decreased Cs. (B, C) The proliferation of VSMCs transfected with miR-22 inhibitor or/and 200 μM PA. (D, E) The apoptos ith miR-22 inhibitor or/and 200 μM PA. (F) The expression of apoptosis associated markers in VSMCs transfected nd 200 μM PA. (G) The migration of VSMCs transfected with miR-22 inhibitor or/and 200 μM PA determined bar=100 μm. (H) Western blot analyses showing that expression of SM22α, calponin, SMMHC, vimentin, col n PA-treated VSMCs transfected with miR-22 inhibitor. n = 3. *P < 0.05, **P < 0.01. Mounting evidence has shown that disorder in fatty acid metabolism plays a casual role in the development of Figure 3. miR-22 inhibitor abolished the effects of PA on VSMC phenotype switch. (A) MiR-22 inhibitor decreased the miR-22 levels in VSMCs. (B, C) The proliferation of VSMCs transfected with miR-22 inhibitor or/and 200 μM PA. (D, E) The apoptosis of VSMCs transfected with miR-22 inhibitor or/and 200 μM PA. (F) The expression of apoptosis associated markers in VSMCs transfected with miR-22 inhibitor or/and 200 μM PA. (G) The migration of VSMCs transfected with miR-22 inhibitor or/and 200 μM PA determined by transwell assay. Scale bar=100 μm. (H) Western blot analyses showing that expression of SM22α, calponin, SMMHC, vimentin, collagen I, and osteopontin in PA-treated VSMCs transfected with miR-22 inhibitor. n = 3. *P < 0.05, **P < 0.01. AGING 8052 www.aging-us.com inhibited VSMC switch to synthetic phenotype, as manifested by inhibiting VSMC proliferation, migration, and synthesis. Mechanistically, PA inhibits VSMC switch to synthetic phenotype through upregulation of miR-22 by targeting EVI1. These findings suggested that PA plays a role in the regulation inhibited VSMC switch to synthetic phenotype, as manifested by inhibiting VSMC proliferation, migration, and synthesis. EVI1 was the target of miR-22 Mechanistically, PA inhibits VSMC switch to synthetic phenotype through upregulation of miR-22 by targeting EVI1. These findings suggested that PA plays a role in the regulation of VSMC phenotype, which may contribute to vascular health and diseases. Several studies have shown that saturated fatty acids increase the risk of cardiovascular diseases [12, 28]. According to the previous study, the saturated fatty www aging-us com 8053 AGING VSMC switch to synthetic phenotype through upregulation of miR-22 by targeting EVI1. These findings suggested that PA plays a role in the regulation Several studies have shown that saturated fatty acid increase the risk of cardiovascular diseases [12, 28 According to the previous study, the saturated fat Figure 4. EVI1 was the target of miR-22. (A) Target genes of miR-22 were predicted by miRDB, TargetScan and ENCORI. (B) MRNA leve of target genes in VSMCs treated with miR-22 mimics. (C, D) Protein levels of SM22α, calponin, SMMHC, vimentin, collagen I, an osteopontin in VSMCs detected by western blot. (E) The complementary sequence between 3′-UTR of EVIL1 and miR-22. (F) Dual-lucifera reporter assay. (G) EVI1 protein level was detected by western blot in VSMCs treated with miR-22 inhibitor or PA. n = 3. *P < 0.05, **P < 0.01 Figure 4. EVI1 was the target of miR-22. (A) Target genes of miR-22 were predicted by miRDB, TargetScan and ENCORI. (B) MRNA levels of target genes in VSMCs treated with miR-22 mimics. (C, D) Protein levels of SM22α, calponin, SMMHC, vimentin, collagen I, and osteopontin in VSMCs detected by western blot. (E) The complementary sequence between 3′-UTR of EVIL1 and miR-22. (F) Dual-luciferase reporter assay. (G) EVI1 protein level was detected by western blot in VSMCs treated with miR-22 inhibitor or PA. n = 3. *P < 0.05, **P < 0.01. AGING 8053 www.aging-us.com focused on different biomarkers of risk of cardiovascular diseases found that not all SFAs exert the same effect, namely, studies do not seem to serve as acids were usually regarded as a singular fatty acid group and they might have the same effects during the metabolism [29, 30]. However, some investigations eatment enhanced miR-22 mimic impacts on aborted the inhibitive effect of EVI1 on VSMC phenotype ession of miR-22 and EVIL1 in VMSCs treated with EVI1 or/and miR-22 overexpression followed by PA treatment. (B) Cell detected by EdU in VSMCs with EVI1/miR-22 overexpression followed by PA treatment. EVI1 was the target of miR-22 (C) Flow cytometry to detect the MCs with EVI1/miR-22 overexpression followed by PA treatment. (D) Western blot to detect the apoptosis associated s. (E, F). Cell migration was detected by transwell assay in EVI1/miR-22-overexpressed VSMCs followed by PA treatment. m. (G) Protein levels of SM22α, calponin, SMMHC, vimentin, collagen I, and osteopontin in VSMC in VSMCs with EVI1/miR- n followed by PA treatment detected by western blot. n = 3. *P < 0.05, **P < 0.01. Figure 5. PA treatment enhanced miR-22 mimic impacts on aborted the inhibitive effect of EVI1 on switch. (A) Expression of miR-22 and EVIL1 in VMSCs treated with EVI1 or/and miR-22 overexpression followed by PA proliferation was detected by EdU in VSMCs with EVI1/miR-22 overexpression followed by PA treatment. (C) Flow cyto apoptosis of VSMCs with EVI1/miR-22 overexpression followed by PA treatment. (D) Western blot to detect the a markers in VSMCs. (E, F). Cell migration was detected by transwell assay in EVI1/miR-22-overexpressed VSMCs followe Figure 5 PA treatment enhanced miR 22 mimic impacts on aborted the inhibitive effect of EVI1 on VSMC phenotype Figure 5. PA treatment enhanced miR-22 mimic impacts on aborted the inhibitive effect of Figure 5. PA treatment enhanced miR-22 mimic impacts on aborted the inhibitive effect of EVI1 on VSMC phenotype switch. (A) Expression of miR-22 and EVIL1 in VMSCs treated with EVI1 or/and miR-22 overexpression followed by PA treatment. (B) Cell proliferation was detected by EdU in VSMCs with EVI1/miR-22 overexpression followed by PA treatment. (C) Flow cytometry to detect the apoptosis of VSMCs with EVI1/miR-22 overexpression followed by PA treatment. (D) Western blot to detect the apoptosis associated markers in VSMCs. (E, F). Cell migration was detected by transwell assay in EVI1/miR-22-overexpressed VSMCs followed by PA treatment. Scale bar = 100 μm. (G) Protein levels of SM22α, calponin, SMMHC, vimentin, collagen I, and osteopontin in VSMC in VSMCs with EVI1/miR- 22 overexpression followed by PA treatment detected by western blot. n = 3. *P < 0.05, **P < 0.01. Figure 5. PA treatment enhanced miR-22 mimic impacts on aborted the inhibitive effect of EVI1 on VSMC phenotype switch. (A) Expression of miR-22 and EVIL1 in VMSCs treated with EVI1 or/and miR-22 overexpression followed by PA treatment. (B) Cell proliferation was detected by EdU in VSMCs with EVI1/miR-22 overexpression followed by PA treatment. (C) Flow cytometry to detect the apoptosis of VSMCs with EVI1/miR-22 overexpression followed by PA treatment. Abbreviations VSMC: Vascular smooth muscle cell; PA: palmitic acid; EVI1: ecotropic virus integration site 1 protein homolog. CONCLUSION Taken together, we found that PA inhibits VSMC switch to synthetic phenotype through upregulation of miR-22 expression. In addition, miR22 inhibits VSMC switch to synthetic phenotype by targeting EVI1. These findings suggested that PA plays a role in regulation of VSMC phenotype, which may contribute to maintenance of vascular health and prevention of vascular diseases. Recent studies support a critical role of miRNAs in regulating VSMC differentiation and phenotype switch, and miR-22 is one of the miRNAs which inhibits VSMC switch to synthetic phenotype [16, 17]. miR-22 is previously demonstrated as a tumor suppressor, but later has been concerned as a prohypertrophic miRNA [34, 35]. A recent study documented that miR-22 playing key role in the regulation role in VSMC biological activity [36]. In addition, it has also been reported that miR-22 involved in VSMC phenotypic modulation, which induces VSMC contractile gene expression, but inhibits VSMC proliferation and migration [17]. These findings indicated that miR-22 serves a key role in regulation of cardiovascular function. Here, we show that PA increased miR-22 expression in VSMCs, and inhibition of miR-22 abolished the PA’s effects on modulation of VSMC phenotype. It has been reported that transforming growth factor-β1 (TGF-β1) transcriptionally modulates miR-22 expression in VSMCs via a P53-dependent mechanism [17]. Whether PA regulates miR-22 expression through TGF-β1 needed further investigation. Indeed, there is evidence that PA treatment increases TGF-β1 in other cells [37]. These findings suggested that PA modulate VSMC phenotype via upregulating miR-22, which serves a crucial role vascular function regulation. EVI1 was the target of miR-22 (D) Western blot to detect the apoptosis associated markers in VSMCs. (E, F). Cell migration was detected by transwell assay in EVI1/miR-22-overexpressed VSMCs followed by PA treatment. Scale bar = 100 μm. (G) Protein levels of SM22α, calponin, SMMHC, vimentin, collagen I, and osteopontin in VSMC in VSMCs with EVI1/miR- 22 overexpression followed by PA treatment detected by western blot. n = 3. *P < 0.05, **P < 0.01. AGING 8054 www.aging-us.com a single role of PA in the development of cardiovascular diseases [31]. PA slightly elevated the LDL- and HDL-cholesterol, which is a significant predictor for cardiovascular disease [32, 33]. Although the role of PA in cardiovascular diseases needs to be further examined, these advances suggest that PA’s effects in cardio-vascular health and disease cannot be easily identified as detrimental or beneficial. Here, we found that PA inhibited VSMC switch to synthetic phenotype, as manifested by inhibiting VSMC proliferation, migration, and synthesis, suggesting that PA may exert beneficial effects on vascular health and diseases, which should be identified by further studies. signaling axis plays a key role in VSMC phenotypic switch and correcting the dysregulation of miR-22/EVI1 or PA could be a potential treatment to vascular diseases. AUTHOR CONTRIBUTIONS Yanchao Hu and Congxia Wang conceived and designed these experiments. Yajie Fan and Chunyan Zhang performed these experiments. Yajie Fan and Chunyan Zhang analyzed and interpreted the data. Yanchao Hu and Congxia Wang wrote the manuscript. All authors read and approved the final manuscript. CONFLICTS OF INTEREST All authors have completed the ICMJE uniform disclosure form. All the authors declare that they have no competing interests. ETHICAL STATEMENT All animal procedures in this study were conducted in accordance with the National Institutes of Health Guidelines on the Use of Laboratory Animals. The study protocol was approved by the Ethics Committee of Xi’an Jiaotong University Second Affiliated Hospital. Animal care and study were approved by the Institutional Animal Care and Use Committee of Xi’an Jiaotong University Second Affiliated Hospital. Previous studies demonstrated that EVI1 functions as a transcriptional regulator to modulate several biological processes, including hematopoiesis, apoptosis, development, differentiation and proliferation [38, 39]. Here, we have found that EVI1 serves as a target gene of miR-22 to modulate VSMC phenotype switch. Further analysis showed that EVI1 transcriptionally inhibits VSMC-specific genes to modulate the VSMC phenotype switch, including SMαA, SM22α, SRF, and Myocd [17]. In addition, inhibiting EVI1 abolished the effects of miR-22 and PA in modulation of VSMC phenotype. These findings suggested that miR-22/EVI1 https://doi.org/10.1161/CIRCRESAHA.115.306361 PMID:26892967 3. Lacolley P, Regnault V, Avolio AP. Smooth muscle cell and arterial aging: basic and clinical aspects. Cardiovasc Res. 2018; 114:513–28. 12. Fattore E, Fanelli R. Palm oil and palmitic acid: a review on cardiovascular effects and carcinogenicity. 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Arterioscler Thromb Vasc Biol. 2006; 26:2244–51. https://doi.org/10.1161/01.ATV.0000240251.50215.50 PMID:16902164 37. Duan NN, Liu XJ, Wu J. Palmitic acid elicits hepatic stellate cell activation through inflammasomes and hedgehog signaling. Life Sci. 2017; 176:42–53. https://doi.org/10.1016/j.lfs.2017.03.012 PMID:28322865 AGING 8058 www.aging-us.com SUPPLEMENTARY MATERIALS Supplementary Table Supplementary Table 1. The primer sequences. Gene Primer (5′–3′) MYH11 F: AGGCGAACCTAGAC R: CTGGATGTTGAGAG SM22α F: TTCTGCCTCAACATG R: CACCTTCACTGG CTT Calponin F: ATGTCCTCTGCTCAC R: CACGTTCACCTTGTT SMMHC F: AGGCGAACCTAGAC R: CTGGATGTTGAGAG Collagen I F: TGACGAGACCAAGA R: GCACCATCATTTCCA Vimentin F: TCGTTTCGAGGTTTT R: GACTAAAACTCGAC Osteopontin F: CAGCCTTCTCAGCCA R: CAAATCACTGCAATT MiR-22 F: AAGCTGCCAGTTGAA R: Universal PCR Reverse (cat. no. B532451; Sangon miR-23b F: ATCACATTGCCAGGG R: Universal PCR Reverse (cat. no. B532451; Sangon miR-125b F: TCCCTGAGACCCTAA R: Universal PCR Reverse (cat. no. B532451; Sangon NET1 F: AAATCCCCGGATGAC R: TGCACTGCTCCTTCT SIRT1 F: GCTCGCCTTGCGGTG R: GACGGCTGGAACTG PTEN F: AGCCTCTTGATGTGT R: CCATTGGTAGCCAAA ERBB3 F: CATCGTGAGGGACCG R: TGTTGCTCGAGTCCA LAMC1 F: GAGGCAAGATATCG R: GTATCTCGCCTGTCC AKT3 F: GGTGCAGAGTCCCCT R: TTGGCGACAGCAGG DPF2 F: TGCCTGTGACATTTG R: TAGCCACGATCGCA F: TCCAACTACTGGTTT Supplementary Table Supplementary Table 1. The primer sequences. Gene Primer (5′–3′) MYH11 F: AGGCGAACCTAGACAAGAATAAG R: CTGGATGTTGAGAGTGGAGATG SM22α F: TTCTGCCTCAACATGGCCAAC3 R: CACCTTCACTGG CTTGGATC Calponin F: ATGTCCTCTGCTCACTTCAAC R: CACGTTCACCTTGTTTCCTTTC SMMHC F: AGGCGAACCTAGACAAGAATAAG R: CTGGATGTTGAGAGTGGAGATG Collagen I F: TGACGAGACCAAGAACTGCC R: GCACCATCATTTCCACGAGC Vimentin F: TCGTTTCGAGGTTTTCGCGTTAGAGAC R: GACTAAAACTCGACCGACTCGCGA Osteopontin F: CAGCCTTCTCAGCCAAACG R: CAAATCACTGCAATTCTCATGGTAGT MiR-22 F: AAGCTGCCAGTTGAAGAACTGT R: Universal PCR Reverse Primer (cat. no. B532451; Sangon Biotech Co., Ltd.) miR-23b F: ATCACATTGCCAGGGATTACCAC R: Universal PCR Reverse Primer (cat. no. (Suppl); 34:S121–4. https://doi.org/10.1007/BF02562257 PMID:10419117 B532451; Sangon Biotech Co., Ltd.) miR-125b F: TCCCTGAGACCCTAACTTGTGA R: Universal PCR Reverse Primer (cat. no. B532451; Sangon Biotech Co., Ltd.) NET1 F: AAATCCCCGGATGACGGC R: TGCACTGCTCCTTCTCTTGG SIRT1 F: GCTCGCCTTGCGGTGGACTT R: GACGGCTGGAACTGTCCGGG PTEN F: AGCCTCTTGATGTGTGCATT R: CCATTGGTAGCCAAACGGAAC ERBB3 F: CATCGTGAGGGACCGAGATG R: TGTTGCTCGAGTCCACAGTC LAMC1 F: GAGGCAAGATATCGCCGTGA R: GTATCTCGCCTGTCCACTCG AKT3 F: GGTGCAGAGTCCCCTAGAGA R: TTGGCGACAGCAGGATCATT DPF2 F: TGCCTGTGACATTTGTGGAA R: TAGCCACGATCGCAGTCATC TGFBR1 F: TCCAACTACTGGTTTACCATTGC R: ACAGCAACTTCTTCTCCCCG F: CGTCTGGGTACCTGAACGAG Primer (5′–3′) F: AGGCGAACCTAGACAAGAATAAG R: CTGGATGTTGAGAGTGGAGATG F: TTCTGCCTCAACATGGCCAAC3 R: CACCTTCACTGG CTTGGATC F: ATGTCCTCTGCTCACTTCAAC R: CACGTTCACCTTGTTTCCTTTC F: AGGCGAACCTAGACAAGAATAAG R: CTGGATGTTGAGAGTGGAGATG F: TGACGAGACCAAGAACTGCC R: GCACCATCATTTCCACGAGC F: TCGTTTCGAGGTTTTCGCGTTAGAGAC R: GACTAAAACTCGACCGACTCGCGA F: CAGCCTTCTCAGCCAAACG R: CAAATCACTGCAATTCTCATGGTAGT F: AAGCTGCCAGTTGAAGAACTGT R: Universal PCR Reverse Primer (cat. no. B532451; Sangon Biotech Co., Ltd.) F: ATCACATTGCCAGGGATTACCAC R: Universal PCR Reverse Primer (cat. no. B532451; Sangon Biotech Co., Ltd.) F: TCCCTGAGACCCTAACTTGTGA R: Universal PCR Reverse Primer (cat. no. B532451; Sangon Biotech Co., Ltd.) F: AAATCCCCGGATGACGGC R: TGCACTGCTCCTTCTCTTGG F: GCTCGCCTTGCGGTGGACTT R: GACGGCTGGAACTGTCCGGG F: AGCCTCTTGATGTGTGCATT R: CCATTGGTAGCCAAACGGAAC F: CATCGTGAGGGACCGAGATG R: TGTTGCTCGAGTCCACAGTC F: GAGGCAAGATATCGCCGTGA R: GTATCTCGCCTGTCCACTCG F: GGTGCAGAGTCCCCTAGAGA R: TTGGCGACAGCAGGATCATT F: TGCCTGTGACATTTGTGGAA R: TAGCCACGATCGCAGTCATC F: TCCAACTACTGGTTTACCATTGC R: ACAGCAACTTCTTCTCCCCG F: CGTCTGGGTACCTGAACGAG R: ACTTCTGTGCCCGTGAGTCT F: GCAGACATTGCGCCTGGGGAA AGING 8059 8059 www.aging-us.com R: CTCACAGCGGTGCTGCCGTT GAPDH F: AACTTTGGCATTGTGGAAGG R: ATTGGGGGTAGGAACA U6 F: TGAGAACTGAATTCCATGGGTT R: ACGCTTCACGAATTTGCGT GAPDH U6 AGING 8060 www.aging-us.com
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A Note-Book on Experimental Mathematics. C. Godfrey and G. M. Bell. (Arnold.)
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Review Source: The Mathematical Gazette, Vol. 3, No. 56 (Mar., 1906), pp. 297-298 Published by: Mathematical Association Stable URL: http://www.jstor.org/stable/3603481 Accessed: 02-12-2015 05:46 UTC Review Review Source: The Mathematical Gazette, Vol. 3, No. 56 (Mar., 1906), pp. 297-298 Published by: Mathematical Association Stable URL: http://www.jstor.org/stable/3603481 Accessed: 02-12-2015 05:46 UTC Your use of the JSTOR archive indicates your acceptance of the Terms & Conditions of Use, available at http://www.jstor.org/page/ info/about/policies/terms.jsp JSTOR is a not-for-profit service that helps scholars, researchers, and students discover, use, and build upon a wide range of content in a trusted digital archive. We use information technology and tools to increase productivity and facilitate new forms of scholarship. For more information about JSTOR, please contact support@jstor.org. Mathematical Association is collaborating with JSTOR to digitize, preserve and extend access to The Mathematical Gazette. http://www.jstor.org This content downloaded from 160.36.178.25 on Wed, 02 Dec 2015 05:46:36 UTC All use subject to JSTOR Terms and Conditions REVIEWS. REVIEWS. 297 297 logarithms, and it is a pity that the authors have not taken advantage of it. logarithms, and it is a pity that the authors have not taken advantage of it. logarithms, and it is a pity that the authors have not taken advantage of it. logarithms, and it is a pity that the authors have not taken advantage of it. In combining logarithms the vertical arrangement is more satis- factory. For instance the writers have In combining logarithms the vertical arrangement is more satis- factory. For instance the writers have log x= 2-0913 + 1 6586 + 0-8318 = 4-5817. log x= 2-0913 + 1 6586 + 0-8318 = 4-5817. g (20913 or logx= 2-0913 This might be written log x= 1-6586 + 1-6586 0 -8318 + 08318 = 45817 = 4-5817 g (20913 or logx= 2-0913 This might be written log x= 1-6586 + 1-6586 0 -8318 + 08318 = 45817 = 4-5817 The theorem of Pythagoras is suggested for discovery (1) by squared paper and the A 3, 4, 5; (2) by construction and calculation. One or two "dissection proofs" might well have been added. In connection with this theorem and the corresponding theorems for acute- and obtuse- angled triangles too much is expected from the student, who is, moreover, asked to discover for the first time that (a - b)2= a2 - 2ab + b2 from a figurecorrespondingto that of Euc. II. 7. The theorem of Pythagoras is suggested for discovery (1) by squared paper and the A 3, 4, 5; (2) by construction and calculation. Review One or two "dissection proofs" might well have been added. In connection with this theorem and the corresponding theorems for acute- and obtuse- angled triangles too much is expected from the student, who is, moreover, asked to discover for the first time that (a - b)2= a2 - 2ab + b2 from a figurecorrespondingto that of Euc. II. 7. figure corresponding The authors speak of the "Equation of a Circle," although they state that it is very important to use the words circumference anil circle in their right senses. figure corresponding The authors speak of the "Equation of a Circle," although they state that it is very important to use the words circumference anil circle in their right senses. right The chapter on Plans, Elevations, etc., is instructive, and should be of great use. It is an important branch which is often neglected in schools. right The chapter on Plans, Elevations, etc., is instructive, and should be of great use. It is an important branch which is often neglected in schools. Density is not defined, but the term relative density is used for specificgravity. Density is not defined, but the term relative density is used for specificgravity. p g y A chapter is given to Trigonometry. It forms a good introduction, but perhaps more attention might have been given to the graphs of the ratios. The examples in this chapter are not well chosen, as they do not give sufficient prominence to practical applications. Moreover, no instructions are given as to the use of trigonometricaltables. p g y A chapter is given to Trigonometry. It forms a good introduction, but perhaps more attention might have been given to the graphs of the ratios. The examples in this chapter are not well chosen, as they do not give sufficient prominence to practical applications. Moreover, no instructions are given as to the use of trigonometricaltables. given trigonometrical The book concludes with a short chapter on Time and Velocity. given trigonometrical The book concludes with a short chapter on Time and Velocity. p y Tables of logarithms, antilogarithms, and of values of sin, cos, and tall for angles of a whole number of degrees are placed in appendices at the end. Answers to the examples are given. This content downloaded from 160.36.178.25 on Wed, 02 Dec 2015 05:46:36 UTC All use subject to JSTOR Terms and Conditions Review p y Tables of logarithms, antilogarithms, and of values of sin, cos, and tall for angles of a whole number of degrees are placed in appendices at the end. Answers to the examples are given. examples given. The book shows much originality, and though exception may perhaps be taken to the order and arrangement, it well fulfils the aims for which it was written. examples given. The book shows much originality, and though exception may perhaps be taken to the order and arrangement, it well fulfils the aims for which it was written. A Note-Book on Experimental Mathematics. C. GODFREY and G. M. BELL. (Arnold.) A Note-Book on Experimental Mathematics. C. GODFREY and G. M. BELL. (Arnold.) A Note-Book on Experimental Mathematics. C. GODFREY and G. M. BELL. (Arnold.) A Note-Book on Experimental Mathematics. C. GODFREY and G. M. BELL. (Arnold.) A Note-Book on Experimental Mathematics. C. GODFREY and G. M. BELL. (Arnold.) A Note-Book on Experimental Mathematics. C. GODFREY and G. M. BELL. (Arnold.) ( ) This book is arranged on the lines of Ashford's Note-Book of Practical Physics, compiled for use in Harrow School, and is intended primarily to cover the requirementsof the army entrance examinations. ( ) This book is arranged on the lines of Ashford's Note-Book of Practical Physics, compiled for use in Harrow School, and is intended primarily to cover the requirementsof the army entrance examinations. requirements army An list of apparatus required in the experiments is given at the beginningof the book. This should provemost useful. requirements army An list of apparatus required in the experiments is given at the beginningof the book. This should provemost useful. beginning prove There are 124 experiments with brief, but generally sufficient instructions to the student. The concrete conceptions which he should gain by working through them should be of great value to him when studying the more theoretical portions of mathematics. It is important, however, that classes, taking practical work should frequently discuss their work with the teacher. It is therefore beginning prove There are 124 experiments with brief, but generally sufficient instructions to the student. The concrete conceptions which he should gain by working through them should be of great value to him when studying the more theoretical portions of mathematics. It is important, however, that classes, taking practical work should frequently discuss their work with the teacher. It is therefore This content downloaded from 160.36.178.25 on Wed, 02 Dec 2015 05:46:36 UTC All use subject to JSTOR Terms and Conditions Fletcher (referred to at the end of the book). Suggestions for further experiments are given in Part IV. The authors hint that the time has come for the retirement of Atwood's machine. It certainly deserves a rest from its old occu- pation of trying to discover "g." p The work in this part is suggestive, and it is almost a pity that there is not more of it, e.g. illustrations of accelerations by means of some apparatus similar to that devised by Mr. W. C. Fletcher (referred to at the end of the book). Suggestions for further experiments are given in Part IV. The authors hint that the time has come for the retirement of Atwood's machine. It certainly deserves a rest from its old occu- pation of trying to discover "g." p y g g Elementary Practical Mathematics. H. A. STERN and W. A. TOPHAM. (G. Bell & Sons.) p y g g Elementary Practical Mathematics. H. A. STERN and W. A. TOPHAM. (G. Bell & Sons.) (G So s ) This contains the first nine chapters of a text book of Practical Mathematics definitely intended to satisfy the new Army Regulations. The po.rtion given in this volume covers very well the syllabus for the " Qualifying" Examination, and its equivalent, the " Practical Measure- ments " of the LeavingCertificate Examination. (G So s ) This contains the first nine chapters of a text book of Practical Mathematics definitely intended to satisfy the new Army Regulations. The po.rtion given in this volume covers very well the syllabus for the " Qualifying" Examination, and its equivalent, the " Practical Measure- ments " of the LeavingCertificate Examination. Leaving The first two chapters on contracted methods and graphs are scarcely necessary, as most of the work is now well done in books on arithmetic and algebra. In the graphs the writers are not uniform in indicating the scales used. It seems desirable that in all cases the numbers should be plainly marked on the axes. For some reason the 'cooling curve" on p. 31 is inverted. Leaving The first two chapters on contracted methods and graphs are scarcely necessary, as most of the work is now well done in books on arithmetic and algebra. In the graphs the writers are not uniform in indicating the scales used. This content downloaded from 160.36.178.25 on Wed, 02 Dec 2015 05:46:36 UTC All use subject to JSTOR Terms and Conditions Part III. commences with instruments of more refined accuracy-the vernier, screw-gauge, spherometer. The vernier is introduced by a neat and convincing experiment, but the principle of a micrometer screw might well have been learnt after experiments with simple screws. The radius of a sphere is to be calculated from formula .2 2R=h+-,, r being obtained by calculating the radius of the circumcircle of the equilateral triangle formed by the three feet of the instrument. For approximate purposes a geometrical construction spring balances perhaps easy up. Part III. commences with instruments of more refined accuracy-the vernier, screw-gauge, spherometer. The vernier is introduced by a neat and convincing experiment, but the principle of a micrometer screw might well have been learnt after experiments with simple screws. The radius of a sphere is to be calculated from formula .2 h di f th spring balances perhaps easy up. Part III. commences with instruments of more refined accuracy-the vernier, screw-gauge, spherometer. The vernier is introduced by a neat and convincing experiment, but the principle of a micrometer screw might well have been learnt after experiments with simple screws. The radius of a sphere is to be calculated from formula .2 h di f th 2R=h+-,, r being obtained by calculating the radius of the 2R=h+-,, r being obtained by calculating the radius of the circumcircle of the equilateral triangle formed by the three feet of the instrument. For approximate purposes a geometrical construction based on A and r (measuredat once) is sufficientlyaccurate. circumcircle of the equilateral triangle formed by the three feet of the instrument. For approximate purposes a geometrical construction based on A and r (measuredat once) is sufficientlyaccurate. (measured once) sufficiently Part III. also contains further work in specific gravity and statics. It ends with a little dynamics-the determination of "g" by means of a lead ball falling against an oscillating bar, and also by the simple pendulum. (measured once) sufficiently Part III. also contains further work in specific gravity and statics. It ends with a little dynamics-the determination of "g" by means of a lead ball falling against an oscillating bar, and also by the simple pendulum. p The work in this part is suggestive, and it is almost a pity that there is not more of it, e.g. illustrations of accelerations by means of some apparatus similar to that devised by Mr. W. C. This content downloaded from 160.36.178.25 on Wed, 02 Dec 2015 05:46:36 UTC All use subject to JSTOR Terms and Conditions This content downloaded from 160.36.178.25 on Wed, 02 Dec 2015 05:46:36 UTC All use subject to JSTOR Terms and Conditions THE MATHEMATICAL GAZErrE. THE MATHEMATICAL GAZErrE. 298 298 advisable, though sometimes difficult to arrange, that the mathematical master should take both the theoretical and practical work of his form. This will generally necessitate a "Practical Mathematics Room" in addition to the Physical Laboratory. Such a room, unless the Physical Laboratory is used for the purpose, will therefore be necessary for forms in schools using this book. In fact at some of the larger schools a Practical Mathematics Room has alreadybeen established. advisable, though sometimes difficult to arrange, that the mathematical master should take both the theoretical and practical work of his form. This will generally necessitate a "Practical Mathematics Room" in addition to the Physical Laboratory. Such a room, unless the Physical Laboratory is used for the purpose, will therefore be necessary for forms in schools using this book. In fact at some of the larger schools a Practical Mathematics Room has alreadybeen established. already The book is divided into four parts; Part I. contains simple experi- ments and measurements connected with lengths, areas, volumes, mass and density, and fluid pressure. Part II. is on statics, with experiments on tensions, moments, and the equilibrium of three forces. The board used to illustrate the parallelogram of forces is placed in a vertical plane, and the three forces are obtained by using hanging weights. The method with three spring-balancesis perhapsmore easy to set up. already The book is divided into four parts; Part I. contains simple experi- ments and measurements connected with lengths, areas, volumes, mass and density, and fluid pressure. Part II. is on statics, with experiments on tensions, moments, and the equilibrium of three forces. The board used to illustrate the parallelogram of forces is placed in a vertical plane, and the three forces are obtained by using hanging weights. The method with three spring-balancesis perhapsmore easy to set up. spring balances perhaps easy up. Part III. commences with instruments of more refined accuracy-the vernier, screw-gauge, spherometer. The vernier is introduced by a neat and convincing experiment, but the principle of a micrometer screw might well have been learnt after experiments with simple screws. The radius of a sphere is to be calculated from formula .2 2R=h+-,, r being obtained by calculating the radius of the circumcircle of the equilateral triangle formed by the three feet of the instrument. For approximate purposes a geometrical construction spring balances perhaps easy up. This content downloaded from 160.36.178.25 on Wed, 02 Dec 2015 05:46:36 UTC All use subject to JSTOR Terms and Conditions It seems desirable that in all cases the numbers should be plainly marked on the axes. For some reason the 'cooling curve" on p. 31 is inverted. g p. The chapters on length, mass, area, and volume are very fair. Mass and weighing are taken before areas and volumes. It is doubtfiu g p. The chapters on length, mass, area, and volume are very fair. Mass and weighing are taken before areas and volumes. It is doubtfiu This content downloaded from 160.36.178.25 on Wed, 02 Dec 2015 05:46:36 UTC All use subject to JSTOR Terms and Conditions
https://openalex.org/W4231506190
http://e-journal.metrouniv.ac.id/index.php/istinbath/article/download/951/783
Indonesian
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FAKTOR PENGHAMBAT DALAM PENEGAKAN QANUN JINAYAT DI ACEH
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cc-by-sa
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Abstrak Tulisan ini membuktikan bahwa pelaksanaan Qanun Aceh No. 14 Tahun 2003 tentang khalwat di Kota Subulussalam belum sepenuhnya berjalan dengan baik. Banyak kendala yang dihadapi baik dari pelaksananya (pemerintah) maupun masyarakat sebagai objek hukum penerapan syariat Islam itu sendiri. Faktor hukum yang merupakan salah satu faktor utama penyebab stagnan-nya penindakan terhadap pelanggar Qanun No. 14 Tahun 2003 di Kota Subulussalam disamping faktor-faktor lainnya. Tulisan ini berusaha menguraikan kendala dalam efektivitas penegakan hukum yang merupakan suatu indikator penilaian terhadap penegakan suatu hukum, dalam artian untuk mengukur keberhasilan atau target yang telah ditetapkan dalam penerapan suatu hukum khususnya terhadap penegakan Qanun No. 14 Tahun 2003 di wilayah hukum Kota Subulussalam, Aceh. Kata kunci: Khalwat, Aceh, Syariat Islam, Jinayat, Subulussalam (Studi Kasus Penerapan Qanun No. 14 Tahun 2003 di Kota Subulussalam) Ali Geno Berutu Sekolah Pascasarjan (SPs) UIN Syarif Hidayatullah Jakarta Ali Geno Berutu Sekolah Pascasarjan (SPs) UIN Syarif Hidayatullah Jakarta Email: ali_geno@ymail.com Ali Geno Berutu Sekolah Pascasarjan (SPs) UIN Syarif Hidayatullah Jakarta Email: ali_geno@ymail.com Keywords: Khalwat, Aceh, Syariat Islam, Jinayat, Subulussalam Abstract This paper proves that the implementation of Qanun Aceh no. 14 of 2003 on khalwat in Subulussalam city has not fully run well. Many obstacles faced both from the executor (government) and the community as the object of the law of the application of Islamic Shari'a itself. The legal factor is one of the main factors causing its stagnant action against the violation of Qanun. 14 Year 2003 in Subulussalam City in addition to other factors. This paper attempts to outline the obstacles in the effectiveness of law enforcement which is an indicator of the assessment of the enforcement of a law, in order to measure the success or targets set in the application of a law especially to the enforcement of Qanun. 14 Year 2003 in the jurisdiction of Subulussalam City, Aceh. Keywords: Khalwat, Aceh, Syariat Islam, Jinayat, Subulussalam Keywords: Khalwat, Aceh, Syariat Islam, Jinayat, Subulussalam 148 1 Haedar Nasir, Islam syariat: Reproduksi Salapiyah Idiologis di Indonesia (Bandung: Mizan 2013), h. 336. 2 Rusjdi Ali Muhammad, Revitalisasi Syari’at Islam di Aceh: Problem, Solusi dan Implementasi, (Ciputat: Logos Wacana Ilmu,2003), h. 49. 1 Haedar Nasir, Islam syariat: Reproduksi Salapiyah Idiologis di Indonesia (Bandung: Mizan 2013 6 3 Khamami Zada, “Sentuhan Adat Dalam Pemberlakuan Syariat Islam di Aceh (1514-1903)”, Karsa, Vol. 20 No. 2, Desember 2012, h. 3. 4 Al Yasa’ Abu Bakar,”Islam, Hukam dan Masyarakat di Aceh Tajdid Syariat dalam Negara Bangsa”, First International Conference of Aceh and Indian Ocean Studies, 24-27 Februari 2007, h. 2. 149 1 Haedar Nasir, Islam syariat: Reproduksi Salapiyah Idiologis di Indonesia (Bandung: Mizan 2013), h. 336. 2 Rusjdi Ali Muhammad, Revitalisasi Syari’at Islam di Aceh: Problem, Solusi dan Implementasi, (Ciputat: Logos Wacana Ilmu,2003), h. 49. 3 Khamami Zada, “Sentuhan Adat Dalam Pemberlakuan Syariat Islam di Aceh (1514-1903)”, Karsa, Vol. 20 No. 2, Desember 2012, h. 3. 4 Al Yasa’ Abu Bakar,”Islam, Hukam dan Masyarakat di Aceh Tajdid Syariat dalam Negara Bangsa”, First International Conference of Aceh and Indian Ocean Studies, 24-27 Februari 2007, h. 2. A. Pendahuluan Masyarakat Aceh memiliki corak kehidupan yang kental dengan agama Islam, hal ini dapat dilacak akar sejarah berdirinya kerajaan–kerajan Islam yang menjadi pusat kekuasaan politik sekaligus agama. Aceh tergolong wilayah yang memiliki sejarah panjang dalam hal kedatangan Islam dan berdirinya kerajaan–kerajaan Islam yang dimulai dari kerajaan Peureulak (840-1291), Samudra Pasai (1042-1427), Teumiang (840-1291), Darussalam (1205-1530) dan Aceh Darussalam (1514-1903) dan kerajaan – kerajaan kecil lainnya.1 Bila ditinjau dari sejarah kerajaan Aceh Darussalam (1514-1903) terdapat pemberlakuan hukum jinayat yang dipengaruhi oleh hukum adat. Pemberlakuan hukum jinayat ini dapat dibuktikan dengan adanya Qanun Meukuta Alam dan keterangan- keterangan tentang praktek pemberlakuan hukum jinayat.2 Dalam Qanun Meukuta Alam ini ditetapkan bahwa dasar kerajaan Acceh Darussalam adalah Islam dan bentuknya adalah kerajaan yang bersuber kepada al-Qur’an, al-Hadist, Ijma’ dan Qiyas.3 Konsep ini sesungguhnya mencerminkan bahwa Islam dan negara adalah integral (al-Isla din wa daulah), tak berlebihan jika kemudian kerajaan Aceh Darussalam merumuskan Qanun al-Asyi sebagai pedoman dasar dalam kehidupan bernegaraa, sosial dan hukum masyarakat. Meskipun Islam telah menjadi sumber undang-undang negara, tetapi tidak dapat dielakkan keterpengaruhan hukum jinayat yang diberlakukan oleh adat istiadat yang dipraktekkan di masyarakat. Dengan kata lain, pelanggaran terhadap pembunahan, perzinaan, pencurian dan khamar dikenakan sanksi yang tegas yang di ambil dari hukum jinayat yang bercampur dengan adat. Hal ini dapat disimpulkan bahwa adat orang Aceh sangat berpengaruh oleh ajaran Islam, bahkan lebih dari sekedar itu, adat orang Aceh terbentuk di atas kesadaran dan keinginan untuk menjalankan syariat Islam dalam kehidupan sehari-hari.4 Masyarakat Aceh memiliki strukur sosial tersendiri yang diatur berdasarkan hukum Qanun Meukuta Alam al-Asyi (Undang-undang kerajaan Aceh). Qanun tersebut menjelaskan bahwa kekuasaan tertinggi berada di tangan sultan, sedangkan kekuasaan agama berada 149 ditangan ulama. Di bawah sultan ada Uleebalang (hulu balang) yang menguasai daerah tertentu dengan hak otonomi luas. Uleebalang membawahi beberapa imum mukim dan dibawah imum mukim terdapat beberapa keuchik.5 Di Aceh, syariat Islam di implementasikan secara formal setelah dikeluarkannya Undang-undang No. 44 Tahun 19996 tentang Penyelenggaraan Keistimewaan Provinsi Daerah Istimewa Aceh dan Undang-undang No. 18 Tahun 2001 tentang Otonomi Khusus bagi Provinsi Daerah Istimewa Aceh7 sebagai Provinsi Nanggroe Aceh Darussalam. Adanya legalitas dari pemerintah untuk menerapkan syariat Islam di Aceh direspon oleh pemerintah daerah dengan mengeluarkan beberapa peraturan daerah (Perda) dalam rangka terlaksananya syariat Islam di Aceh. 5 Hasan Basri, ”A. Hasjmy (1914-1998) Kajian Sosial-Intlektual Pemikiran Tentang Politik Islam”, (Disertasi Sps UIN Jakarta, 2008), h. 53. Lihat juga, Asma Uddin, "Religious Freedom Implications of Sharia Implementation in Aceh, Indonesia," University of St. Thomas Law Journal: Vol. 7: Iss. 3 (2010), Article Available at: http://ir.stthomas.edu/ustlj/vol7/iss3/8(diakses pada tanggal 02 Mei 2016) h. 618. 9 Asma Uddin, "Religious Freedom Implications of Sharia ..., h. 632. 8 Syamsul Bahri, “Pelaksanaan Syariat Islam di Aceh Sebagai Bagian dari Wlayah NKRI”, inamika Hukum Vol. 12 No. 2 Mei 2012. p j p gg 6 Haidar Nassir, Islam Syariat Reproduksi Salafiyah Idiologis di Indonesia (Bandung: Mizan, 2013),h. 332. 7 Topo Santoso, Membumikan Hukum Pidana Islam: Penegakan Syariat dalam Wacana dan Agenda, 7 Topo Santoso, Membumikan Hukum Pidana Islam: Penegakan Syariat dalam Wacana dan Agenda, : GIP, 2003),h. 106. , y p f y g ( g , 2013),h. 332. 7 Topo Santoso, Membumikan Hukum Pidana Islam: Penegakan Syariat dalam Wacana dan Agenda, ( Jakarta: GIP, 2003),h. 106. p j p gg 6 Haidar Nassir, Islam Syariat Reproduksi Salafiyah Idiologis di Indonesia (Bandung: Mizan, 2013),h. 332. 5 Hasan Basri, ”A. Hasjmy (1914-1998) Kajian Sosial-Intlektual Pemikiran Tentang Politik Islam”, (Disertasi Sps UIN Jakarta, 2008), h. 53. Lihat juga, Asma Uddin, "Religious Freedom Implications of Sharia Implementation in Aceh, Indonesia," University of St. Thomas Law Journal: Vol. 7: Iss. 3 (2010), Article Available at: http://ir.stthomas.edu/ustlj/vol7/iss3/8(diakses pada tanggal 02 Mei 2016) h. 618. 6 Haidar Nassir, Islam Syariat Reproduksi Salafiyah Idiologis di Indonesia (Bandung: Mizan, 2013),h. 332. 7 Topo Santoso, Membumikan Hukum Pidana Islam: Penegakan Syariat dalam Wacana dan Agenda, ( Jakarta: GIP, 2003),h. 106. 8 Syamsul Bahri, “Pelaksanaan Syariat Islam di Aceh Sebagai Bagian dari Wlayah NKRI”, Jurnal Dinamika Hukum Vol. 12 No. 2 Mei 2012. 9 Asma Uddin, "Religious Freedom Implications of Sharia ..., h. 632. 8 Syamsul Bahri, “Pelaksanaan Syariat Islam di Aceh Sebagai Bagian da Jurnal Dinamika Hukum Vol. 12 No. 2 Mei 2012. 14 Wilayatul Hisbah adalah lembaga atau badan yang berwenang memberitahukan kepada masyarakat tentang peraturan-peraturan yang sudah berlaku dan menyadarkan anggota masyarakat tersebut, agar mematuhi aturan-aturan supaya tidak dikenakan sanksi atau denda dari peraturan- peraturan terebut (law enforcement). Lihat, Al Yasa’ Abu Bakar, Wilayatul Hisbah, Polisi Pamong Praja Dengan Kewenangan Khusus di Aceh, (Banda Aceh: Dinas Syari‘at Islam Aceh, 2009), h. 22. A. Pendahuluan Dari perda-perda itu selanjutnya dikembangkan lagi menjadi peraturan-peraturan daerah yang menyangkut tata laksana syariat Islam yang pada gilirannya melahirkan Qanun Aceh.8 Lima qanun disahkan antara tahun 2002-2004 yang berisi hukuman pidana atas pelanggaran syariah yakni: Qanun No. 11 Tahun 2002 tentang Penerapan Syariah Dalam Aspek Kepercayaan (aqidah), Ritual (ibadah), dan Penyebaran (syi’ar) Islam yang meliputi persyaratan busana islami; Qanun No. 12 Tahun 2003 tentang larangan Menkonsumsi dan Menjual Minuman ber–alkohol (khamar); Qanun No. 13 Tahun 2003 tentang larangan Judi (maisir); Qanun No. 14 Tahun 2003 melarang “perbuatan bersunyi-sunyian” (khalwat)9 dan Qanun No. 7 Tahun 2004 tentang Pembayaran Zakat. Dari penjelasan latar belakang di atas, maka tulisan ini akan mencoba mengungkapkan faktor-faktor yang mempengaruhi implementasi Qanun No. 14 Tahun 2003 Tentang khalwat di Kota Subulussalam, baik foktor dari segi hukum maupun non hukum, sehingga dapat tergambar dengan jelas apa saja yang menjadi penghalang efektifitas Qanun No. 14 di di Kota Subulussalam dan di Aceh pada umumnya. Penelitian ini bersumber dari penelitian lapangan (field research) dan penelitian kepustakaan (library research) dengan metode kualitatif dan menggunakan pendekatan sosio-legal-historis. 150 10 Soerjono Soekanto, Faktor-Faktor yang Mempengaruhi Penegakan Hukum (Jakarta: PT. Raja Grafindo Persada, 2008), h. 8. Lihat juga, Jainuddin Ali, Sosiologi Hukum (Jakarta: Sinar Grafika, 2007), h. 62. B. Pembahasan Ada banyak faktor yang mempengaruhi dalam efektivitas penegakan Qanun Kahalwat di Subulussalam. Merujuk kepada teori Soerjono Soekanto yang menjelaskan bahwa masalah pokok dalam penegakan hukum adalah terletak pada hukumnya sendiri (peraturan perundang-undangan yang berlaku), penegak hukumnya yakni pihak-pihak yang mengawal penerapan hukum, sarana atau fasilitas yang mendukung penerapan hukum, masyarakat di mana hukum tersebut diberlakukan dan budaya hukum dalam masyarakatnya.10 Kelima faktor tesebut saling memiliki kaitan yang erat dikarenakan esensi dari penegakan hukum. Kelima faktor tersebut merupakan tolak ukur daripada efektifitas penerapan hukum. 11 Pasal 4 Qanun Nomor 14 Tahun 2003 Tentang Khalwat. Lihat juga Zulkarnain Lubis, Menyambut Qanun Jinayah Nomor 6 Tahun 2014, https://drive. google. com/file/d/0B5UQVc J8Df8WenBNSWNfZU VMcTQ/view? pli=1 (diakses 20 Oktober 2015). Q/ p ( ) 12 Pasal 22 Qanun Nomor 14 Tahun 2003 Tentang Khalwat. / p ( 12 Pasal 22 Qanun Nomor 14 Tahun 2003 Tentang Khalwat. l 22 Qanun Nomor 14 Tahun 2003 Tentang Khalwat. l 23 ayat (1) Qanun Nomor 6 Tahun 2014 Tentang Hukum Jinayat. Pasal 22 Qanun Nomor 14 Tahun 2003 Tentang Khalwat. Pasal 23 ayat (1) Qanun Nomor 6 Tahun 2014 Tentang Hukum Jinayat. J8Df8WenBNSWNfZU VMcTQ/view? pli=1 (diakses 20 Oktober 2015). 12 Pasal 22 Qanun Nomor 14 Tahun 2003 Tentang Khalwat. 1. Faktor Undang-undang (Qanun) Bila kita mengkaji Qanun Nomor 14 Tahun 2003 tentang khalwat, maka kita akan menemui suatu kepastian bentuk ‘uqubat bagi mereka yang melanggar ketentuan-ketentuan khalwat di Aceh. Qanun ini tegas mengtakan bahwa perbuatan khalwat adalah haram,11 dan barang siapa yang melanggar ketentuan tersebut dapat di kenakan ‘uqubat ta’zir berupa cambuk paling banyak 9 (sembilan) kali cambukan.12 Begitu juga dengan Qanun Nomor 6 Tahun 2014 tenntang hukum jinayat yang merupa pembaharu terhadap Qanun Nomor 14 Tahun 2003, dikatakan bahwa hukum berkhalwat adalah haram dan setiap orang yang melanggar ketentuan tersebut di ancam dengan ‘uqubat ta’zir berupa cambuk sebanyak 10 (sepuluh kali) cambukan.13 Sebenarnya kelemahan utama yang terdapat dalam Qanun 14 tahun 2003 adalah tidak adanya kewenangan yang diberikan kepada wilayatul hisbah (WH)14 untuk melakukan penahanan. WH tidak berwenang menahan dan memeriksa, sehingga penahanan yang dilakukan WH merupakan tindakan melanggar ketentuan hukum dan 151 melampaui batas kewenangan. Kewenangan yang dimiliki WH hanya sebatas melakukan pengawasan terhadap penegakan syariah Islam sekaligus melakukan pembinaan terhadap para pelanggar syariah Islam yang sedang berlaku di Aceh. Dan tidak ada kewenangan WH melakukan penahanan terhadap para pelanggar pelaku Syariah di Aceh bila berdasarkan kepada Qanun Nomor 14 Tahun 2003.15 Apabila WH menangkap pelaku pelanggaran khalwat akan dilimpahkan perkaranya ke pihak penyidik di kepolisian, dan pihak kepolisianlah yang berwenang melakukan penahanan terhadap mereka.16 Setelah di sahkannya Qanun Nomor 7 Tahun 2013 Tentang Hukum Acara Jinayat sepertinya telah memberi angin segar bagi penegakan syariat Islam di Aceh. Hal ini ditandai dengan penambahan kewenangan yang diberikan kepada Wilayatul Hisbah dalam melakukan tindakan yang nyata terhadap penegakan Qanun Jinayat di Aceh, dimana pada qanun-qanu sebelumnya (Qanun 12, 13 dan 14 Tahun 2003) kewenangan WH hanya berupa pengawsan terhadap pemberlakuan syariat Islam di Aceh. Sebagai mana di jelaskan dalam Pasal 7 dan 9 Qanun 2013 sebagai berikut: Penyelidik merupakan Pejabat Polri dan PPNS yang telah diberi wewenang oleh Undang-Undang dan/atau Qanun untuk melakukan penyelidikan (Pasal 6). Dalam Pasal 7 dan pasal 9 ayat (2) dijelaskan: Dalam Pasal 7 dan pasal 9 ayat (2) dijelaskan: (1) Penyelidik sebagaimana dimaksud dalam Pasal 6, karena kewajibannya berwenang: a. menerima laporan atau pengaduan dari seseorang tentang adanya Jarimah; (1) Penyelidik sebagaimana dimaksud dalam Pasal 6, karena kewajibannya berwenang: a. menerima laporan atau pengaduan dari seseorang tentang adanya Jarimah; b i k t d b b kti a. menerima laporan atau pengaduan dari seseorang tentang adanya Jarimah; b. mencari keterangan dan barang bukti; c. p 16 Keputusan Gubernur Aceh No. 01 tahun 2004 tentang kewenangan pembentukan Organisasi Wilayatul Hisbah. 15 Wawancara dengan Sarkani, Kasi Ketentraman dan Ketertiban Pol PP dan WH Kota Subulussalam, 23 April 2015. 17 Pasal 10 Qanun Nomor 7 Tahun 2013 Tentang Hukum Acara Jinayat. 1. Faktor Undang-undang (Qanun) menyuruh berhenti seseorang yang dicurigai dan menanyakan serta memeriksa tanda pengenal diri; dan d. mengadakan tindakan lain menurut hukum secara bertanggung jawab dan sesuai dengan prinsip-prinsip Syariat Islam. (2) Penyelidik sebagaimana dimaksud dalam Pasal 6, atas perintah penyidik dapat melakukan tindakan: a. penangkapan, larangan meninggalkan tempat, penggeledahan dan penyitaan; b. pemeriksaan dan penyitaan surat; a. penangkapan, larangan meninggalkan tempat, penggeledahan dan penyitaan; b. pemeriksaan dan penyitaan surat; c. mengambil sidik jari dan memotret seseorang; dan/atau c. mengambil sidik jari dan memotret seseorang; dan/atau d. membawa dan menghadapkan seseorang kepada penyidik. 15 Wawancara dengan Sarkani, Kasi Ketentraman dan Ketertiban Pol PP dan WH Kota Subulussalam, 23 April 2015. 16 Keputusan Gubernur Aceh No. 01 tahun 2004 tentang kewenangan pembentukan 152 (3) Penyelidik membuat dan menyampaikan laporan hasil pelaksanaan tindakan sebagaimana dimaksud pada ayat (1) dan ayat (2) kepada Penyidik. Pasal 9 Ayat (2): (2) Penyidik PPNS sebagaimana dimaksud dalam Pasal 8 ayat (1) huruf b, berwenang: a. menerima laporan atau pengaduan dari seseorang tentang adanya tindak pidana pelanggaran Qanun dan/atau Peraturan Perundang-undangan lainnya; b. melakukan tindakan pertama di Tempat Kejadian Perkara (TKP) dan melakukan pemeriksaan; c. menyuruh berhenti seorang Tersangka dan memeriksa tanda pengenal diri Tersangka; d. melakukan pengeledahan, penangkapan, dan penahanan; e. melakukan penyitaan benda dan/atau surat sesuai dengan Peraturan Perundang-undangan; f. mengambil sidik jari dan memotret seorang Tersangka; f. mengambil sidik jari dan memotret seorang Tersangka; g. memanggil orang untuk didengar dan diperiksa sebagai Tersangka atau Saksi; h. mendatangkan Saksi ahli yang diperlukan dalam hubungannya dengan pemeriksaan perkara; i. membuat Surat Perintah Dimulainya Penyidikan (SPDP) kepada Penuntut Umum dengan tembusan kepada korwas PPNS setelah terlebih dahulu berkoordinasi dengan korwas; j. melakukan penghentian penyidikan setelah berkoordinasi dengan penuntut umum bahwa tidak terdapat cukup bukti atau peristiwa tersebut bukan merupakan tindak pidana dan tembusannya disampaikan kepada Korwas PPNS dan selanjutnya memberitahukan hal tersebut kepada Tersangka atau kuasanya; dan/atau k. mengadakan tindakan lain sesuai dengan Peraturan Perundang-undangan. (3) Dalam melaksanakan kewenangan sebagaimana dimaksud pada ayat (1) dan ayat (2) penyidik wajib menjunjung tinggi nilai-nilai Syariat Islam dan Peraturan Perundang- undangan. PPNS dalam melaksanakan kewenangan penyelidikan sebagimana dimaksud dalam Pasal 7 dan Pasal 9 ayat (2) dibantu oleh anggota kepolisian dan polisi WH.17 Inilah yang menjadi dasar bagi WH dalam melakukan penahan terhadap setiap orang yang melanggar PPNS dalam melaksanakan kewenangan penyelidikan sebagimana dimaksud dalam Pasal 7 dan Pasal 9 ayat (2) dibantu oleh anggota kepolisian dan polisi WH.17 Inilah yang menjadi dasar bagi WH dalam melakukan penahan terhadap setiap orang yang melanggar 153 ketentuan-ketentuan qanun jina>ya>t di Aceh, dimana ketentuan seperti ini sebelumya tidak pernah di atur dalam qanun-qanun jinayat terdahulu (Qanun 12, 13 dan 14 Tahun 2003). Jadi kendala yang selama ini menjadi alasan utama bagi WH dalam menindak pelaku jinayat di Aceh sudah terjawab dengan disahkannya Qanun No. 7 Tahun 2013 Tentang Hukum Acara Jinayat. Kendala selanjutnya adalah adanya benturan antara qanun dalam penanganan kasus khalwat. Permasalahan khalwat di Aceh, selain di atur dalam Qanun Nomor 14 Tahun 2003 dan ditegaskan kemabali dalam Qanun Nomor 6 Tahun 2014 juga di atur dan menjadi salah satu kewenangan dari peradilan adat di Aceh, hal ini di jelaskan dalam Qanun Nomor 9 Tahun 2008 Tentang Pembinaan Kehidupan Adat dan Adat Istiadat. 18 Wawancara dengan Kalidin, Kasi Penegakan Syariat Islam, WH Kota Subulussalam, 23 April 2015. 19 Putri Sahadat Bancin, Penyelesaian Tindak Pidana Khalwat/Mesum Berdasarkan Qanun Nomor 14 Tahun 2003 (Suatu Penelitian Di Kota Subulussalam), (Banda Aceh: Skripsi Fak. Hukum Univ. Syiah Kuala, 2015), h. 55. p 19 Putri Sahadat Bancin, Penyelesaian Tindak Pidana Khalwat/Mesum Berdasarkan Qanun 14 Tahun 2003 (Suatu Penelitian Di Kota Subulussalam), (Banda Aceh: Skripsi Fak. Hukum Syiah Kuala, 2015), h. 55. 18 Wawancara dengan Kalidin, Kasi Penegakan Syariat Islam, WH Kota Subulussa April 2015. Pasal 9 Ayat (2): Dalam Pasal 13 ayat (1) dijelaskan bahwa khalwat merupakan salah satu dari 18 kewenangan yang di berikan kepada mahkamah adat di Aceh. Hal inilah yang menjadi celah permasalahan baru dalam penerapan Qanun Khalwat di Aceh dan khususnya Kota Subulussalam. Dengan adanya Qanun Nomor 9 Tahun 2008, setiap pelanggaran khalwat yang terjadi Aceh harus diselesaikan melalui peradilan adat terlebih dahulu. Apabila bisa diselesaikan dalam peradilan adat, maka pihak berwenang dalam hal ini WH tidak berwenang lagi untuk menindaklanjuti kasus tersebut.18 Mengenai mekanisme penyelesaian kasus khalwat di lembaga adat akan disesuaikan dengan kebiasaan adat di wilayah masyarakat setempat, bararti hukum adat di Aceh tidak memiliki standar tertentu dalam mekasnisme penyelesaiannya, hal ini dikarenakan masyarakat Aceh yang majemuk dan terdiri dari beberapa suku yang berbeda.19 Adanya dualisme lembaga dalam peneyelesaian kasus khalwat di Aceh merupakan kendala tersendiri bagi WH dalam menegakkan Qanu Khalwat. Satu penelitian terkait dengan khalwat/mesum di kota Lhokseumawe yang dilakukan Abdullah, seorang dosen Sekolah Tinggi Agama Islam Negeri (STAIN) Lhokseumawe menyebutkan, berkurangnya penyelesaian kasus khalwat/mesum melalui WH dan mahkamah syar’iyah dikarena penyelesaian kasus khalwat/mesum telah bergeser. Kasus khalwat/mesum saat ini lebih mengutamakan penyelesaian secara adat ketimbang kepada jalur WH dan mahkamah syar’iyah. 154 Hasil penelitian itu telah dipaparkan dalam Aceh Development International Conference (ADIC) yang berlangsung pada 27 Maret 2013 di Kuala Lumpur. Apa yang disimpulkan Abdullah, barangkali ada benarnya. Sebab sejak Majelis Adat Aceh20 (MAA) melakukan sosialisasi penyelesaian kasus masyarakat melalui adat dalam beberapa tahun terakhir, maka kasus-kasus yang masuk dalam kategori 18 kasus adat sesuai dengan amanah Qanun Nomor 9 Tahun 2008, bisa diselesaikan melalui mahkamah adat di gampong ataupun mukim.21 Untuk mengatasi hal tersebut, sudah saatnya para pemangku kepentingan untuk duduk bersama dan merumuskan mekanisme penyelesaian kasus khalwat di Aceh, khususnya di Subulussalam. Hal ini penting dilakukan guna mempertegas mengenai ketentuan-ketentuan khalwat seperti apa yang menjadi wilayah kewenangan masing-masing lembaga. Karena selama ini penangangan kasus khalwat menurun penyelesaiannya di tingkat mahkamah syar’iyah. Disamping itu ada yang beranggapan bahwa hukuman22 yang diberikan lembaga adat terhadap pelaku khalwat di nilai kurang adil, karena bentuk hukumannya kurang begitu tegas dan tidak menimbulkan efek jera bagi para pelakunya. Di Kota Subulussalam sendiri penyelesaian kasus khalwat di tempuh dengan jalur kekeluargaan. Sebagaimana yang dijelaskan Kasi Penegak Kebijakan Daearah Dan Syariat Islam Kota Subulussalam bahwa, setiap pelanggaran kasus khalwat diwilayah hukum Kota Subulussalam terlebih dahulu dilakukan penyelesaian dengan cara kekeluargaan dan adat istiadat yang berlaku di Subulussalam. 20 Di Aceh disamping adanya lembaga hukum formal yang menangani pelanggaran syariat Islam, juga terdapat lembaga informal yaitu lembaga adat yang sangat berpengaruh pada pola kehidupan masyarakat Aceh. Lembaga adat memiliki landasan hukum yakni Qanun No. 9 Tahun 2008 tentan Pembinaan Kehidupan Adat dan Adat Istiadat serta Qanun Nomor 10 Tahun 2008 tentang Lembaga Adat. p g ( p gg g ) 22 Pasal 16 (1) Qanun Nomor 9 Tahun 2008 dijelaskan bahwa jenis-jenis sanksi yang dapat dijatuhkan dalam penyelesaian sengketa adat sebagai berikut: nasehat; teguran; pernyataan maaf; sayam; diyat; denda; ganti kerugian; dikucilkan oleh masyarakat gampong atau nama lain; dikeluarkan dari masyarakat gampong atau nama lain; pencabutan gelar adat; dan bentuk sanksi lainnya sesuai dengan adat setempat. g g 21 Teuku Muttaqin Mansur, “Penyelesaian Kasus Mesum Melalui Mahkamah Adat atau Mahkamah Syar’iyah?”, http://aceh.tribunnews.com/2013/09/04 /peny elesaian-kasus-mesum- melalui-ma-atau-ms?page3 (dikases pada tanggal 24 Agustus 2015). Pasal 9 Ayat (2): Setelah pihak Satpol PP dan WH memberikan teguran dan peringatan kepada para pihak yang melanggar qanun tentang khalwat tersebut, lalu menyerahkannya kepada geucik (kepala desa) untuk diselesaikan secara adat dan aturan kampong tersebut, setelah dilakukan proses hukum adat tapi belum menghasilkan suatu 155 kesepakatan, baru diselesaikan secara qanun yang berlaku.23 Pada dasarnya penyelesaian kasus khalwat ditempuh dengan jalan damai dimana kedua keluarga yang tersangka khalwat/mesum duduk bersama untuk mencari solusi terhadap penyelesaian kasus tersebut dengan hukumuan yang sering di capai adalah berupa pemberian nasehat, perdamaian, pernyataan maaf, ganti rugi/denda dan di nikahkan.24 gg p 24 Wawancara dengan H.M Layari Kombih, selaku Ketua Majelis Adat Aceh (MAA) Kota Subulussalam, bertempat dikantor MAA Kota Subulussalam pada tanggal 27 April 2015. 23 Wawancara dengan Bapak Kalidin, Kasie Penegak Kebijakan Daearah Dan Syariat Islam Kota Subulussalam. Kantor Satuan Polisis Pamong Praja dan Wilayatul Hisbah Kota Subulussalam, pada tanggal 23 April 2015. , J y y , , , 26 Wawancara dengan Abdul Malik, selaku Kepala Satpol PP dan WH Kota Subulussalam. p p gg p 25 Danial, “Efektifitas ‘Uqūbat Dalam Qanun No. 14 Tahun 2003 dan DQHR Tentang Khalw khtilath”, Jurnal Asy-Syir’ah Vol. 45, No. I, 2011, h. 1004. 25 Danial, Efektifitas Uqūbat Dalam Qanun No. 14 Tahun 2003 dan DQHR Tentang K dan Ikhtilath”, Jurnal Asy-Syir’ah Vol. 45, No. I, 2011, h. 1004. tanggal 23 April 2015. 24 Wawancara dengan H.M Layari Kombih, selaku Ketua Majelis Adat Aceh (MAA) Kota Subulussalam, bertempat dikantor MAA Kota Subulussalam pada tanggal 27 April 2015. 25 Danial “Efektifitas ‘Uqūbat Dalam Qanun No 14 Tahun 2003 dan DQHR Tentang Khalwat 2. Faktor Penegak Hukum Penegak hukum dilakukan oleh institusi yang diberi wewenang untuk itu (penegakan qanun syariat Islam), dalan hal ini adalah wilayatul hisbah. Sejak hukum itu mengandung perintah dan pemaksaan, maka sejak itu pula hukum membutuhkan bantuan untuk mewujudkan perintah tersebut. Hukum menjadi tidak ada artinya, bila perintahnya tidak dapat dilaksanakan. Karena itu, diperlukan usaha dan tindakan manusia agar perintah dan paksaan yang secara potensial ada di dalam peraturan itu menjadi nyata dalam praktek hukum. Donald Black, sebagaimana dikutip Sadjipto Rahardjo mengatakan bahwa dimensi keterlibatan manusia dalam penegakan hukum tersebut dinamakan dengan mobilisasi hukum. Dalam mobilisasi hukum inilah manusia turut campur sehingga hukum tidak hanya mengancam dan berjanji di atas kertas.25 Wilayatul Hisbah dan Sat Pol PP Kota Subulussalam saat ini memiliki 139 orang anggota dan keseluruhan dari anggota tersebut berpusat di Kantor Pol PP dan WH Kota Subulussalam di Subulussalam. Adapun status dari 139 orang anggota tersebut, baru 15 orang yang diangkat menjadi pegawai negeri sipil (PNS) dan sisanya masih berstatus sebagai pegawai honorer.26 Selain itu WH Kota Subulussalam juga belum mempunyai Penyidik Pegawai Negeri Sipil (PPNS), padahal keberadaan PPNS ini sangat di butuhkan guna untuk proses penyelidikan dan penyidikan yang dilakukan WH terhadap pelaku pelanggaran qanun jinayat di Kota Subulussalam. Ketersediaan sumber daya manusia yang memadai sangat mempengaruhi efektivitas suatu hukum. Berdasarkan keterangan di atas bahwa saat ini Wilayatul Hisbah dan Pol PP Kota Subulussalam memiliki 139 anggota dimana 15 belas di antaranya adalah PNS, sisanya pengawai honorer yang kebayakan memiliki latar belakang pendidikan Sekolah Menengah 156 Atas (SMA).27 Pendidikan para penegak hukum ini (WH) sangat vital dalam penegakan hukum syariah di Aceh, karena hukum yang akan di tegakkan nantinya bukanlah hukum yang berdasarkan kepada UUD sebaggai landasar utama pembuatan hukumnya, akan tetapi al-Qur’an dan al-Hadist. Maka untuk itu setiap anggota WH di tuntut untuk memiliki kecapan dalam bidang hukum Islam (fikih). Inilah yang menjadi permaslahan selama ini, dimana masyarakat menilai, para penegak hukum syariah di Kota Subulussalam belum layak untuk dijadikan sebagai penegak qanun jinayat. Hal ini dikarenakan sikap dan perilaku keseharian anggota WH yang belum mencerminkan nilai-nilai ke-Islaman dalam kehidupan masyarakat sehari-hari di luar masa tugasnya.28 Memang dalam perekrutan anggota WH selam ini belum ada standar yang khusus mengenai latar belakang pendidikan bagi anggota WH. 2. Faktor Penegak Hukum Menurut Wildan Sastra selaku pemerhati syariat Islam dan gender kota Subulussalam, mengkritik bahwa sudah seharusnya WH Kota Subulussalam membuat standar khusus dalam perekrutan anggota WH dimana dinai-nilai ke-Islaman harus menjadi standar utama dalam perekerutan tersebut, sehingga dalam penegakan qanun-qanun syariat anggota WH bisa mengetahui dengan baik dan benar apa yang menjadi tugasnya dan begitu juga dalam kehidupan bermasyarakat sehari-hari, anggota WH di harapkan bisa memberikan contoh yang nyata kepada masyarakat akan perlunya menegakkan syariat Islam di Kota Subulussalam. Wildan mengatakan, anggota WH seharusnya menjadi garda terdepan dalam penegakan dan pencotohan syariat Islam bukan malah sebaliknya.29 g 29 Wawancara dengan Wildan Sastra, selaku pemerhati syariat Islam di Kota Subulussalam. 28 Wawancara dengan Arifin Sarbaini, selaku Ketua FPI Kota Subulussalam. 27 Wawancara dengan Bambang Aprianto, Selaku Subag TU Pol PP dan WH Kota Subulussalam. 3. Faktor Fasilitas Penunjang Penegakan Hukum Keterpusatan WH di Kota Subulussalam sebenarnya menjadi masalah tersendiri terhadap penegakan qanun jinayat di Subulusslam. Saat ini Kota Subulussalam memiliki 5 kecamatan yang terdiri dari Kecamatan Simpang Kiri, Penanggalan, Sultan Daulad, Runding dan Longkib. Tiga dari lima kecamatan ini pada dasarnya jauh dari Kantor Pol PP dan WH Kota Subulussalam yang terletak di pusat Pemerintahan Kota Subulussalam di Kecamatan Simpang Kiri. Ketiga kecamatan tersebut adalah, Kecamatan Sultan Daulad, Kecamatan Rundeng dan Kecamatan Longkib. Sedangkan dua lainnya yakni Kecamatan Simpang Kiri dan Kecamatan Penanggalan relatif berdekatan. Maka tidak heran bila selama ini kasus yang di tangani WH Kota Subulussalam berasal dari Kecamatan Simpang Kiri dan 157 Kecamatan Penanggalan, karena dua kecamatan inilah yang paling memungkinkan untuk dilakukan pengontrolan setiap saat, karena letak dan jaraknya dari kantor Pol PP dan WH tidak terlalu jauh. Keberadaan Kantor WH seharusnya tidak hanya berada di satu titik tertentu, tapi harus ada di setiap kecamatan-kecamatan di Kota Subulusssalam. Hal ini dilakukan supaya keberadaan WH dapat dirasakan langsung oleh masyarakat bukan hanya sebagai simbol dari pelengkap lembaga-lembaga daerah di Aceh. Dalam Keputusan Gubernur No. 1 Tahun 2004 Tentang Pembentukan Organisasi dan Tata Kerja Wilayatul Hisbah disebutkan bahwa susunan organisasi Wilayatul Hisbah, terdiri atas; Wilayatul Hisbah Tingkat Provinsi; Wilayatul Hisbah Tingkat Kabupaten/Kota; Wilayatul Hisbah Tingkat Kecamatan, dan Wilayatul Hisbah Tingkat Kemukiman.30 Berarti pembentukan kantor WH di setiap kecamatan sangat mungkin untuk di bentuk karena telah mempunyai landasan hukum yang kuat. Ketika penulis menanyakan hal tersebut, Kepala Satuan Polisi Pamong Praja dan Wilayatul Hisbah Kota Subulussalam Bapak Abdul Malik mengatakan, hal itu mungkin saja dilakukan tapi tidak dalam waktu dekat ini, karena menurut Abdul Malik saat ini Pol PP dan WH masih berfokus kepada kelengkapan fasilitas penunjang lainnya yang menurutnya sangat belum memadai/ideal untuk sebuah kantor WH tingkat kota di Aceh. Selain itu, Kantor WH kota Subulussalam sekarang ini juga belum memiliki kantor yang permanen tapi masih dalam bentuk sewa yang dilakukan oleh Pemerintah Kota Subulussalam. Memang inilah kendala utama mengapa WH kota Subulussalam belum membentuk kantor WH di tingkat kecamatan, karena semua itu tentunya membutuhkan anggaran yang tidak sedikit. Alokasi anggaran yang disediakan APBK Kota Subulussalam terhadap Kantor Satpol PP dan WH memang sangat terbatas. Sebagai gambaran, pada tahun 2013 Pol PP dan WH mendapat alokasi anggaran sebesar Rp. 4.215.000.000,00 (empat milyar dua ratus lima belas juta rupiah) sedangkan pada tahun 2014 alokasi anggaran Pol PP dan WH adalah Rp. 30 Hirwan Jack, “Efektivitas Wilayatul Hisbah Dalam Pencegahan Aliran Sesat Di Aceh”, http://bkpp.acehprov .go.id/ simp egbrr /Art ikel/Artikel05-02-2015/Wilayatul H i s bah _Aceh.pdf ,h. 2. (diakses pada tanggal 16/03/2015). 3. Faktor Fasilitas Penunjang Penegakan Hukum 4.300.000.000,00 (empat milyar tiga ratus juta rupiah) dan pada tahun 2015 alokasi anggaran yang disediakan adalah Rp. 4.350.000.000,00 (empat milyar tiga ratus lima puluh juta rupiah). Dengan anggaran tersebut WH dikatakan Abdul Malik belum bisa berbuat banyak mengenai pengawasan syariat Islam di Kota Subulussalam, belum lagi wilayah 158 subulussalam yang menjadi daerah tugasnya juga luas dan berjauhan antar satu kecamatan dengan kecamatan yang lainnya. Untuk saat ini Pol PP dan WH Kota Subulussalam memiliki bebera fasilitas untuk keperluan dinas seperti, satu buah kenderaan mobil patroli, 16 (enam belas) kenderaan sepada motor, 8 (delapan) koputer/laptop.31 Dengan anggaran dan fasilitas seperti itu, bisa di bayangkan bagaimana proses pengawasan yang di lakukan oleh WH Kota Subulussalam terhadap qanun-qanun syariat Islam Kota Subulussalam, belum lagi status anggota WH yang mayoritas masih sebagai pegawai honorer, tentu hal ini juga bisa mempengaruhi konsentrasi mereka dalam menjalankan tugasnya sehari-hari. Memang dalam hal ini, keseriusan Pemerintahan Kota Subulussalam dalam menerapkan syariat Islam masih sangat mungkin untuk dipertanyakan, pemerintah seharusnya sepenuh hati dalam menerapkan syariat Islam di kota ini dengan menyediakan alokasi anggaran untuk WH yang memadai. Maka wajar saja kalau selama ini banyak kalangan yang menilai proses penerapan syariat Islam di Kota Subulusssalam masih satgnan dan jalan di tempat. Belum lagi ketiadaan Mahkamah Syar’iyah Kota Subulussalam, karena saat ini Mahkamah Syar’iyah Kota Subulussalam masih menginduk kepada Mahkamah Syar’iyah Singkil di Kabupaten Aceh Singkil. Maka wajar-wajar saja jika selama ini kasus khalwat, maisir maupun khamar hanya di tindak di sekitar wilayah Kecamatan Simpang Kiri dan Penanggalan, karena kedua kecamatan tersebut merupakan berdekatan dengan kantor WH yang tersentral di Simpang Kiri Kota Subulussalam. Hal ini dapat di lihat dari data tempat terjadinya pelanggaran Qanun 12, 13 dan 14 Tahun 2003 yang di miliki oleh Kantor Satpoll PP dan WH Kota Subulussalam sebagai berkut: 31 Wawancara dengan Bambang Aprianto, Selaku Subag TU Pol PP dan WH Kota Subulussalam. Tabel Lokasi Tempat Pelanggaran Qanun Nomor 12, 13 dan 14 di Kecamatan Simpang Kiri dan Penanggalan 31 Wawancara dengan Bambang Aprianto, Selaku Subag TU Pol PP dan WH Kota Subulussalam. No Kecamatan Lokasi Jenis Kegiatan 1 Simpang Kiri 1. Terminal Terpadu Kota Subulussalam 2. Cafe Remang- Mesum, Khamar dan Maisir No Kecamatan Lokasi Jenis Kegiatan 1 Simpang Kiri 1. Terminal Terpadu Kota Subulussalam 2. Cafe Remang- Mesum, Khamar dan Maisir Jenis Kegiatan 31 Wawancara dengan Bambang Aprianto, Selaku Subag TU Pol PP dan WH Kota Subulussalam. 31 Wawancara dengan Bambang Aprianto, Selaku Subag TU Pol PP dan WH Kota Subulussalam. 159 remang di Suka Makmu dan Bakal Buah. 3. Taman Kota Subulussalam di Lae Oram. Penanggalan 1. Hotel Hermes One 2. Penginapan Jambu Alas 3. Jalan Unaya Penanggalan 4. Cafe di Banataran Kali Lae Kombih Gampong Dasan Raja. 5. Cafe Remang- remang di Cirep-rep 6. Cafe Remang- remang di Lae Mbersih. 7. Cafe Remang- remang di Kuta Tengah 8. Cafe Remang- remang di Sikelang. 9. Cafe Remang- remang di Jontor. 10. Warung- warung di Karoke, Mesum, Kahmar dan Maisir remang di Suka Makmu dan Bakal Buah. 3. Taman Kota Subulussalam di Lae Oram. Penanggalan 1. Hotel Hermes One 2. Penginapan Jambu Alas 3. Jalan Unaya Penanggalan 4. Cafe di Banataran Kali Lae Kombih Gampong Dasan Raja. 5. Cafe Remang- remang di Cirep-rep 6. Cafe Remang- remang di Lae Mbersih. 7. Cafe Remang- remang di Kuta Tengah 8. Cafe Remang- remang di Sikelang. 9. Cafe Remang- remang di Jontor. 10 W Karoke, Mesum, Kahmar dan Maisir 2 2 1. Hotel Hermes One Karoke, Mesum, Kahmar dan Maisir 2. Penginapan Jambu Alas 3. Jalan Unaya Penanggalan 4. Cafe di Banataran Kali Lae Kombih Gampong Dasan Raja. 5. Cafe Remang- remang di Cirep-rep 6. Cafe Remang- remang di Lae Mbersih. 7. Cafe Remang- remang di Kuta Tengah 8. Cafe Remang- remang di Sikelang. 160 *Berdasarkan data dari Kantor Pol PP dan WH Kota Subulussalam tahun 2015. sekitar Objek Wisata Pemandian air terjun Nantampuk Mas, SKPC dan Lae Kombih di Sikelang. 11. Gang Barto di Jln. Swadaya Penanggalan. *Berdasarkan data dari Kantor Pol PP dan WH Kota Subulussalam tahun 2015. 4. Faktor Kesadaran Hukum Masyarakat Yang Masih Rendah Kesadaran hukum masyarakat secara umum masih rendah. Sebagian warga masyarakat menganggap kewajiban taat pada syariat Islam merupakan beban yang memberatkan. Perasaan terbebani ini di samping dipengaruhi oleh kondisi sosial ekonomi yang sulit, juga karena sifat masyarakat lokal berlakunya qanun-qanun jinayat di Subulussalam. Kondisi sosial ekonomi yang sulit banyak berpengaruh pada terjadinya tindak pidana perjudian, perdagangan miras, prostitusi dan lain-lain. Keterlibatan masyarakat dalam mengawal Qanun Khalwat sangat mempengaruhi efektivitas pemeberlakuan qanun tersebut. Dengan segala keterbatsan yang dimiliki oleh WH Kota Subulussalam, keterlibatan aktif masyarakat sangat di butuhkan, karena bagaimanpun juga perbuatan pelanggaran qanun khalwat dilakukan dan terjadi di tengah- tengah kalangan masyarakat Kota Subulussalam. Mengingat pentingnya peran dan kesadaran hukum masyarakat ini, Qanun Nomor 14 Tahun 2003 jauh-jauh hari telah memberikan ruang berupa peran aktif masyarakat dalam mengawal pemberlakuan qanun ini, seperti yang di jelaskan dalam BAB IV Pasal 8-12 sebagai berikut: Masyarakat berperanserta dalam membantu upaya pencegahan dan pemberantasan perbuatan khalwat/mesum; Masyarakat wajib melapor kepada pejabat yang berwenang baik secara lisan maupun tulisan apabila mengetahui adanya pelanggaran terhadap larangan khalwat/mesum; Dalam hal pelaku pelanggaran tertangkap tangan oleh warga masyarakat, maka pelaku beserta barang bukti segera diserahkan kepada pejabat yang berwenang; Pejabat yang berwenang wajib memberikan perlindungan dan jaminan keamanan kepada pelapor sebagaimana dimaksud dalam 161 pasal 8 dan/atau orang yang menyerahkan pelaku sebagaimana dimaksud dalam pasal 9; Warga masyarakat dapat menuntut pejabat yang berwenang sebagaimana dimaksud dalam pasal 9 apabila lalai memberikan perlindungan dan jaminan keamanan bagi pelapor dan/atau orang yang menyerahkan pelaku; Tata cara penuntutan sebagaimana dimaksud dalam pasal 11 dilakukan sesuai peraturan perundang-undangan yang berlaku dan diajukan ke mahkamah. Kesadaran dan pengetahuan tentang qanun inilah yang dirasa masih kurang di tengah-tengah masyarakat. Di beberapa tempat, seperti Sultan Daulad, Runding dan Penanggalan, ketika penulis berdiskusi dengan masyarakat dan pemuda setempat mengatakan, tindak pelanggaran terhadap qanun jinayat, khususnya qanun 14/2003 sebenarnya sangat sering terjadi di daerah mereka masing-masing, masyarakat juga sudah merasa bosan untuk selalu mengingatkan bahwa perbuatan mereka bertentangan dengan qanun jinayat di Aceh. Pada akhirnya masyarakatpun cuek dan berharap petugas WH melakukan patroli ke wilayah mereka.32 Sebenarnaya apabila kita memperhatikan Pasal 8-12 Qanun No. 14 Tahun 2003, seharusnya tidak ada lagi keraguan bagi masyarakat untuk melaporkan setiap orang yang melanggar ketentuan Qanun 14/2003. Karena dalam Pasal 10 dijelaskan pejabat yang berwenang wajib memberikan perlindungan dan jaminan keamanan kepada pelapor sebagaimana dimaksud dalam Pasal 8 dan/atau orang yang menyerahkan pelaku sebagaimana dimaksud dalam Pasal 9. g ( ) g p g g ( ) 33 Dinas Syariat Islam adalah perangkat daerah sebagai unsur pelaksana syariat Islam di lingkungan Pemerintah Daerah Aceh yang berada di bawah Gubernur. Pembentukan Dinas Syariat Islam di Aceh ini merupakan suatu bentuk tindak lanjut terhadap UU No. 44 tahun 1999 tentang Penyelenggaraan Keistimewaan Daerah Istimewa Aceh, untuk meningkatkan kelancaran pelaksanaan tugas operasional Pemerintah Provinsi Daerah Istimewa Aceh di bidang pelaksanaan 32 Wawancara dengan S (nama disamarkan) Warga Gampong Sikelang Kecamatan Penanggalan. HL (nama disamarkan) warga Gampong Rindeng, Kecamatan Rundeng. Wawancara dengan HM (nama disamarkan) warga Gampong Sigrun, Kec. Sultan Daulad. (27 Mei 2015). syariat Islam yang lebih efisien dan berolehguna, maka dipandang sangat perlu untuk membentuk susunan organisasi dan tata kerja Dinas Syariat Islam Provinsi Daerah Istimewa Aceh yang sesuai dengan karakteristik, potensi dan kebutuhan daerah. Lihat, Konsideran Peraturan Daerah Propinsi Daerah Istimewa Aceh No. 33 Tahun 2001 Tentang Pembentukan Susunan Organsasi Dan Tata Kerja Dinas Syariat Islam Propinsi Daerah Istimewa Aceh, Lembaran Daerah Propinsi Daerah Istimewa Aceh Tahun 2001 No. 65. 34 Wawancara dengan Hotma Capah, selaku Sekretaris DSI Kota Subulussalam. 21 April 2015. 35 Hukum Online, “Fiksi Hukum Harus Didukung Sosialisasi Hukum”, http://www. hukumonline.com/berita/baca/hol19115/fiksi-hukum-harus-didukung (diakses pada tanggal 25 Nov. 2015). 34 Wawancara dengan Hotma Capah, selaku Sekretaris DSI Kota Subulussalam. 21 April 2015 37 Pasal 13 Qanun Nomor 14 Tahun 2003 Tentang Khalwat. g 38 Pasal 13 Qanun Nomor 14 Tahun 2003 Tentang Khalwat. 4. Faktor Kesadaran Hukum Masyarakat Yang Masih Rendah Tapi permasalahannya kembali kepada kepedulian dan kesadaran masyarakat juga, apakah masyarakat sudah mengetahui mengenai peransertanya dalam penegakan qanun-qanun syariah di Aceh. Kesadaran hukum masyarakat tidak dapat dipisahkan dari intensitas disseminasi dan penyuluhan yang dilakukan para penyelenggara pemerintah daerah (Aceh) kepada masyarakat. Setiap penyelenggara negara berkewajiban memberikan penyuluhan hukum sebagai bagian dari proses edukasi dan pembudayaan hukum. Penyuluhan hukum merupakan tanggung jawab setiap penyelenggara negara. Kesadaran hukum masyarakat ini sebenarnaya bisa dibagun melalui bentuk/kegiatan sosialisi tentang qanun-qanun jinayat khususnya Qanun Nomor 14 Tahun 2003. Peran Kantor Dinas Syariat Islam33 (DSI) Kota 162 Subulussalam dalam hal ini sangat di harapkan kontribusinya. Hotma Capah selaku Sekretasis DSI Kota Subulussalam menjelaskan bahwa, selama ini DSI telah berupa semaksimal mungkin dalam mensosialisasikan qanun syariat Islam di Aceh, mulai dari remaja-remaja masjid, tokoh masyarakat dan sekolah-sekolah. DSI Kota Subulussalam juga menghimbau kepada seluruh da’i perbatasan yang ada di gampong-gampong untuk ikut mensosialisasikan qanun-qanun syariat Islam di Aceh.34 Lain dengan keterangan pemerintah (DSI), masyarakat menilai bahwa sosialisasi yang dilakukan DSI sekarang ini masih disara sangat kurang. DSI Kota Subulussalam di nilai terkesan setengah hati dalam melakukan sosialisasi kepada masyarakat, terlebih-lebih Parlemen Aceh telah mengesahkan Qanun No 6/2014 tentang Hukum Jinayat pada 27 September 2014, lalu dimasukkan ke dalam lembaran daerah pada 23 Oktober 2014. Qanun itu baru bisa berlaku setelah setahun sejak pengesahan. Pemerintah diamanatkan untuk mensosialiasikan keberadaan Qanun Pidana itu ke segala lapisan masyarakat. DSI Kota Subulussalam dituntut untuk lebih giat lagi dalam melakukan sosialisasi semua qanun syariat Islam di Kota Subulussalah, sehingga asas fiksi hukum yang menganggap semua orang tahu hukum (presumptio iures de iure). Semua orang dianggap tahu hukum, tak terkecuali petani yang tak lulus sekolah dasar, atau warga yang tinggal di pedalaman. Dalam bahasa latin dikenal pula adagium ignorantia jurist non excusat, ketidaktahuan hukum tidak bisa dimaafkan. Seseorang tidak bisa mengelak dari jeratan hukum dengan berdalih belum atau tidak mengetahui adanya hukum dan peraturan perundang-undangan tertentu,35 dapat dijalankan ditengah-tegah masyarakat Kota ubulussalam. Dibutuhkan kesadaran yang tinggi dari masyarakat Kota Subulussalam untuk melaksanakan Syariat Islam, disamping itu juga harus adanya political will pemerintah dan peran serta stakeholders dan tentunya dilakukan secara bertahap atau gradual. Nov. 2015). 163 36 Lihat Bab IV Pasal 8, 9 dan 10 Qanun Nomor 14 Tahun 2003. 36 Lihat Bab IV Pasal 8, 9 dan 10 Qanun Nomor 14 Tahun 2003. A. Pengawasan dan Pembinaan Terhadap Qanun Nomor 14 Tahun 2003 Untuk meningkatkan efektivitas menerapan qanun ini, peran serta masyarakat sangat dibutuhkan, sama halnya dengan Qanun 12, 13 Tahun 2003 dimana masyarakat diberi peran dalam membantu upaya pencegahan dan pemberantasan perbuatan khalwat dengan kewajiban melapor kepada pejabat yang berwenang baik lisan maupun tulisan apabila masyarakat mengetahui adanya orang atau kelompok masyarakat yang melakukan pelanggaran terhadap larangan khalwat/mesum di Aceh. Guna untuk menjamin keamanan masyarakat yang melaporkan tindakan perlanggaran terhadap qanun khalwat/mesum, pejabat bewenang wajib memberikan perlindungan dan jaminan keamanan kepada masyarakat yang melapor.36 Mengenai pengwasan dan pembinaan, penyidikan dan penuntutan sebagai mana tertera dalam bab V dan Bab VI Qanun Nomor 14 Tahun 2003 memiliki kandungan yang sama seperti dua qanun sebelumnya. Untuk melakukan pengawasan dan pembinaan terhadap pelaksanaan qanun ini, Gubernur, Bupati/Walikota membentuk Wilayatul Hisbah; Susunan dan kedudukan Wilayatul Hisbah diatur lebih lanjut dengan surat Keputusan Gubernur dan/atau Bupati/Walikota setelah mendengar pendapat Majelis Permusyawaratan Ulama.37 Dalam melaksanakan fungsi pengawasannya, Pejabat Wilayatul Hisbah sebagaimana dimaksud dalam pasal 13 bila menemukan pelaku pelanggaran terhadap larangan sebagaimana dimaksud dalam pasal 5 dan 6, menyampaikan laporan secara tertulis kepada penyidik; Dalam melaksanakan fungsi pembinaannya, Pejabat Wilayatul Hisbah yang menemukan pelaku jarimah khalwat/mesum dapat memberi peringatan dan pembinaan terlebih dahulu kepada pelaku sebelum menyerahkannya kepada penyidik; Pejabat Wilayatul Hisbah wajib menyampaikan laporan kepada penyidik tentang telah dilakukan peringatan dan pembinaan sebagaimana dimaksud dalam ayat (2).38 Menurut hemat penulis, pengawasan terhadap qanun pidana di Kota Subulussalam baik khamar, masir maupun khalwat selama ini memang belum begitu berjalan dengan baik. Hal ini bukan semata menajdi salah dari pemerintah saja, tetapi semua aspek tatanan kehidupan sosial kemasyarakatan di Kota Subulussalam, baik pemerintah maupun masyarakatnya. Kelemahan dalam melaukan penegakan dan pengawasan yang dilakukan pihak berwenag selama ini, seperti yang telah teridentifikasi di atas seperti keterbatasan 164 anggota WH, sarana dan fasilitas, anggaran dan lainnya sebenarnya bisa disiasati dengan melibatkan peran serta masyarakat dalam menanggulangi terjadinya perbuatan khalwat (mesum) di Kota Subulussalam. Peran masyarakat inilah yang dirasakan masih kurang selama ini dalam proses penegakan qanun-qanun jinayat di Kota Subulussalam.39 Untuk meningkatkan pengawasan terhadap pemberlakuan Qanun Nomor 14 Tahun 2003 di Kota Subulussalam, ada beberapa hal yang harus di perhatikan pemerintah sebagai berikut: 1. Melibatkan Peran Aktif Masyarakat di Gampong-gampong Peran aktif masyarakat memang sangat di harapkan dalam penerapan setiap qanun di Aceh, begitu juga halnya dengan qanun khalwat yang diatur dalam Qanun 14 Tahun 2003. Pemerintah dalam hal ini Dinas Syariat Islam dan Wilayatul Hisbah sudah saatnya meningkatkan hubungan dengan masyarakat dan khususnya para tokoh masyarakat di tiap-tiap gampong yang ada di Kota Subulussalam dan menjadikan mereka menjadi salah unsur pengawas utama setiap qanun di gampongnya masing-masing. Kenapa harus menjadikan tokoh masyarakat gampong menjadi pengawas utama dalam penegakan Qanun Nomor 14/2003? Hal ini dikarenakan para tokoh masyarakat gampong pada dasarnya sangat disegani dan para pelaku khalwat diharapakan berpikir berulangkali ketika hendak melakukan perbuatan yang bertentangan dengan ketentuan Qanun Nomor 14 Tahun 2003. Jika tiap-tiap gampong telah diterapkan sistem pengawasan yang seperti itu, maka prilaku menyimpang masyarakat khususnya khalwat dapat di tekan ketitik yang paling rendah. Selama ini peran masyarakat bukan berarti tidak ada, hanya saja peran masyarakat sangat sulit diwujudkan, karena tidak memiliki ruang yang legal dan tidak memiliki wewenang formal yang dapat dipertanggung jawabkan.40 Dengan demikian apabila ada kontrak kerja antara pemerintah dan tokoh masyarakat gampong, maka di harapkan peran masyarakat tersebut dapat teraktualisasi di lapangan. 39 Wawancara dengan Ust. H. Qaharuddin Kombih, selaku Ketua MPU Kota Subulussalam (20 April 2015). b b k d “ d b h l d l 40 Abubakar dan Anwar, “Strategi dan Hambatan Penerapan Qanun Khalwat dalam Pencegahan Prilaku Khalwat pada Remaja Kota Banda Aceh”, JURNAL PENDIDIKAN SERAMBI ILMU, 2011.68-90. 39 Wawancara dengan Ust. H. Qaharuddin Kombih, selaku Ketua MPU Kota Subulussalam (20 April 2015). 40 Abubakar dan Anwar, “Strategi dan Hambatan Penerapan Qanun Khalwat dalam Pencegahan Prilaku Khalwat pada Remaja Kota Banda Aceh”, JURNAL PENDIDIKAN SERAMBI ILMU, 2011.68-90. 2. Megaktifkan Remaja Masjid Masa remaja adalah masa yang paling rentan, karena pada masa-masa seperti ini anak yang baru menuju kepada dewasa sering mencari tau tentang jati dirinya di tengah- tengah masyarakat. Maka untuk itu perlu adanya suatu konsep dan sistem yang 165 memusatkan pencegahan penyimpangan syariat Islam pada remaja. Remaja masjid dipandang sebagai suatu sarana/sistem dalam membentuk karakter para para remaja, sehingga dengan adanya kegiatan keremajaan di masjid di harapkan dapat memberi pengetahuan yang baik tentang keislaman itu sendiri kepada setiap remaja. 4. Membuat Pos Pengamanan WH Seperti yang telah dijelaskan pada bab sebelumnya, bahwa salah satu kelemahan WH dalam mengawasi pemberlakuan Qanun 14/2003 adalah dikarenakan keterpusatan kantor WH yang hanya ada di pusat kota Subulussalam, walupun sebenarnya idealnya kantor WH ada di setiap kecamatan di Kota Subulussalam, tapi karena angggaran yang terbatas, maka untuk saat ini pemerintah seharusnya membentuk pos-pos pengawasan WH di setiap kecamatan, mengingat antar kecamatan di Kota Subulussalam sangat berjauhan secara geografis dan hal ini dianggap sangat efektif karena anggaran yang dibutuhkan juga tidak terlalu besar. 3. Kerjasama Dengan Pengusaha Seperti Hotel dan Cafe Perbuatan khalwat yang terjadi di Kota Subulussalam kebanyakan terjadi di Hotel (penginapan) dan cafe-cafe yang tersebar di Kota Subulussalam, seperti contoh kasus bahwa pelaku khalwat di tangkap di sebuah penginapan di kawasan Penanggalan dan di salah satu cafe di bantaran Lae Kombih. Maka untuk itu pemerintah seharusnya menjalin kerjasama dengan para pengusaha tersebut supaya tidak memberikan fasilitas kepada mereka yang melakukan perbuatan yang bertentangan dengan Qanun Aceh. C. Kesimpulan Dari penjelasan di atas dapat diambil satu kesimpulan besar bahwa banyak faktor yang melatarbelakangi kurang efektifnya pemberlakuan qanun jinayat di Kota Subulussalam khususnya terhadap Qanun No. 14 Tahun 2003 tentang khalwat, baik dari segi faktor hukum maupun faktor non-hukum. Kedua faktor tersebut tentu saling bertalian satu sama lain yang diharapkan dapat segera meneukan formulasi yang tepat untuk mengatasi masalah-maslaah tersebut. Mengenai adanya dualisme dalam penanganan kasus khalwat di Aceh memang menjadi polemik tersendiri bagi para penegak qanun syariah di Aceh (wilayatul hisbah). Sudah seharusnya para pemangku kepentingan duduk bersama untuk menentukan batasan dan kadar khalwat seperti apa dan bagaimana yang dapat diselesaikan melalui Mahkamah 166 Adat Aceh dan Mahkamah Syar’iyah. Seperi yang sudah dijelaskan di atas bahwa tindak pidana khalwat (mesum) tidak semuanya bisa dilimpahkan ke mahkamah syar’iyah. Ada beberapa faktor yang mempengaruhinya, antara lain: Adat Aceh dan Mahkamah Syar’iyah. Seperi yang sudah dijelaskan di atas bahwa tindak pidana khalwat (mesum) tidak semuanya bisa dilimpahkan ke mahkamah syar’iyah. Ada beberapa faktor yang mempengaruhinya, antara lain: 1. Karena proses hukumnya di selesaikan secara hukum adat berdasarkan kekeluargaan dengan cara menikahkan pelaku khalwat (mesum), membayar denda dan perdamaian; 2. Karena adanya tuntutan dari masyarakat untuk diselesaikan secara damai melalui hukum yang berlaku dalam masyarakat dengan cara mendidik dan membimbing masyarakat yang telah berbuat khalwat (mesum); 3. Karena perbuatan khalwat (mesum) yang dilakukan hanya bersifat ringan dan sedang, kecuali khalwat (mesum) yang sifatnya berat. 3. Karena perbuatan khalwat (mesum) yang dilakukan hanya bersifat ringan dan sedang, kecuali khalwat (mesum) yang sifatnya berat. Di Aceh disamping adanya lembaga hukum formal yang menangani pelanggaran syariat Islam, juga terdapat lembaga informal yaitu lembaga adat yang sangat berpengaruh pada pola kehidupan masyarakat Aceh. Lembaga adat memiliki landasan hukum yakni Qanun No. 9 Tahun 2008 tentan Pembinaan Kehidupan Adat dan Adat Istiadat serta Qanun Nomor 10 Tahun 2008 tentang Lembaga Adat. Dengan demikian, hukum adat yang berlaku dalam masyrakat merupakan salah satu hukum yang membentengi hukum-hukum yang lainnya. Walaupun hukum adat tidak betentangan dengan hukum-hukum atau peraturan perundang-rundangan yang lain yang dibuat oleh pemeintah, karena tujuan hukum adat mendidik masyarakat dengan jalan menasehati dan membimbing masyarakat untuk kembali kepada jalan yang benar, tapi pada kenyataannya hukum adat sedikit banyaknya telah mempengaruhi proses penerapan hukum Islam di Aceh seperti halnya kasus khalwat. li, Jainuddin. Sosiologi Hukum. Jakarta: Sinar Grafika, 2007. D. PUSTAKA RUJUKAN Abu Bakar, Al Yasa’. ”Islam, Hukam dan Masyarakat di Aceh Tajdid Syariat dalam Negara Bangsa”, First International Conference of Aceh and Indian Ocean Studies, 24-27 Februari 2007. Abu Bakar, Al Yasa’. ”Islam, Hukam dan Masyarakat di Aceh Tajdid Syariat dalam Negara Bangsa”, First International Conference of Aceh and Indian Ocean Studies, 24-27 Februari 2007. Abu Bakar, Al Yasa’. Wilayatul Hisbah, Polisi Pamong Praja Dengan Kewenangan Khusus di Aceh. Banda Aceh: Dinas Syari‘at Islam Aceh, 2009. Abu Bakar, Al Yasa’. Wilayatul Hisbah, Polisi Pamong Praja Dengan Kewenangan Khusus di Aceh. Banda Aceh: Dinas Syari‘at Islam Aceh, 2009. Abubakar dan Anwar. “Strategi dan Hambatan Penerapan Qanun Khalwat dalam Pencegahan Prilaku Khalwat pada Remaja Kota Banda Aceh”. JURNAL PENDIDIKAN SERAMBI ILMU, 2011. Abubakar dan Anwar. “Strategi dan Hambatan Penerapan Qanun Khalwat dalam Pencegahan Prilaku Khalwat pada Remaja Kota Banda Aceh”. JURNAL PENDIDIKAN SERAMBI ILMU, 2011. Ali, Jainuddin. Sosiologi Hukum. Jakarta: Sinar Grafika, 2007. Ali, Jainuddin. Sosiologi Hukum. Jakarta: Sinar Grafika, 2007. Ali, Jainuddin. Sosiologi Hukum. Jakarta: Sinar Grafika, 2007. 167 Bahri, Syamsul. “Pelaksanaan Syariat Islam di Aceh Sebagai Bagian dari Wlayah NKRI”. Jurnal Dinamika Hukum Vol. 12 No. 2 Mei 2012. Basri, Hasan. ”A. Hasjmy (1914-1998) Kajian Sosial-Intlektual Pemikiran Tentang Politik Islam”. Disertasi Sps UIN Jakarta, 2008. Danial. “Efektifitas ‘Uqūbat Dalam Qanun No. 14/ 2003 dan DQHR Tentang Khalwat dan Ikhtilath, “Jurnal Asy-Syir’ah” Vol. 45, No. I, 2011. Hukum Online, Fiksi Hukum Harus Didukung Sosialisasi Hukum, http://www. hukumonline.com/berita/baca/hol19115/fiksi-hukum-harus-didukung (diakses pada tanggal 25 Nov. 2015). Jack, Hirwan. “Efektivitas Wilayatul Hisbah Dalam Pencegahan Aliran Sesat Di Aceh”, http://bkpp.acehprov .go.id/ simp egbrr /Art ikel/Artikel05-02-2015/Wilayatul H i s bah _Aceh.pdf , 2. (diakses pada tanggal 16/03/2015). Keputusan Gubernur Aceh No. 01 tahun 2004 tentang kewenangan pembentukan Organisasi Wilayatul Hisbah. Konsideran Peraturan Daerah Propinsi Daerah Istimewa Aceh No. 33 Tahun 2001 Tentang Pembentukan Susunan Organsasi Dan Tata Kerja Dinas Syariat Islam Propinsi Daerah Istimewa Aceh, Lembaran Daerah Propinsi Daerah Istimewa Aceh Tahun 2001 No. 65. Lubis, zulkarnain. Menyambut Qanun Jinayah Nomor 6 Tahun 2014, https://drive. google. Com/file/d/0B5UQVcJ8Df8WenBNSWNfZU VMcTQ/view? pli=1 (diakses 20 Oktober 2015). Mansur, Teuku Muttaqin. Penyelesaian Kasus Mesum Melalui Mahkamah Adat atau Mahkamah Syar’iyah?, http://aceh.tribunnews.com/2013/09/04 /peny elesaian- kasus-mesum-melalui-ma-atau-ms?page=3 (dikases pada tanggal 24 Agustus 2015). Muhammad, Rusjdi Ali. Revitalisasi Syari’at Islam di Aceh: Problem, Solusi dan Implementasi. Ciputat: Logos Wacana Ilmu,2003. r, Haedar. Islam syariat: Reproduksi Salapiyah Idiologis di Indonesia. Bandung: Mizan 2013. Soekanto, Soerjono. Faktor-Faktor yang Mempengaruhi Penegakan Hukum. Jakarta: PT. Raja Grafindo Persada, 2008. Zada, Khamami. “Sentuhan Adat Dalam Pemberlakuan Syariat Islam di Aceh (1514-1903)”, Karsa, Vol. 20 No. 2, Desember 2012. Uddin, Asma. "Religious Freedom Implications of Sharia Implementation in Aceh, Indonesia," University of St. Thomas Law Journal: Vol. 7: Iss. 3 (2010). D. PUSTAKA RUJUKAN Putri Sahadat Bancin, Penyelesaian Tindak Pidana Khalwat/Mesum Berdasarkan Qanun Nomor 14 Tahun 2003 (Suatu Penelitian Di Kota Subulussalam). Banda Aceh: Skripsi Fak. Hukum Univ. Syiah Kuala, 2015. Qanun No. 9 Tahun 2008 tentan Pembinaan Kehidupan Adat dan Adat Istiadat. Qanun Nomor 10 Tahun 2008 tentang Lembaga Adat. Santoso, Topo. Membumikan Hukum Pidana Islam: Penegakan Syariat dalam Wacana dan Agenda. Jakarta: GIP, 2003. 168 Soekanto, Soerjono. Faktor-Faktor yang Mempengaruhi Penegakan Hukum. Jakarta: PT. Raja Grafindo Persada, 2008. Uddin, Asma. "Religious Freedom Implications of Sharia Implementation in Aceh, Indonesia," University of St. Thomas Law Journal: Vol. 7: Iss. 3 (2010). Zada, Khamami. “Sentuhan Adat Dalam Pemberlakuan Syariat Islam di Aceh (1514-1903)”, Karsa, Vol. 20 No. 2, Desember 2012. Soekanto, Soerjono. Faktor-Faktor yang Mempengaruhi Penegakan Hukum. Jakarta: PT. Raja Grafindo Persada, 2008. 169
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Microphthalmia in Texel Sheep Is Associated with a Missense Mutation in the Paired-Like Homeodomain 3 (PITX3) Gene
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Abstract 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: Cord.Droegemueller@itz.unibe.ch * E-mail: Cord.Droegemueller@itz.unibe.ch . These authors contributed equally to the work. . These authors contributed equally to the work. final shape of the eye [1,3]. Genetic studies have identified some of the critical determinants of eye formation. A set of putative transcription factors required for the earliest step of eye development were identified in Drosophila. The involvement of homologous proteins in vertebrate lens development was subsequently elucidated by the characterization of mutations that cause congenital human or murine ocular disorders and their comparison to mutations in model organisms [1]. Analyzing inherited isolated microphthalmia/anophthalmia in humans revealed a total of eight genes (SOX2, PAX6, OTX2, RAX, CHX10, FOXE3, PITX3, CRYBA4) carrying causative mutations [4–11]. For some human non-syndromic microphthalmia cases the underlying mutation has not yet been found [2]. The role of the eight genes during lens development was confirmed by studying spontaneous mouse mutants and genetically engineered mice with more or less similar ocular phenotypes as in human [12]. Besides CRYBA4, encoding a lens specific structural protein, seven of these genes encode transcription factors which are required for appropriate lens formation during eye development [1]. Microphthalmia in Texel Sheep Is Associated with a Missense Mutation in the Paired-Like Homeodomain 3 (PITX3) Gene Doreen Becker1., Jens Tetens2., Adrian Brunner1., Daniela Bu¨ rstel3, Martin Ganter3, James Kijas4 for the International Sheep Genomics Consortium, Cord Dro¨ gemu¨ ller1* 1 Institute of Genetics, Vetsuisse Faculty, University of Berne, Berne, Switzerland, 2 Institute for Animal Breeding and Husbandry, Christian-Albrechts-University Kiel, Kiel, Germany, 3 Clinic for Pigs and Small Ruminants, Forensic Medicine and Ambulatory Service Small Animals, University of Veterinary Medicine Hannover, Hannover, Germany, 4 Commonwealth Scientific and Industrial Research Organisation Livestock Industries, St Lucia, Brisbane, Queensland, Australia Abstract Microphthalmia in sheep is an autosomal recessive inherited congenital anomaly found within the Texel breed. It is characterized by extremely small or absent eyes and affected lambs are absolutely blind. For the first time, we use a genome-wide ovine SNP array for positional cloning of a Mendelian trait in sheep. Genotyping 23 cases and 23 controls using Illumina’s OvineSNP50 BeadChip allowed us to localize the causative mutation for microphthalmia to a 2.4 Mb interval on sheep chromosome 22 by association and homozygosity mapping. The PITX3 gene is located within this interval and encodes a homeodomain-containing transcription factor involved in vertebrate lens formation. An abnormal development of the lens vesicle was shown to be the primary event in ovine microphthalmia. Therefore, we considered PITX3 a positional and functional candidate gene. An ovine BAC clone was sequenced, and after full-length cDNA cloning the PITX3 gene was annotated. Here we show that the ovine microphthalmia phenotype is perfectly associated with a missense mutation (c.338G.C, p.R113P) in the evolutionary conserved homeodomain of PITX3. Selection against this candidate causative mutation can now be used to eliminate microphthalmia from Texel sheep in production systems. Furthermore, the identification of a naturally occurring PITX3 mutation offers the opportunity to use the Texel as a genetically characterized large animal model for human microphthalmia. Citation: Becker D, Tetens J, Brunner A, Bu¨rstel D, Ganter M, et al. (2010) Microphthalmia in Texel Sheep Is Associated with a Missense Mutation in the Paired-Like Homeodomain 3 (PITX3) Gene. PLoS ONE 5(1): e8689. doi:10.1371/journal.pone.0008689 Editor: Alfred Lewin, University of Florida, United States of America Received August 19, 2009; Accepted December 14, 2009; Published January 13, 2010 Received August 19, 2009; Accepted December 14, 2009; Published January 13, 2010 Copyright:  2010 Becker et al. This is an open-access article distributed under the terms of the Creative Commons Attributi unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited. ker 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: This study was financed by research grants of the Swiss National Science Foundation (SNSF), Berne, Switzerland (grant no. 3100A0-116247), and the H.W. Schaumann Stiftung, Hamburg, Germany, to CD. PLoS ONE | www.plosone.org January 2010 | Volume 5 | Issue 1 | e8689 Introduction Human microphthalmia, characterized by small eyes and other ocular abnormalities in newborns, is highly variable with the most severe cases anophthalmic [1,2]. Anophthalmia and microphthalmia cause congenital blindness and affect up to 30 per 100,000 people worldwide [2]. Both anophthalmia and microphthalmia may occur in isolation or as part of a syndrome, as in one-third of cases [2]. Morphological studies showed that impaired lens formation seems to be the major cause of anophthalmia and microphthalmia, although the precise path- ogenesis of these phenotypes remains unknown [3]. Lens development is a critical embryonic period in vertebrate eye development during which many inductive signals are exchanged between the optic vesicle and surface ectoderm [1,3]. This stage is characterized by formation of the lens placode, a thickening of the surface ectoderm that comes into contact with the optic vesicle [1,3]. Coordinated invagination of the lens placode and the optic vesicle results in the formation of the lens vesicle and a double-layered optic cup and provides the first indication of the PLoS ONE | www.plosone.o January 2010 | Volume 5 | Issue 1 | e8689 January 2010 | Volume 5 | Issue 1 | e8689 1 PLoS ONE | www.plosone.org PLoS ONE | www.plosone.org PITX3 Mutation in Sheep Isolated congenital microphthalmia occurs in various mamma- lian species [1,3,13] including the Texel breed of sheep (Figure 1) [14–20]. In Texel sheep microphthalmia behaves as a monogenic autosomal recessive trait [16,20]. An abnormal development of the lens vesicle was shown to be the primary event [19], but so far the underlying genetic defect has not been elucidated. In an initial analysis, we performed a partial genome scan and observed genetic linkage to microsatellite markers on sheep chromosome 23 [21]. After significant extension of the available material we were not able to confirm these results. from our experimentally established pedigree [20,21]. Parents of affected offspring were classified as obligate carriers (n = 133). Partial pedigree records were available and allowed us to establish 73 two-generation spanning sheep families segregating for microphthalmia containing 254 of the individuals collected (Figure S1). Due to incomplete pedigree records, it was impossible to determine if affected sheep from the Texel breeding population share common ancestors and trace back to a single common founder. Besides the experimental mating of an affected male to known disease carriers [20,21], the parents of all available cases were healthy (Figure S1). Sample Collection We collected samples from 134 microphthalmia affected lambs and 269 unaffected sheep from different sheep farms with Texel purebred or Texel/Whiteheaded mutton crossbred sheep, and Figure 1. Microphthalmia phenotype in Texel sheep. (A) Normal newborn lamb. (B) Microphthalmia affected lamb. doi:10.1371/journal.pone.0008689.g001 Subsequently, we applied a homozygosity mapping approach to narrow the region containing the microphthalmia mutation. Based on the reported occurrence of microphthalmia some generations after the introgression of Texel sheep from the Netherlands we hypothesized that the affected sheep most likely were inbred to one single founder animal. Under this scenario the affected lambs were expected to be identical by descent (IBD) for the causative mutation and flanking chromosomal segments. We analyzed the cases for extended regions of homozygosity with simultaneous allele sharing. Only one genome region fulfilled our search criteria (Table S1). On OAR 22 all 23 affected genotyped sheep were homozygous and shared identical alleles over 39 SNP markers corresponding to a 2.4 Mb interval from 24.5–26.9 Mb (Figure 2C). In order to further examine the critical interval defined using SNP data, we genotyped three microsatellite markers derived from the surrounding virtual genome sequence of OAR 22 (Table S2). The analysis of the microsatellite genotypes confirmed an increased homozygosity within the microphthalmia affected lambs compared to the controls. The observed microsatellite heterozy- gosity in the cases ranged from 3–52% compared to 84–92% in the controls (Table S2). A total of 130 out of 134 microphthalmia affected lambs showed homozygosity at microsatellite INRA81 located at 24.9 Mb on the virtual genome map of OAR 22 (Table S1). The obtained LOD score of 10.5 conclusively confirmed the linkage of microphthalmia to the candidate gene region in the family material. Mapping of the Causative Mutation We genotyped approximately 50’000 evenly spaced SNPs from 23 microphthalmia affected lambs and 23 control sheep. A genome-wide significant association was shown for SNPs on sheep chromosome 22 (OAR 22) (Figure 2A). Of the remaining 44,865 SNPs with a genotyping rate .99% and minor allele frequency .5%, eights SNPs, over the region 24,529,089 to 28,147,610 on OAR 22, showed strongest association with the microphthalmia phenotype, with a genome-wide corrected p,0.01 (Figure 2B). No other region in the genome showed genome-wide associated SNPs. The OAR 22 SNP at position 24,952,721 showed strongest association with the microphthalmia phenotype, with an asymp- totic raw p of 7.6610211 and a genome-wide corrected of 9.961025. Introduction The pedigrees were consistent with a monogenic autosomal recessive inheritance. Therefore, we hypothesized that a more comprehensive genome-wide mapping strategy may lead to the identification of the microphthalmia locus in Texel sheep. The ability to assay 50,000 evenly spaced SNP across the sheep genome was recently made possible by development of Illumina’s OvineSNP50 BeadChip. This study demonstrates the effectiveness of using this SNP array for the finescale mapping of an inherited defect when testing only a modest number of cases and controls. Identification and Characterization of Functional Candidate Genes As the resolution of the virtual sheep genome annotation is still far from perfect [22], we inferred the gene annotation of the mapped interval from the corresponding human interval (Figure 2D). The sheep microphthalmia interval corresponds to a segment from 102.2–104.8 Mb on HSA 10. This human interval contains 46 annotated genes and 5 hypothetical loci (NCBI MapViewer, build 36.3). A careful inspection of these genes and Figure 1. Microphthalmia phenotype in Texel sheep. (A) Normal newborn lamb. (B) Microphthalmia affected lamb. doi:10.1371/journal.pone.0008689.g001 January 2010 | Volume 5 | Issue 1 | e8689 PLoS ONE | www.plosone.org 2 PITX3 Mutation in Sheep Figure 2 Genome wide association mapping of microphthalmia (A) Case control whole genome association analysis finds si PITX3 Mutation Figure 2. Genome-wide association mapping of microphthalmia. (A) Case-control whole genome association analysis finds significa association to SNPs on chromosome 22. (B) Single SNP association statistic across OAR 22. (C) Homozygosity mapping of the microphthalm mutation. The analysis of SNP genotypes from affected sheep indicated that they had extended overlapping homozygous regions on OAR 2 (indicated as black blocks). Thus – assuming that it resides on the common haplotype block – the causative mutation is located within a 2.4 M interval on OAR 22 (indicated as red box). All 23 affected sheep had homozygous identical by state intervals with shared alleles between 24.5 Mb an 26.9 Mb. (D) Gene content of the corresponding human chromosome 10 segment. doi:10.1371/journal.pone.0008689.g002 PL S ONE | l 3 J 2010 | V l 5 | I 1 | 868 Figure 2. Genome-wide association mapping of microphthalmia. (A) Case-control whole genome association analysis finds significant association to SNPs on chromosome 22. (B) Single SNP association statistic across OAR 22. (C) Homozygosity mapping of the microphthalmia mutation. The analysis of SNP genotypes from affected sheep indicated that they had extended overlapping homozygous regions on OAR 22 (indicated as black blocks). Thus – assuming that it resides on the common haplotype block – the causative mutation is located within a 2.4 Mb interval on OAR 22 (indicated as red box). All 23 affected sheep had homozygous identical by state intervals with shared alleles between 24.5 Mb and 26.9 Mb. (D) Gene content of the corresponding human chromosome 10 segment. doi:10.1371/journal.pone.0008689.g002 Figure 2. Genome-wide association mapping of microphthalmia. (A) Case-control whole genome association analysis finds significant association to SNPs on chromosome 22. Identification and Characterization of Functional Candidate Genes (B) Single SNP association statistic across OAR 22. (C) Homozygosity mapping of the microphthalmia mutation. The analysis of SNP genotypes from affected sheep indicated that they had extended overlapping homozygous regions on OAR 22 (indicated as black blocks). Thus – assuming that it resides on the common haplotype block – the causative mutation is located within a 2.4 Mb interval on OAR 22 (indicated as red box). All 23 affected sheep had homozygous identical by state intervals with shared alleles between 24.5 Mb and 26.9 Mb. (D) Gene content of the corresponding human chromosome 10 segment. doi:10.1371/journal.pone.0008689.g002 January 2010 | Volume 5 | Issue 1 | e8689 PLoS ONE | www.plosone.org 3 PITX3 Mutation in Sheep database searches of their presumed function revealed PAX2 and PITX3 as possible functional candidate genes within the critical interval at 102.5 Mb and 103.9 Mb on HSA 10, respectively. The paired box gene 2 (PAX2) is a transcription factor expressed during embryonic eye development and mutations in the human and mouse homologs are accompanied by eye malformations [1]. PITX3 encodes the paired-like homeodomain 3 (PITX3) tran- scription factor, expressed during early vertebrate lens develop- ment [23]. Deletions in the promoter of this gene cause abnormal lens development in the aphakia mouse mutant, which has only rudimentary lenses [24,25]. An abnormal development of the lens vesicle was shown to be the primary event in ovine microphthal- mia as well [19]. Therefore, we initially investigated whether mutations in the ovine PITX3 gene might be responsible for the microphthalmia phenotype. As neither mRNA nor genomic sequence data of ovine PITX3 was publicly available, we determined the complete sequence from an ovine BAC clone containing the PITX3 gene. In order to further evaluate PITX3 as a positional candidate, we analyzed its expression in head tissue of d30 sheep fetuses because PITX3 shows a lens specific expression in human, mice, and cattle embryos, respectively. We detected three transcripts, which were verified by direct DNA-sequencing of the RT-PCR products. Full-length PITX3 cDNAs obtained by RACE differed in their 59 untranslated regions, whereas they shared a common open reading frame (Figure 3A). These cDNA sequences were used for comparison with the genomic sequence. These analyses indicated that the ovine PITX3 gene consists of five exons separated by four introns. Identification and Characterization of Functional Candidate Genes In accordance to the human gene annotation, we numbered the two alternatively spliced 59 exons 1 and 1A and the three coding exons 2–4 (Figure 3A). All splice donor/splice acceptor sites conform to the GT/AG rule. The experimentally verified existence of the alternatively spliced exon 1A is in agreement with an identified cattle 59-EST sequence (GenBank: EG705801). The coding sequence of ovine PITX3 displays 97%, 90% and 88% similarity to the bovine, human and murine homologue, respectively. Ovine PITX3 encodes a protein of 302 amino acids containing a DNA binding homeobox domain and, like in other paired-like homeodomain containing proteins, an otp, aristaless, and rax (OAR) domain of 14 amino acids within the C-terminal region. PITX3 along with PITX1 and PITX2 form the PITX/RIEG sub-family of the Paired-like class of homeobox proteins which are highly conserved across species [26,27]. In addition, we obtained cDNA sequence of ovine PAX2 by sequencing RT-PCR products of head tissue of d30 sheep fetuses. We detected a single PAX2 transcript with an open reading frame of 1182 bp encoding a protein of 393 amino acids containing a N- terminal paired box (PAX) domain which is 98% identical to human PAX2 isoform b. within a population sample of Texel sheep was 2.2%. The mutant C-allele was absent from 115 control sheep from 14 diverse sheep breeds. RT-PCR on cDNA and subsequent sequencing confirmed that the PITX3 RNA is normally spliced in d30 fetuses of both available genotypes, homozygous C/C and heterozygous G/C, respectively. The c.338G.C substitution is predicted to result in an exchange of an arginine to a proline in the homeodomain of PITX3 protein sequence (p.R113P). This suggests that the G.C SNP is located in a functionally important region. The potential impact of this microphthalmia associated substitution was evaluated by multiple species alignment of PITX3 homeodomain (Figure 3C). PITX3 belongs to the paired-like homeodomain containing transcription factor group and a bicoid-like subgroup [25]. The bicoid-like homeodomains are characterized by a lysine at position 50 in the homeodomain, which is known to selectively recognize the 39 CC dinucleotide adjacent to the TAAT core DNA binding sequence. The microphthalmia associated p.R113P substitution is located at the perfectly conserved position 52 of the homeodomain. To evaluate possible functional consequences we searched for experimentally determined protein structures of PITX family members. Identification and Characterization of Functional Candidate Genes The structure of the human PITX2 homeodomain-DNA complex showed that the p.R113P mutation is indeed likely to affect the DNA-binding function of PITX3 [27]. The mutant proline shows an uncharged side chain and it cannot act as a hydrogen bond donor (Figure S2). Therefore, as proline disrupts alpha helixes in general, it may act as a structural disruptor in the middle of third alpha helix of the PITX3 homeodomain. Furthermore, the putative consequences of the polymorphisms on the modified proteins were analyzed using two independent computer based amino acid conservation analysis software. These programs are sequence homology-based tools that sort intolerant from tolerant amino acid substitutions and predict whether an amino acid substitution in a protein has a possible phenotypic effect. According to PolyPhen, the PITX3 p.R113P substitution is damaging with a high probability (score 2.6) and the PMut calculation predicts a highly reliable pathological effect. To test for possible PAX2 mutations we sequenced RT-PCR products of the experimentally derived d30 sheep fetuses. No size difference or alternative splicing was detected and analyzing the ORF of four individuals, two homozygous and two heterozygous for the associated INRA81 microsatellite allele, respectively, revealed no polymorphisms affecting the amino acid sequence of PAX2. Discussion Historically, the development of genomic tools for the sheep genome has lagged behind those of other major livestock species such as the cattle and chicken. This has limited the ability to identify genes controlling specific traits of interest [28]. The development of low density microsatellite based linkage maps [29] have lead to the mapping of Mendelian diseases [30–32] and subsequent discovery of mutations underlying at least three genetic diseases in sheep [32–34], however many others remain uncharacterized [35]. The recent development of a set of SNP markers distributed across the sheep genome has changed the prerequisites for such gene mapping projects. For the first time, we demonstrate the use of a genome-wide ovine SNP array for efficient positional cloning of a Mendelian trait in sheep. The result illustrates the power of genome-wide association analysis in domestic animals for the genetic dissection of trait loci [36]. PLoS ONE | www.plosone.org Mutation Analysis We designed PCR primers for the amplification of a 14.5 kb segment containing the entire PITX3 gene and determined the genomic sequence of four microphthalmia affected and four healthy control sheep. This analysis revealed just a single sequence polymorphism (Figure 3A). This non-synonymous SNP located in PITX3 exon 4 (c.338G.C; Figure 3B) showed perfect association to the microphthalmia phenotype (Table 1). All 134 affected sheep were homozygous C/C and all 133 known carriers were heterozygous G/C. Twelve out of 47 healthy full- and half-sibs of microphthalmia affected sheep were also heterozygous G/C. Testing a population sample of 89 healthy Texels which had unknown relatedness revealed none carried the homozygous C/C genotype, while four were presumed carriers with the G/C genotype. Thus the allele frequency of the deleterious C-allele Twenty-three affected and 23 controls were selected for genome-wide association and were genotyped using Illumina’s OvineSNP50 BeadChip. Both, case-control association analysis PLoS ONE | www.plosone.org January 2010 | Volume 5 | Issue 1 | e8689 January 2010 | Volume 5 | Issue 1 | e8689 4 PITX3 Mutation in Sheep Figure 3. PITX3 mutation analysis. (A) Ovine PITX3 gene structure. (B) Electropherograms of the PITX3 c. 338G.C mutation. Representative sequence traces of PCR products amplified from genomic DNA of three sheep with the different genotypes are shown. (C) Multispecies alignment of the evolutionary conserved homeodomain of the PITX3 protein sequence. The p.R113P mutation in PITX3is indicated by an arrow. It affects an arginine residue, which is perfectly conserved from human to zebrafish across all investigated species. The sequences for the alignment were taken from the following accessions: (sheep, NP_005020 (human), XP_589431 (cattle), XP_001499185 (horse), EDL41981 (mouse), NP_062120 (rat), XP_421631 (chicken), NP_001082023 (Xenopus laevis), NP_991238 (Danio rerio). doi:10.1371/journal.pone.0008689.g003 Figure 3. PITX3 mutation analysis. (A) Ovine PITX3 gene structure. (B) Electropherograms of the PITX3 c. 338G.C mutation. Representative sequence traces of PCR products amplified from genomic DNA of three sheep with the different genotypes are shown. (C) Multispecies alignment of the evolutionary conserved homeodomain of the PITX3 protein sequence. The p.R113P mutation in PITX3is indicated by an arrow. It affects an arginine residue, which is perfectly conserved from human to zebrafish across all investigated species. The sequences for the alignment were taken from the following accessions: (sheep, NP_005020 (human), XP_589431 (cattle), XP_001499185 (horse), EDL41981 (mouse), NP_062120 (rat), XP_421631 (chicken), NP_001082023 (Xenopus laevis), NP_991238 (Danio rerio). doi:10.1371/journal.pone.0008689.g003 Figure 3. PITX3 mutation analysis. Figure 3. PITX3 mutation analysis. (A) Ovine PITX3 gene structure. (B) Electropherograms of the PITX3 c. 338G.C mutation. Representative sequence traces of PCR products amplified from genomic DNA of three sheep with the different genotypes are shown. (C) Multispecies alignment of the evolutionary conserved homeodomain of the PITX3 protein sequence. The p.R113P mutation in PITX3is indicated by an arrow. It affects an arginine residue, which is perfectly conserved from human to zebrafish across all investigated species. The sequences for the alignment were taken from the following accessions: (sheep, NP_005020 (human), XP_589431 (cattle), XP_001499185 (horse), EDL41981 (mouse), NP_062120 (rat), XP_421631 (chicken), NP_001082023 (Xenopus laevis), NP_991238 (Danio rerio). doi:10.1371/journal.pone.0008689.g003 Mutation Analysis wildtype Arg113 is located in the middle of an alpha helix and binds to the DNA that is highly likely not possible with the imino acid proline as possible helix disruptor (Figure S2). Thus it is conceivable that this mutation affects the proper folding and stability of the native conformation, possibly inactivating the transcription factor significantly. Apparently, one copy of the PITX3 wildtype allele is sufficient for regulation an undisturbed embryonic lens development, because no visible eye phenotype has been reported in heterozygous carriers of the microphthalmia mutation [19,20]. The p.R113P mutation in microphthalmia affected sheep probably does not affect skeletal muscular development in newborn lambs, although Pitx3 expression during myogenesis has been reported [41]. In mice it was speculated that the lacking PITX3 function in muscles is perhaps completely compensated by the maintenance of PITX2 expression [41]. A microRNA was identified that regulates the maturation and function of midbrain dopaminergic neurons within a negative feedback circuit including the transcription factor PITX3 [42]. The authors propose a role for this feedback circuit in the fine- tuning of dopaminergic behaviors such as locomotion. We found no clinical evidence for conspicuous behavior in microphthalmia affected Texel sheep [20]. p [ ] The PITX3 gene was the most compelling functional candidate in the 2.4 Mb critical interval (Figure 2C). It was not, however, the only plausible positional candidate as the paired box gene 2 (PAX2) is located within the region. While it has been associated with eye malformations in human and mouse, these congenital phenotypes are usually accompanied by kidney anomalies [1] which have not been observed in the ovine form of the disease [20,21]. Sequencing of the coding sequence of PAX2 showed no evidence for disease causing mutations in sheep. However, DNA sequencing revealed a non-synonymous mutation in the PITX3 gene, which is perfectly associated with the microphthalmia phenotype in Texel sheep. We confirmed the presence of this mutation on the genomic DNA and mRNA level. Although we cannot provide functional proof of the causality of the mutation at this time, the wealth of functional data, which are available for the PITX3 gene, strongly supports the hypothesis that p.R113P is indeed the causative mutation. Human patients with point mutations in PITX3 demonstrate congenital cataracts along with anterior segment defects (ASD) in some cases when one allele is affected and microphthalmia with brain malformations when both copies are mutated [10,37]. Mutation Analysis ASD includes a spectrum of developmental abnormalities of the cornea (Peters anomaly), iris (Axenfeld-Rieger syndrome), iridocorneal angle, and ciliary body, which are caused by mutations in the homeodomain of PITX2, encoding a closely related transcription factor [27,38]. A morphological description of microphthalmia in Texel sheep showed an abnormal development of the lens vesicle [19]. These authors also concluded that hereditary microphthal- mia in Texel sheep closely resembles autosomal recessive lens aplasia in mice [39]. A similar phenotype is also the key feature in the Pitx3 loss of function aphakia mouse, where the lens begins to form, but its development is abnormal [24,25]. Morpholino- induced knockdown of pitx3 at early embryonic stages in zebrafish resulted in a lens and retinal phenotype similar to the one seen in the aphakia mouse [40]. Many inherited diseases of domestic animals are analogous to human hereditary disorders and have proven to be valuable for the investigation of the pathogenesis and therapeutic trials of rare human phenotypes with identical molecular basis [43]. Recently, a mutation causing an autosomal recessive inherited disorder characterized by dysplasia of the lens, retinal detachment, persistence of the hyaloid artery, and microph- thalmia in cattle was identified [44]. This large animal model demonstrated the essential role of WFDC1, a small secretory protein specifically expressed in the lens, retina, and optic nerves of embryonic and adult mouse eyes, in mammalian eye development for the first time. Our finding of a PITX3 p.R113P mutation in sheep with microphthalmia provides a valuable large animal model for human medicine and confirms PITX3 as a microphthalmia gene. Our study indicates that coding mutations of the PITX3 gene might also be responsible for rare recessive forms of human isolated microphthalmia. The naturally occurring microphthalmia sheep model may represent a better model for human microphthalmia than aphakia mice because of its eye size and structure and the resulting similarity to the human situation. In addition, the longer life expectancy of sheep allows for investigations over a longer time period. Domestic production animals have the additional advantages of being economic to maintain and having been bred for easy management. Moreover, a high level of expertise in reproductive technology and veterinary care is available for them. The known PITX3 mutations in mouse and humans don’t affect the homeodomain, which is different from that seen in other homeodomain proteins including its close family member, PITX2 [26]. Mutation Analysis (A) Ovine PITX3 gene structure. (B) Electropherograms of the PITX3 c. 338G.C mutation. Representative sequence traces of PCR products amplified from genomic DNA of three sheep with the different genotypes are shown. (C) Multispecies alignment of the evolutionary conserved homeodomain of the PITX3 protein sequence. The p.R113P mutation in PITX3is indicated by an arrow. It affects an arginine residue, which is perfectly conserved from human to zebrafish across all investigated species. The sequences for the alignment were taken from the following accessions: (sheep, NP_005020 (human), XP_589431 (cattle), XP_001499185 (horse), EDL41981 (mouse), NP_062120 (rat), XP_421631 (chicken), NP_001082023 (Xenopus laevis), NP_991238 (Danio rerio). doi:10.1371/journal.pone.0008689.g003 confirmed an increased homozygosity within a total of 134 microphthalmia affected lambs compared to 212 controls. In addition, the availability of segregating families allowed us to confirmed an increased homozygosity within a total of 134 microphthalmia affected lambs compared to 212 controls. In addition, the availability of segregating families allowed us to with genome-wide significance and homozygosity mapping identified the same OAR 22 region for microphthalmia in Texel sheep. The analysis of three flanking OAR 22 microsatellites January 2010 | Volume 5 | Issue 1 | e8689 January 2010 | Volume 5 | Issue 1 | e8689 PLoS ONE | www.plosone.org 5 PITX3 Mutation in Sheep Table 1. Association of the PITX3 mutation with the microphthalmia phenotype. Texel or Texel/Whiteheaded mutton crossbred sheep Other breeds PITX3 microphthalmia affected 1 (n = 134) microphthalmia carrier 2 (n = 133) Control, related 3 (n = 47) Control, unknown relationship (n = 89) Controls (n = 115) c.338G.C GG 12 85 115 (p.R113P) GC 133 35 4 CC 134 1Thirty not closely related cases and 104 affected lambs from the family material. 2Thirty not closely related sheep recorded as parent of affected lambs and 103 parents from the family material. 3Healthy littermates of affected lambs. doi:10.1371/journal.pone.0008689.t001 Texel or Texel/Whiteheaded mutton crossbred sheep 1Thirty not closely related cases and 104 affected lambs from the family material. 2Thirty not closely related sheep recorded as parent of affected lambs and 103 parents from the family material. 3Healthy littermates of affected lambs. doi:10.1371/journal.pone.0008689.t001 confirm the mapping on OAR 22 by linkage analysis. Taken together, the presented statistical support showed that the previously published genetic linkage to microsatellite markers on sheep chromosome 23 using a subset of the family material now did not prove to be correct [21]. PLoS ONE | www.plosone.org Fetal Tissue Tissues were collected in accordance with the animal care and use protocols approved by the Lower Saxony (Germany) governmental animal rights protection authorities (Ref. No. 509.6- 42502/3-04/851). A total of six d30 fetuses were surgically obtained after targeted mating of an affected male to two known disease carriers (Figure S1). Upon collection, fetuses were divided in front (head) and back section and stored in RNAlater (Qiagen). For mutation analysis, PCR products were amplified from four microphthalmia affected and four healthy control sheep using AmpliTaq Gold 360 Master Mix (Applied Biosystems). The sequences of the primers are listed in Table S3. The subsequent re- sequencing of the PCR products was performed after rAPid alkaline phosphatase (Roche) and exonuclease I (New England Biolabs) treatment using both PCR primers with the ABI BigDye Terminator Sequencing Kit 3.1 (Applied Biosystems) on an ABI 3730. Sequence data were analyzed with Sequencher 4.9 (GeneCodes). DNA and RNA extraction. Genomic DNA was isolated from blood or tissue using the Nucleon Bacc2 kit (GE Healthcare). Total RNA was isolated using Trizol reagent according to the manufacturer’s instructions (Invitrogen). Mapping of the microphthalmia mutation. Genomic DNA from 23 cases and 23 controls, mostly selected as discordant sib-pairs, was genotyped using Illumina’s OvineSNP50 BeadChip (49,034 SNPs) [47]. Two small nuclear families segregating for microphthalmia were included to check SNPs for Mendelian inheritance. The results were analyzed with PLINK [48]. After removing 5 SNPs with low genotyping success (failed calls .0.1) the average genotyping rate per individual was 99.9%. A total of 4,164 SNPs had a minor allele frequency (MAF),0.05. A case-control analysis using the options –assoc was applied. Genome-wide corrected empirical p-values were determined applying the max(T) permutation procedure implemented in PLINK with 10,000 permutations. To identify extended homozygous regions with allele sharing across all affected animals the options –homozyg- group and –homozyg-match were applied. All given positions correspond to the virtual sheep genome v 2 [22]. The corresponding human chromosome segment was identified by BLASTN searches of ovine SNP flanking sequences to the human genome sequence. Mapping of the microphthalmia mutation. Genomic DNA from 23 cases and 23 controls, mostly selected as discordant sib-pairs, was genotyped using Illumina’s OvineSNP50 BeadChip (49,034 SNPs) [47]. Two small nuclear families segregating for microphthalmia were included to check SNPs for Mendelian inheritance. The results were analyzed with PLINK [48]. Mutation Analysis The reported p.R113P mutation in ovine microphthalmia lies within the conserved homeodomain and the wildtype arginine is conserved across all PITX3 sequences (Figure 3C). Therefore, it is likely that the mutation p.R113P results in an impaired lens development. In addition, the arginine residue at the similar position of PITX2 was found to be mutated to cysteine in patients with Axenfeld-Rieger syndrome [26,27]. Furthermore, in the PLoS ONE | www.plosone.org January 2010 | Volume 5 | Issue 1 | e8689 January 2010 | Volume 5 | Issue 1 | e8689 6 PITX3 Mutation in Sheep In conclusion, we have identified the p.R113P mutation in the ovine PITX3 gene as the candidate causative mutation for microphthalmia in Texel sheep. In comparison to the commer- cially available DNA test which relies on linked markers [45] this result allows direct genetic testing and improved power to eradicate this common genetic disease from the worldwide Texel breeding population. Our study also provides a defined animal model for similar human hereditary diseases and confirms PITX3 critical function for eye development. 4.0 software (Applied Biosystems). Twopoint parametric linkage analysis under the assumption of microphthalmia segregating as a biallelic autosomal recessive trait with complete penetrance was performed with Merlin software version 1.1.2 [49]. The frequency of the mutant allele in the considered population was unknown and there were no data available that would have made it possible to estimate the frequency in a reliable manner. For the calculations a frequency of 0.001 for the mutant allele was assumed. The LOD score test statistic was used to estimate the proportion of linked families and the corresponding maximum heterogeneity LOD score. Within the available family material, a maximum LOD score of 11.512 would have been possible. Animals Later, the exact ovine genomic structure was determined using the experimentally derived ovine mRNA sequences. Animals Analysis of the ovine PITX3 gene and mutation identification. The BAC clone CH243-315I22 containing the ovine PITX3 gene was identified by BLASTN searches of ovine BAC end sequences to the HSA 10 sequence [22]. The BAC DNA was prepared using the Qiagen Midi plasmid kit according to the modified protocol for BAC clones (Qiagen). The insert sequence of 127 kb of the CH243-315I22 BAC clone has been determined by generating 1,149,790 read pairs using the Chrysalis 36cycles v 2.0 kit on a Genome Analyzer II (Illumina) and de novo assembly with Velvet [50]. Remaining gaps were closed by a primer walking strategy and an ABI 3730 capillary sequencer (Applied Biosystems) until both strands were completely sequenced. The obtained sequence was submitted under accession FN432136 to the EMBL nucleotide database. We collected samples from 134 microphthalmia affected lambs (59 male, 75 female), their available healthy siblings (n = 47), sires (n = 10), and dams (n = 93) from different sheep farms with Texel purebred or Texel/Whiteheaded mutton crossbred sheep and our experimentally established pedigree [20,21]. In addition, we collected 30 Texel sheep recorded as parents of microphthalmia affected offspring. We also collected 89 healthy Texel sheep with unknown relatedness resulting in a total of 403 samples. Furthermore, we sampled unrelated control sheep from different breeds (Whiteheaded mutton (n = 50), Swiss White Alpine (n = 24), Bu¨ndner Oberla¨nder (n = 4), Engadine Red (n = 4), Swiss Black-Brown Mountain (n = 4), Swiss Mirror (n = 4), Valais Blacknose (n = 4), Valais Red (n = 4), Suffolk (n = 1)) and 16 founder animal of the International Mapping Flock (Texel, Coopworth, Perendale, Romney, Merino) [46] for the re-sequencing of PITX3 exon 4. The human reference PITX3 mRNA (GenBank: NM_005029) was used as query in cross-species BLAST searches identifying corresponding bovine EST entries (GenBank: EG705801, EG707045, EG707660). Initially, the putative ovine genomic structure was determined by alignment of bovine PITX3 EST and human PITX3 mRNA to the determined ovine genomic sequence using Spidey [51]. Later, the exact ovine genomic structure was determined using the experimentally derived ovine mRNA sequences. The human reference PITX3 mRNA (GenBank: NM_005029) was used as query in cross-species BLAST searches identifying corresponding bovine EST entries (GenBank: EG705801, EG707045, EG707660). Initially, the putative ovine genomic structure was determined by alignment of bovine PITX3 EST and human PITX3 mRNA to the determined ovine genomic sequence using Spidey [51]. References 18. Roe WD, West DM, Walshe MT, Jolly RD (2003) Microphthalmia in Texel lambs. NZ Vet J 51: 194–195. 1. Graw J (2003) The genetic and molecular basis of congenital eye defects. Nat Rev Genet 4: 876–888. 2. Verma AS, Fitzpatrick DR (2007) Anophthalmia and microphthalmia. Orphanet J Rare Dis 2: 47. 19. van der Linde-Sipman JS, van den Ingh TS, Vellema P (2003) Morphology and morphogenesis of hereditary microphthalmia in Texel sheep. J Comp Pathol 128: 269–275. 3. Chow RL, Lang RA (2001) Early eye development in vertebrates. Annu Rev Cell Dev Biol 17: 255–296. 20. Tetens J, Bu¨rstel D, Ganter M, Do¨pke C, Mu¨ller G, et al. (2007) Untersuchungen zur kongenitalen Mikrophthalmie beim Texelschaf. Tiera¨rztl Prax 35(G): 211–218. 4. Fantes J, Ragge NK, Lynch SA, McGill NI, Collin JR, et al. (2003) Mutations in SOX2 cause anophthalmia. Nat Genet 33: 461–462. 21. Tetens J, Ganter M, Mu¨ller G, Dro¨gemu¨ller C (2007) Linkage mapping of ovine microphthalmia to chromosome 23, the sheep orthologue of human chromo- some 18. Invest Ophthalmol Vis Sci 48: 3506–3515. 5. Glaser T, Jepeal L, Edwards JG, Young SR, Favor J, et al. (1994) PAX6 gene dosage effect in a family with congenital cataracts, aniridia, anophthalmia and central nervous system defects. Nat Genet 7: 463–471. 6. Ragge NK, Brown AG, Poloschek CM, Lorenz B, Henderson RA, et al. (2005) Heterozygous mutations of OTX2 cause severe ocular malformations. Am J Hum Genet 76: 1008–1022. 22. Dalrymple BP, Kirkness EF, Nefedov M, McWilliam S, Ratnakumar A, et al. (2007) Using comparative genomics to reorder the human genome sequence into a virtual sheep genome. Genome Biol 8: R152. 23. Medina-Martinez O, Shah R, Jamrich M (2009) Pitx3 controls multiple aspects of lens development. Developmental Dynamics, early view DOI 10.1002/ dvdy.21924. 7. Voronina VA, Kozhemyakina EA, O’Kernick CM, Kahn ND, Wenger SL, et al. (2004) Mutations in the human RAX homeobox gene in a patient with anophthalmia and sclerocornea. Hum Mol Genet 13: 315–322. 24. Semina EV, Murray JC, Reiter R, Hrstka RF, Graw J (2000) Deletion in the promoter region and altered expression of Pitx3 homeobox gene in aphakia mice. Hum Mol Genet 9: 1575–1585. 8. Percin E, Ploder LA, Yu JJ, Arici K, Horsford DJ, et al. (2000) Human microphthalmia associated with mutations in the retinal homeobox gene CHX10. Nat Genet 25: 397–401. 25. Acknowledgments The authors are grateful that Hauke Peters and all other sheep breeders donated samples and shared pedigree data. The authors would like to thank Brigitta Colomb for expert technical assistance and Ulrich Baumann for helpful discussions. Supporting Information Figure S1 Pedigrees of families in study. Filled symbols represent microphthalmia affected sheep, open symbols represent normal sheep. DNA samples were available from numbered sheep. Two females, which appear as mothers in different families, are marked with a rectangle. Sheep that were used for the initial whole genome association study are marked with an asterisk. The genotypes for the PITX3 c.338G.C mutation are given below the symbols. A single affected and two healthy offspring from the experimental family 73 were used to obtain d30 fetuses (E4-E6). Found at: doi:10.1371/journal.pone.0008689.s001 (0.04 MB PDF) Figure S1 Pedigrees of families in study. Filled symbols represent microphthalmia affected sheep, open symbols represent normal sheep. DNA samples were available from numbered sheep. Two females, which appear as mothers in different families, are marked with a rectangle. Sheep that were used for the initial whole genome association study are marked with an asterisk. The genotypes for the PITX3 c.338G.C mutation are given below the symbols. A single affected and two healthy offspring from the experimental family 73 were used to obtain d30 fetuses (E4-E6). Found at: doi:10.1371/journal.pone.0008689.s001 (0.04 MB PDF) Table S1 Results of homozygosity mapping. Found at: doi:10.1371/journal.pone.0008689.s003 (1.66 MB PDF) Table S2 Microsatellites. Found at: doi:10.1371/journal.pone.0008689.s004 (0.01 MB PDF) Table S3 Primer sequences for the amplification of ovine PITX3 gene. g Found at: doi:10.1371/journal.pone.0008689.s005 (0.02 MB PDF) Figure S2 Impact of the microphthalmia associated PITX3 substitution. (A) Alignment of human PITX3 (NP_005020) and human PITX2 isoform a, b, and c (NP_700476; NP_700475; NP_000316) protein sequences. The conserved domains (homeo- domain and the C-terminal OAR domain) are indicated in bold face type. The mutated arginine at PITX3 position 113 is shown in red. (B) Structure of the PITX2 homeodomain-DNA complex (Code 1YZ8 taken from PDB [54]). The tertiary structure of the PITX2 homeodomain (96% identical to sheep PITX3 homeodo- main) is composed of three alpha helices (shown in light grey). The wildtype PITX3 Arg113 equivalent PITX2 Arg90 (shown with carbon atoms in pink) is located in the third alpha helix and binds with its positively charged capped guanidinium group (shown with blue nitrogen atoms) via two hydrogen bonds (indicated in black) to the DNA backbone (oxygens are shown in red, nitrogen atoms in blue, and carbon atoms in green and light blue). Figure S2 Impact of the microphthalmia associated PITX3 substitution. Fetal Tissue After removing 5 SNPs with low genotyping success (failed calls .0.1) the average genotyping rate per individual was 99.9%. A total of 4,164 SNPs had a minor allele frequency (MAF),0.05. A case-control analysis using the options –assoc was applied. Genome-wide corrected empirical p-values were determined applying the max(T) permutation procedure implemented in PLINK with 10,000 permutations. To identify extended homozygous regions with allele sharing across all affected animals the options –homozyg- group and –homozyg-match were applied. All given positions correspond to the virtual sheep genome v 2 [22]. The corresponding human chromosome segment was identified by BLASTN searches of ovine SNP flanking sequences to the human genome sequence. RT-PCR. Aliquots of 1 mg total RNA were reverse transcribed into cDNA using 20 pmol (T)24V primer and Superscript III reverse transcriptase (Invitrogen). Two microliters of the cDNA were used as a template in PCR. RT-PCR reactions were performed as described above and primer sequences are given in Table S4. The human reference PAX2 mRNA (GenBank: NM_003987) was used as query in cross-species Spidey alignment to identifying corresponding cattle sequences. Primers for RT-PCR in sheep were derived from the bovine PAX2 59UTR and 39UTR sequence. Isolation of full length cDNA for the ovine PITX3 gene was achieved by a rapid amplification of cDNA ends (RACE) protocol with the FirstChoice RNA ligase- mediated (RLM)-RACE kit (Applied Biosystems). The ovine cDNA sequences were deposited in the EMBL nucleotide database (PITX3: FN432137, FN432138, and FN432139; PAX2: FN600706). Linkage analysis in candidate genes. Microsatellite markers were amplified using the Multiplex PCR Kit (Qiagen) and fragment size analyses were determined on an ABI 3730 capillary sequencer (Applied Biosystems) and analyzed with the GeneMapper PLoS ONE | www.plosone.org January 2010 | Volume 5 | Issue 1 | e8689 7 PITX3 Mutation in Sheep Found at: doi:10.1371/journal.pone.0008689.s002 (0.53 MB PDF) Supporting Information (A) Alignment of human PITX3 (NP_005020) and human PITX2 isoform a, b, and c (NP_700476; NP_700475; NP_000316) protein sequences. The conserved domains (homeo- domain and the C-terminal OAR domain) are indicated in bold face type. The mutated arginine at PITX3 position 113 is shown in red. (B) Structure of the PITX2 homeodomain-DNA complex (Code 1YZ8 taken from PDB [54]). The tertiary structure of the PITX2 homeodomain (96% identical to sheep PITX3 homeodo- main) is composed of three alpha helices (shown in light grey). The wildtype PITX3 Arg113 equivalent PITX2 Arg90 (shown with carbon atoms in pink) is located in the third alpha helix and binds with its positively charged capped guanidinium group (shown with blue nitrogen atoms) via two hydrogen bonds (indicated in black) to the DNA backbone (oxygens are shown in red, nitrogen atoms in blue, and carbon atoms in green and light blue). Table S4 Primer sequences for the amplification of ovine PITX3 and PAX2 cDNA. Found at: doi:10.1371/journal.pone.0008689.s006 (0.01 MB PDF) Author Contributions Conceived and designed the experiments: CD. Performed the experiments: DB JT AB DB. Analyzed the data: DB JT AB JK CD. Contributed reagents/materials/analysis tools: MG JK. Wrote the paper: DB JT CD. Conceived and designed the experiments: CD. Performed the experiments: DB JT AB DB. Analyzed the data: DB JT AB JK CD. Contributed reagents/materials/analysis tools: MG JK. Wrote the paper: DB JT CD. 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Optimizing photo-Fenton like process for the removal of diesel fuel from the aqueous phase
Journal of environmental health science & engineering
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5,275
Abstract Background: In recent years, pollution of soil and groundwater caused by fuel leakage from old underground storage tanks, oil extraction process, refineries, fuel distribution terminals, improper disposal and also spills during transferring has been reported. Diesel fuel has created many problems for water resources. The main objectives of this research were focused on assessing the feasibility of using photo-Fenton like method using nano zero-valent iron (nZVI/UV/H2O2) in removing total petroleum hydrocarbons (TPH) and determining the optimal conditions using Taguchi method. Results: The influence of different parameters including the initial concentration of TPH (0.1-1 mg/L), H2O2 concentration (5-20 mmole/L), nZVI concentration (10-100 mg/L), pH (3-9), and reaction time (15-120 min) on TPH reduction rate in diesel fuel were investigated. The variance analysis suggests that the optimal conditions for TPH reduction rate from diesel fuel in the aqueous phase are as follows: the initial TPH concentration equals to 0.7 mg/L, nZVI concentration 20 mg/L, H2O2 concentration equals to 5 mmol/L, pH 3, and the reaction time of 60 min and degree of significance for the study parameters are 7.643, 9.33, 13.318, 15.185 and 6.588%, respectively. The predicted removal rate in the optimal conditions was 95.8% and confirmed by data obtained in this study which was between 95-100%. Conclusion: In conclusion, photo-Fenton like process using nZVI process may enhance the rate of diesel degradation in polluted water and could be used as a pretreatment step for the biological removal of TPH from diesel fuel in the aqueous phase. Keywords: Photo-Fenton like process, nZVI, Taguchi method, TPH, Diesel fuel Keywords: Photo-Fenton like process, nZVI, Taguchi method, TPH, Diesel fuel lesser amount of aromatic compounds and includes mol- ecules containing sulfur, nitrogen and metal oxides [2]. The toxicity of diesel fuel is mostly due to BTEX aro- matic hydrocarbons. The carcinogen property of diesel fuel is due to C10 and C20 alkenes and alkylated benzene. Therefore, developing an efficient method for the re- moval of diesel fuel from contaminated water resources is very crucial [4]. Different physical, chemical, and bio- logical techniques have been used to degrade the contami- nated soils and water [6-10]. The aromatic hydrocarbons with high toxic nature cannot be degraded simply by conventional treatment methods [11]. Most conventional techniques such as evaporation, oil phase separation, filtration, dissolved air flotation, coagulation, flocculation, absorption, and ultrafiltration only transfer pollutions from one media to another [12,13]. RESEARCH ARTICLE Open Access * Correspondence: mahdifarzadkia@gmail.com 2Department of Environmental Health Engineering, Iran University of Medical Sciences, Tehran, Iran Full list of author information is available at the end of the article Abstract Therefore, another method should be developed to remove hydrocarbon pol- lutions [14]. In other words, these methods do not remove Optimizing photo-Fenton like process for the removal of diesel fuel from the aqueous phase Mansooreh Dehghani1, Esmaeel Shahsavani1, Mahdi Farzadkia2* and Mohammad Reza Samaei © 2014 Dehghani et al.; licensee BioMed Central Ltd. This is an Open Access article distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/by/2.0), which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly credited. JOURNAL OF ENVIRONMENTAL HEALTH SCIENCE & ENGINEERING JOURNAL OF ENVIRONMENTAL HEALTH SCIENCE & ENGINEERING Dehghani et al. Journal of Environmental Health Science & Engineering 2014, 12:87 http://www.ijehse.com/content/12/1/87 Dehghani et al. Journal of Environmental Health Science & Engineering 2014, 12:87 http://www.ijehse.com/content/12/1/87 JOURNAL OF ENVIRONMENTAL HEALTH SCIENCE & ENGINEERING Chemicals and analytical method In this research, regular diesel fuel from a gas station in Shiraz was used as a pollutant model. Water-oil emulsion was prepared using emulsifier SDS. Nano zero-valent iron (nZVI) was supplied by Iran Oil Industry Research Center. The rest of the chemicals were purchased from Merck (Germany). UV lamp, 125 Kw, 247.3 nm wave length, (ARDA, Netherland) was used as the radiation source. In this study, photo-Fenton like process which is based on electrochemical system using nano zero-valent iron (nZVI) and peroxide hydrogen as reductive and oxidative reagents, respectively, was used. In photo-Fenton process, the formation of ferrous ion reduces the process effective- ness and eventually it will halt the reaction. In photo- Fenton like process (Fe°/UV/H2O2), ferrous (Fe+2) and ferric (Fe+3) are formed, respectively. These ions practic- ally enhance the efficiency of the process [19]: The standard method No. 8015B of Environment Pro- tection Agency at the United States (EPA) was used to detect petroleum hydrocarbons especially gasoline range organics (GROs, C6-C10) with the boiling point of 60- 170°C, and diesel fuel range organics (DROs, C10-C28) with the boiling point of 170-430°C [20]. DRO was prepared using liquid-liquid extraction method with n- hexane as solvent [21]. The recovery of the sample from the aqueous phase with this method of extraction was 98%. The method provides the chromatographic condi- tions for detecting non-halogen volatile organic com- pounds. Diesel standard was supplied by Merck (Germany, analytical grade GC). For TPH detection in diesel fuel in the aqueous phase a Varian Model gas chromatography (Australia) system with fused silica capillary columns (CP- SIL 5 CB column model, 30 m*0.25 mm, 25 um) was calibrated and tested prior to injection of the samples. A Flame Ionization Detector (FID) was used to detect TPH in the samples. The injector and detector temperatures were set at 200°C and 340°C, respectively. The column temperature was maintained at 45°C for 3 min, and then increased to 275°C (at a rate of 12°C/min), where it was kept for 12 min. Introduction In recent years, pollution of soil and groundwater caused by fuel leakage from old underground storage tank, re- fineries, fuel distribution terminals, improper disposal and also spills during transferring, has been reported [1-3]. The accidental spills of more than 2 million tons of refined oil products per year into the environment are a worldwide problem [4]. Large amounts of benzene, toluene, ethyl benzene, and xylenes (BTEX) have been detected in polluted water resources [5]. The leaching of petroleum hydrocarbons such as diesel fuel into water resources causes many serious environmental problems [6]. Diesel fuel consists of a complex compound includ- ing paraffin, olefins, aliphatic hydrocarbons, as well as a * Correspondence: mahdifarzadkia@gmail.com 2Department of Environmental Health Engineering, Iran University of Medical Sciences, Tehran, Iran Full list of author information is available at the end of the article Page 2 of 7 Dehghani et al. Journal of Environmental Health Science & Engineering 2014, 12:87 http://www.ijehse.com/content/12/1/87 the pollutants, but generally produce highly concentrated wastes in lower volume. In addition, these methods have lower efficiency in removing smaller oil droplets and emulsions [15]. Nowadays, biological and bioremediation techniques are used to treat polluted soils and sewages, but these methods need improvement for enhancing the enzymatic activity in microbial population [16]. Therefore, it is very important to use advanced methods in order to remove oily compounds from groundwater resources. Advanced oxidation processes (AOPs) is an efficient environment-friendly method in which hydroxyl radi- cals (OH°) are used to oxidize recalcitrant organic pol- lutants and convert them to harmless end-products such as H2O and CO2 [17,18]. to (i) evaluate the feasibility of using photo-Fenton like method (nZVI/UV/H2O2) in removing total petroleum hydrocarbons (TPH) and (ii) determine the optimal condi- tions using Taguchi method so that the standard limit can be achieved by further complementary treatment. Chemicals and analytical method The flow rate of helium carrier gas and Fe∘þ H2O2 →Fe2þ þ OH∘þ OH− ð1Þ Fe2þ þ H2O2 →Fe3þ þ OH∘þ OH− ð2Þ Organicmaterial þ OH∘→Oxidizedcompound þ H2O ð3Þ Fe∘þ H2O2 →Fe2þ þ OH∘þ OH− ð1Þ Fe2þ þ H2O2 →Fe3þ þ OH∘þ OH− ð2Þ Organicmaterial þ OH∘→Oxidizedcompound þ H2O ð3Þ ð1Þ ð2Þ Organicmaterial þ OH∘→Oxidizedcompound þ H2O ð3Þ ð3Þ Previous studies in Iran have reported soil and water pollution caused by petroleum compounds near Shiraz, Esfahan and Tehran refineries [6]. Since Fars (in Southern part of Iran) enjoys a top rank in oil refinery, storage, and distribution of oil products in the country in recent years, there is a concern regarding the effect of petroleum hy- drocarbons in water resources on people's health and the environment. Therefore, the objectives of the study were 5 10 15 20 25 30 Minutes -0.10 0.00 0.25 0.50 0.75 1.00 Volts X: Y: 0.0765 Minutes -0.0000400 Volts WI:8 WI:16 Figure 1 GC chromatograms for the initial sample of diesel fuel. Figure 1 GC chromatograms for the initial sample of diesel fuel. 5 10 15 20 25 30 Minutes -0.10 0.00 0.25 0.50 0.75 1.00 Volts X: Y: 0.6123 Minutes -0.000131 Volts FP+ FP- WI:8 WI:16 Figure 2 GC chromatograms for the treated sample of diesel fuel. Dehghani et al. Journal of Environmental Health Science & Engineering 2014, 12:87 Page 3 of 7 http://www.ijehse.com/content/12/1/87 Page 3 of 7 Dehghani et al. Journal of Environmental Health Science & Engineering 2014, 12:87 http://www.ijehse.com/content/12/1/87 5 10 15 20 25 30 Minutes -0.10 0.00 0.25 0.50 0.75 1.00 Volts X: Y: 0.6123 Minutes -0.000131 Volts FP+ FP- WI:8 WI:16 Figure 2 GC chromatograms for the treated sample of diesel fuel. Figure 2 GC chromatograms for the treated sample of diesel fuel. nitrogen make up gas, were set at 5-7 mL/min and 30 mL/ min, respectively. The detection limit for the sample was 0.01 mg/L. GC chromatograms for the initial and treated samples of diesel fuel are presented in Figures 1 and 2. TPH reduction rate was analyzed using QT4 software. The most effective parameters for removing diesel fuel from the aqueous phase, the rate of efficiency, and the level of precision, and optimal conditions were determined. The effects of nZVI particles concentration According to Figure 4, the optimal nZVI concentration and the reduction rate of TPH were 20 mg/L and 77.7%, respectively. The TPH reduction rate in diesel fuel in- creased from 65.9 to 77.7% as the nZVI concentration increased from 10 to 20 mg/L. However, an increase in the concentration from 20 to 100 mg/L, caused a decrease in the reduction rate of diesel degradation (58.3%). In the current study (nZVI/UV/H2O2), increasing nZVI increased the production rate of hydroxyl radical. Due to the fact that by increasing nZVI the metal active surface was increased to make better contact with peroxide hydrogen and UV ra- diation. The reduction rate was decreased by increasing the nZVI concentration above the optimal concentration; in this condition the process is in favor of producing more ferrous ions rather than producing hydroxyl radical [15]. Therefore, TPH reduction rate increased with nZVI con- centration up to a specific level (10-20 mg/L) and then began to decrease (20-100 mg/L). The reduction of the pollutant is basically proportional to the formation of hy- droxyl radicals on the surface of the catalyst. Due to high turbidity of the solution at higher concentrations of nZVI (20-100 mg/L), UV radiation cannot penetrate into the Sample preparation Diesel fuel (commercial grade) at the concentration of 100 mL was added gradually to 1 liter of distilled water using 2.5 mL of 0.1 g/L SDS as emulsifier. The water- diesel emulsion was mixed using Jar test at the speed of 200 rpm for 24 h. In order to separate non-dispersed diesel in oil-water emulsion, the solution was kept at rest for 1 h. Then, the emulsion was transferred to a separ- ator funnel and the supernatant was removed and the rest was collected and passed through Whatman filter paper (20 μm in diameter). The prepared emulsion was used as stock solution and different concentrations 0.1- 1 mg/L were prepared by diluting with distilled water. The specification of photochemical reactor is shown in Figure 3. One liter samples were used in 2 liter volume reactor. Test was performed in a closed reactor with ad- justable mixer. The UV radiation source, 1020 μw/Cm2, was embedded at the bottom of reactor. The UV lamp was protected by Quartz tube. A thermometer was placed inside the photochemical cell to record the temperature. The temperature inside the reactor was kept at the range of 24-26°C using cooling water recirculation system. Experimental setup Five parameters including the initial concentration of TPH, H2O2 concentration, nZVI concentration, pH, and reaction time were selected in 4 levels to analyze the re- moval efficiency of TPH in diesel fuel in the aqueous phase (Table 1). Taguchi’s statistical method and Qualitek-4 (QT4) software were used for the experimental design. Using this software, 16 tests were designed randomly to reduce the errors. Two replications were done for each sample. Table 1 Parameters and the selected levels of photo-Fenton like treatment process (nZVI/UV/H2O2) for the reduction of TPH from diesel fuel in the aqueous phase Table 1 Parameters and the selected levels of photo-Fenton like treatment process (nZVI/UV/H2O2) for the reduction of TPH from diesel fuel in the aqueous phase Variables level Level 1 Level 2 Level 3 Level 4 Concentration TPH (mg/L) 0.1 0.4 0.7 1 nZVI Concentration (mg/L) 10 20 40 100 H2O2 Concentration (mmole/L) 5 10 15 20 pH 3 5 7 9 Reaction time (min) 15 60 90 120 Table 1 Parameters and the selected levels of photo-Fenton like treatment process (nZVI/UV/H2O2) for the reduction of TPH from diesel fuel in the aqueous phase Dehghani et al. Journal of Environmental Health Science & Engineering 2014, 12:87 http://www.ijehse.com/content/12/1/87 Page 4 of 7 Dehghani et al. Journal of Environmental Health Science & Engineering 2014, 12:87 http://www.ijehse.com/content/12/1/87 Figure 3 Photochemical reactor. and biological processes especially advanced oxidation efficiency. In addition, pH has a considerable effect on the solubility of petroleum hydrocarbon, catalyst surface charge, as well as the mechanism of hydroxyl radical production [11]. The Fenton and photo-Fenton reactions depend on the pH. The feasibility of hydroxyl radical pro- duction and oxidation efficiency also depend on pH [23]. The reduction rate of TPH reduced in higher pH, because of the formation of ferric hydroxide which in turn reduced the intensity of UV radiation and the potential of hydroxyl radical production as well [24]. Additionally, high pH values intensify the formation of HO2 −2 ions and de- struction of hydroxyl radicals by carbonate and bicar- bonate ions (Figure 5). solution and mostly adsorbed by the particles. In addition, the rate of H2O2 photolysis depends on the intensity of UV radiation. Therefore, lower UV radiation results in redu- cing the hydroxyl radical production as well as the deg- radation rate [20]. The additional Fe ions react with the hydroxyl radical and therefore reduce the efficiency of the process [22]. pH effect D Data regarding the effect of pH shows that as pH in- creased from 3.0 to 9, the rate of TPH reduction de- creased. Based on the data obtained in the present study, pH of 3 is optimal for TPH degradation. The reduction rate was more than 83% in this case. Generally, pH is one of the most important factors affecting chemical The effect of H2O2 concentration The effect of H2O2 concentration mostly tend toward a semi-Fenton process and producing more hydroxyl radicals [26]. Many advanced processes such as ultrasonic and photochemical reaction were used to remove organic pollutants (e.g. diesel) from aqueous solution [27-30]. 2 2 The effect of H2O2 concentration on TPH reduction was shown in Figure 7. Based on the data obtained in the present study, H2O2 concentration of 5 mmol/L is opti- mal for diesel fuel degradation and the reduction rate was 81.7%. As H2O2 concentration increased from 5 to 20 mmol/L, TPH reduction rate decreased to 42.5%. Add- ing extra H2O2 concentration (more than 5 mmol/L) will act as the scavenger for hydroxyl radical and form HO2° which has lower oxidative ability and longer lifetime comparing to OH° [15]. The decomposition of hydrogen peroxide into oxygen and water occurred at H2O2 concen- tration of more than optimal. Therefore, it can be concluded that high concentrations of H2O2 act as an in- hibitor for the formation of hydroxyl radicals' formation and consequently reduced the efficiency of the process [22]. Adding hydrogen peroxide to nZVI considerably re- duces production of free electrons and the reaction will The effects of reaction time h ff f The effect of reaction time on TPH reduction rate from diesel fuel in the aqueous phase was studied at four levels 15, 60, 90, and 120 min (Figure 6). Data regarding the effect of reaction time shows that as the time in- creased from 15 to 60 min, the rate of reduction in- creased by 75%. However, from 60 to 120 min there was a reduction rate in TPH degradation (63.5%). Based on our findings, 60 min reaction time is optimal for TPH degradation (Figure 6). The optimization of reaction time is one of the most important parameters in studying the removal processes. Basically, an optimal contact time is a very important parameter for any chemical reactions. At equilibrium, TPH degradation reached a plateau. If the re- action time exceeds equilibrium, the process will be no longer economical [25]. Coelho et al. showed that the op- timal reaction time for the removal of TPH from petrol- eum wastewater was equal to one hour [26]. Figure 4 The effect of nZVI on the reduction rate of TPH from diesel fuel in the aqueous phase using photo- Fenton like process. Figure 4 The effect of nZVI on the reduction rate of TPH from diesel fuel in the aqueous phase using photo- Fenton like process. Figure 4 The effect of nZVI on the reduction rate of TPH from diesel fuel in the aqueous phase using photo- Fenton like process. Dehghani et al. Journal of Environmental Health Science & Engineering 2014, 12:87 Page 5 of 7 http://www.ijehse.com/content/12/1/87 Dehghani et al. Journal of Environmental Health Science & Engineering 2014, 12:87 http://www.ijehse.com/content/12/1/87 Dehghani et al. Journal of Environmental Health Science & Engineering 2014, 12:87 http://www.ijehse.com/content/12/1/87 Page 5 of 7 Figure 5 The effect of pH on the reduction rate of TPH from diesel fuel in the aqueous phase using photo- Fenton like process. Figure 7 The effect of H2O2 on the reduction rate of TPH from diesel fuel in the aqueous phase using photo- Fenton like process. Figure 7 The effect of H2O2 on the reduction rate of TPH from diesel fuel in the aqueous phase using photo- Fenton like process. Figure 7 The effect of H2O2 on the reduction rate of TPH from diesel fuel in the aqueous phase using photo- Fenton like process. The effects of reaction time h ff f Figure 7 The effect of H2O2 on the reduction rate of TPH from diesel fuel in the aqueous phase using photo- Fenton like process. Figure 5 The effect of pH on the reduction rate of TPH from diesel fuel in the aqueous phase using photo- Fenton like process. Figure 7 The effect of H2O2 on the reduction rate of TPH from diesel fuel in the aqueous phase using photo- Fenton like process. The effects of initial TPH concentration Wang S: A comparative study of Fenton and Fenton-like reaction kinetics in decolorization of wastewater. Dyes Pigm 2008, 76:714–720. 6. Wang S: A comparative study of Fenton and Fenton-like reaction kinetics in decolorization of wastewater. Dyes Pigm 2008, 76:714–720. 7. Dehghani M, Naseri S, Zamanian Z: Biodegradation of alachlor in liquid and soil cultures under variable carbon and nitrogen sources by bacterial consortium isolated from corn field soil. J Environ Health Sci Eng 2013, 10(1):21. doi:10.1186/1735-2746-10-21. 7. Dehghani M, Naseri S, Zamanian Z: Biodegradation of alachlor in liquid and soil cultures under variable carbon and nitrogen sources by bacterial consortium isolated from corn field soil. J Environ Health Sci Eng 2013, 10(1):21. doi:10.1186/1735-2746-10-21. Conclusion Our results suggest that the photo-Fenton like process (nZVI/UV/H2O2) can be used as a pre-treatment step for the biological removal of TPH from diesel fuel in the aqueous phase. In the conventional photo-Fenton like methods, high concentrations of ferrous salts were used. Therefore, the large amount of ferrous sludge was formed. The current study revealed that a lower amount of Fe is needed and the nZVI particles can be reused in a mag- netic field. The variance analysis suggests that the optimal conditions for TPH reduction rate from diesel fuel in the aqueous phase using photo-Fenton like method (nZVI/ UV/H2O2) are as follows: the initial TPH concentration equals to 0.7 mg/L, nZVI concentration 20 mg/L, H2O2 concentration equals to 5 mmol/L, pH 3, and the reaction time of 60 min and degree of significance for the study pa- rameters are 7.643, 9.33, 13.318, 15.185 and 6.588 percent, respectively. The predicted removal rate in optimal condi- tions was 95.8%, confirmed by the results of our study which was between 95-100%. 8. Dehghani M, Naseri S, Karamimanesh M: Removal of 2,4-Dichlorophenolyxacetic acid (2,4-D) herbicide in the aqueous phase using modified granular activated carbon. J Environ Health Sci Eng 2014, 12(28). doi:10.1186/2052-336X-12-28. 9. Dehghani M, Naseri S, Ahmadi M, Samaei MR, Anushiravani A: Removal of penicillin G from aqueous phase by Fe+3-TiO2/UV-A process. J Environ Health Sci Eng 2014, 12(56). doi:10.1186/2052-336X-12-56. 10. Dehghani M, Naseri S, Hashemi H: Study of the Bioremediation of Atrazine under Variable Carbon and Nitrogen Sources by Mixed Bacterial Consortium Isolated from Corn Field Soil in Fars Province of Iran. J Environ Public Health 2013, 7. Article ID 973165. 10. Dehghani M, Naseri S, Hashemi H: Study of the Bioremediation of Atrazine under Variable Carbon and Nitrogen Sources by Mixed Bacterial Consortium Isolated from Corn Field Soil in Fars Province of Iran. J Environ Public Health 2013, 7. Article ID 973165. J Environ Public Health 2013, 7. Article ID 973165. 11. Saien J, Shahrezari F: Organic pollutants removal from petroleum refinery. Photoenergy 2012, 27:1–5. 12. Portela JRS, Lopez J, Nebot E, Ossa EM: Hydrothermal oxidation of oily wastes: An alternative to conventional treatment methods. Eng Life Sci 2003, 3(2):85–89. 13. Li YSY, Xiang CB, Hong LJ: Treatment of oily wastewater by organic–inorganic composite tubular ultrafiltration (UF) membranes. Desalination 2006, 196:76–83. 13. Li YSY, Xiang CB, Hong LJ: Treatment of oily wastewater by organic–inorganic composite tubular ultrafiltration (UF) membranes. Competing interests Th h d l h p g The authors declare that they have no competing interests. 16. Stepnowski P, Siedlecka EM, Behrend P, Jastorff B: Enhanced photo- degradation of contaminants in petroleum refinery wastewater. Water Res 2002, 36(9):2167–2172. 16. Stepnowski P, Siedlecka EM, Behrend P, Jastorff B: Enhanced photo- degradation of contaminants in petroleum refinery wastewater. Water Res 2002, 36(9):2167–2172. Authors’ contributions The overall implementation of this study including design, experiments and data analysis, and manuscript preparation were the results of the corresponding author's efforts. All authors have made extensive contribution into the review and finalization of this manuscript. All authors read and approved the final manuscript. 17. Will I: Photo-Fenton degradation of wastewater containing organic compounds in solar reactors. Separ Purif Technol 2004, 34(1–3):51–57. 17. Will I: Photo-Fenton degradation of wastewater containing organic compounds in solar reactors. Separ Purif Technol 2004, 34(1–3):51–57. 18. Farzadkia M, Dehghani M, Moafian M: The effects of Fenton process on the removal of petroleum hydrocarbons from oily sludge in Shiraz oil refinery, Iran. J Environ Health Sci Eng 2014, 12(31). doi:10.1186/2052-336X-12-31. 19. Kusic H, Koprivanac N, Srsan L: Azo dye degradation using Fenton type processes assisted by UV irradiation: A kinetic study. Photochem Photobiol 2006, 181:195–202. Acknowledgments This article is extracted from the Master's thesis and the authors appreciate the Deputy of Research and Technology of Shiraz University of Medical Sciences for its financial support for the research project of 91-6170. 20. USEPA: Total Petroleum Hydrocarbons (TPH) as gasoline and diesel. 1996, Available from: http://www.epa.gov/region9/qa/pdfs/8015.pdf. 21. Cheng R, Wang JL, Zhang WX: Comparison of reductive dechlorination of p-chlorophenol using Fe° and nanosized Fe°. J Hazard Mater 2007, 144:334–339. Author details 1 f 1Department of Environmental Health Engineering, School of Health, Shiraz University of Medical Sciences, Shiraz, Iran. 2Department of Environmental Health Engineering, Iran University of Medical Sciences, Tehran, Iran. 22. Tony M, Zhao YQ, Purcell PJ, El-Sherbiny MF: Evaluating the photo- catalytic application of Fenton's reagent augmented with TiO2 and ZnO for the mineralization of an oil-water emulsion. J Environ Sci Health Part A 2009, 44(5):488–493. Received: 13 June 2013 Accepted: 17 May 2014 Published: 23 May 2014 Published: 23 May 2014 23. Oliveira R, Almeida MF, Santos L, Madeira LM: Experimental design of 2, 4- dichlorophenol oxidation by Fenton's reaction. Ind Eng Chem Res 2006, 45:1266–1276. The effects of initial TPH concentration The effects of initial TPH concentration on the reduc- tion rate of diesel fuel in the aqueous phase have been shown in Figure 8. As initial TPH concentration of diesel fuel increased from 0.1 to 0.7 mg/L, TPH reduc- tion rate increased from 57.8 to 75.8% (Figure 8). How- ever, as the initial TPH concentration increased from 0.7 to 1.0 mg/L, a decrease in the reduction rate in diesel fuel degradation (72.7%) was seen. The decrease in the TPH reduction rate at more than 0.7 mg/L was possibly the result of increasing the turbidity of the solution and consequently decreasing the UV radiation permeability. Figure 6 The effect of reaction time on the reduction rate of TPH from diesel fuel in the aqueous phase using photo- Fenton like process. Figure 8 The effect of initial TPH concentration on the reduction rate of TPH from diesel fuel in the aqueous phase using photo- Fenton like process. Figure 8 The effect of initial TPH concentration on the reduction rate of TPH from diesel fuel in the aqueous phase using photo- Fenton like process. Figure 6 The effect of reaction time on the reduction rate of TPH from diesel fuel in the aqueous phase using photo- Fenton like process. Figure 6 The effect of reaction time on the reduction rate of TPH from diesel fuel in the aqueous phase using photo- Fenton like process. Figure 8 The effect of initial TPH concentration on the reduction rate of TPH from diesel fuel in the aqueous phase using photo- Fenton like process. Figure 8 The effect of initial TPH concentration on the reduction rate of TPH from diesel fuel in the aqueous phase using photo- Fenton like process. Page 6 of 7 Page 6 of 7 Dehghani et al. Journal of Environmental Health Science & Engineering 2014, 12:87 http://www.ijehse.com/content/12/1/87 Besides, H2O2 photolysis depends on UV radiation in- tensity. There is a direct relation between UV radiation, the potential formation of hydroxyl radical and diesel fuel decomposition rate. 5. Galvão SAO, Mota ALN, Silva DN, Moraes JEF, Nascimento CAO, Chiavone-Filho O: Application of the photo-Fenton process to the treatment of wastewaters contaminated with diesel. Sci Total Environ 2006, 367(1):42–49. 5. Galvão SAO, Mota ALN, Silva DN, Moraes JEF, Nascimento CAO, Chiavone-Filho O: Application of the photo-Fenton process to the treatment of wastewaters contaminated with diesel. Sci Total Environ 2006, 367(1):42–49. 6. Conclusion Desalination 2006, 196:76–83. 14. Diya`udeen BH, Wan Daud WM, Abul Aziz AR: Treatment Technologies for Petroleum Refinery Effluent. A Review. Process Saf Environ 2011, 89:95–105. 15. Tony MA, Purcell PJ, Zhao YQ, Tayeb AM, El-Sherbiny MF: Photo-catalytic degradation of oil-water emulsion using the photo-fenton treatment process: effects and statistical optimization. J Environ Sci Health Pt 2009, 44(2):179–187. Dehghani et al. Journal of Environmental Health Science & Engineering 2014, 12:87 http://www.ijehse.com/content/12/1/87 Dehghani et al. Journal of Environmental Health Science & Engineering 2014, 12:87 http://www.ijehse.com/content/12/1/87 References 24. Tamimi M, Qourzal S, Barka N, Assabbane A, Ait-Ichou Y: Methomyl degrad- ation in aqueous solutions by Fenton's reagent and the photo-Fenton system. Separ Purif Technol 2008, 61(1):103–108. 1. Tiburtius ERL, Peralta-Zamora P, Emmel A: Treatment of gasoline-contaminated waters by advanced oxidation processes. J Hazard Mater 2005, 126(1–3):86–90. 25. Seok-Young OH, Byung PCC, Kim J, Cha DK: Zero-valent iron pretreatment for enhancing the biodegradability of RD. Water Res 2005, 39:5027–5032. 2. Vieira PA, Vieira RB, de France FP, Cardoso VL: Biodegradation of effluent contaminated with diesel fuel and gasoline. J Hazard Mater 2006, 140:52–59. 26. Coelho AC, Dezotti AV, Sant M, Anna GL: Treatment of petroleum refinery sourwater by advanced oxidation processes. J Hazard Mater 2006, B137:178–184. 3. Volke-Sepulveda T, Ernesto Favela-Torres MGR: Biodegradation of high concentrations of hexadecane by Aspergillus niger in a solid-state system: Kinetic analysis. Bioresource Technol 2005, 97(14):1583–1591. 27. Marc Pera-Titus V, Miguel GM, Baños A, Giménez J, Esplugas S: Degradation of chlorophenols by means of advanced oxidation processes: a general review. Appl Catal B Environ 2004, 47:219–256. 4. Lohi A, Alvarez Cuenca M, Anania G, Upreti SR, Wan L: Biodegradation of diesel fuel-contaminated wastewater using a three-phase fluidized bed reactor. J Hazard Mater 2007, 154:105–111. Page 7 of 7 Page 7 of 7 Dehghani et al. Journal of Environmental Health Science & Engineering 2014, 12:87 http://www.ijehse.com/content/12/1/87 28. Mahvi AH, Maleki A, Rezaee R, Safari M: Reduction of humic substances in water by application of ultrasound waves and ultraviolet irradiation. Iranian J Environ Health Sci En 2009, 6(4):233–240. 29. Dobaradran S, Nabizadeh R, Mahvi AH, Mesdaghinia AR, Naddafi K, Yunesian M, Rastkari N, Nazmara S: Survey on degradation rates of trichloroethylene in aqueous solutions by ultrasound. Iranian J Environ Health Sci En 2010, 7(4):307–312. 30. Mahvi AH: Application of ultrasonic technology for water and wastewater treatment. Iranian J Environ Health Sci En 2009, 38(2):1–17. doi:10.1186/2052-336X-12-87 Cite this article as: Dehghani et al.: Optimizing photo-Fenton like process for the removal of diesel fuel from the aqueous phase. Journal of Environmental Health Science & Engineering 2014 12:87. 28. Mahvi AH, Maleki A, Rezaee R, Safari M: Reduction of humic substances in water by application of ultrasound waves and ultraviolet irradiation. Iranian J Environ Health Sci En 2009, 6(4):233–240. 29. Dobaradran S, Nabizadeh R, Mahvi AH, Mesdaghinia AR, Naddafi K, Yunesian M, Rastkari N, Nazmara S: Survey on degradation rates of trichloroethylene in aqueous solutions by ultrasound. References Iranian J Environ Health Sci En 2010, 7(4):307–312. 30. Mahvi AH: Application of ultrasonic technology for water and wastewater treatment. Iranian J Environ Health Sci En 2009, 38(2):1–17. doi:10.1186/2052-336X-12-87 Cite this article as: Dehghani et al.: Optimizing photo-Fenton like process for the removal of diesel fuel from the aqueous phase. Journal of Environmental Health Science & Engineering 2014 12:87. 28. Mahvi AH, Maleki A, Rezaee R, Safari M: Reduction of humic substances in water by application of ultrasound waves and ultraviolet irradiation. Iranian J Environ Health Sci En 2009, 6(4):233–240. 29. Dobaradran S, Nabizadeh R, Mahvi AH, Mesdaghinia AR, Naddafi K, Yunesian M, Rastkari N, Nazmara S: Survey on degradation rates of trichloroethylene in aqueous solutions by ultrasound. Iranian J Environ Health Sci En 2010, 7(4):307–312. 30. Mahvi AH: Application of ultrasonic technology for water and wastewater treatment. Iranian J Environ Health Sci En 2009, 38(2):1–17. doi:10.1186/2052-336X-12-87 Cite this article as: Dehghani et al.: Optimizing photo-Fenton like process for the removal of diesel fuel from the aqueous phase. Journal of Environmental Health Science & Engineering 2014 12:87. 28. Mahvi AH, Maleki A, Rezaee R, Safari M: Reduction of humic substances in water by application of ultrasound waves and ultraviolet irradiation. Iranian J Environ Health Sci En 2009, 6(4):233–240. 29. Dobaradran S, Nabizadeh R, Mahvi AH, Mesdaghinia AR, Naddafi K, Yunesian M, Rastkari N, Nazmara S: Survey on degradation rates of trichloroethylene in aqueous solutions by ultrasound. Iranian J Environ Health Sci En 2010, 7(4):307–312. doi:10.1186/2052-336X-12-87 Cite this article as: Dehghani et al.: Optimizing photo-Fenton like process for the removal of diesel fuel from the aqueous phase. Journal of Environmental Health Science & Engineering 2014 12:87. 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Biological control of weeds in the 22 Pacific island countries and territories: current status and future prospects
NeoBiota
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Abstract Biological control of introduced weeds in the 22 Pacific island countries and territories (PICTs) began in 1911, with the lantana seed-feeding fly introduced into Fiji and New Caledonia from Hawaii. To date, a to­ tal of 62 agents have been deliberately introduced into the PICTs to control 21 weed species in 17 countries. A further two agents have spread naturally into the region. The general impact of the 36 biocontrol agents now established in the PICTs ranges from none to complete control of their target weed(s). Fiji has been most active in weed biocontrol, releasing 30 agents against 11 weed species. Papua New Guinea, Guam, and the Federated States of Micronesia have also been very active in weed biocontrol. For some weeds such as Lantana camara, agents have been released widely, and can now be found in 15 of the 21 PICTs in which the weed occurs. However, agents for other commonly found weeds, such as Sida acuta, have been released in only a few countries in which the weed is present. There are many safe and effective biocontrol agents already in the Pacific that could be utilised more widely, and highly effective agents that have been released elsewhere in the world that could be introduced following some additional host specificity testing. This pa­ per discusses the current status of biological control efforts against introduced weeds in the 22 PICTs and re­ views options that could be considered by countries wishing to initiate weed biological control programmes. Biological control of weeds in the 22 Pacific island countries and territories: current status and future prospects Michael D. Day1, Rachel L. Winston2 1 Department of Agriculture and Fisheries, Ecosciences Precinct, GPO Box 267, Brisbane, Qld 4001 Australia 2 MIA Consulting, 316 N. Hansen Ave., Shelley, ID 83274 USA Corresponding author: Michael D. Day (michael.day@daf.qld.gov.au) Academic editor: C. Daehler  |  Received 6 November 2015  |  Accepted 28 March 2016  |  Published 23 June 2016 Academic editor: C. Daehler  |  Received 6 November 2015  |  Accepted 28 March 2016  |  Published 23 June 2016 Citation: Day MD, Winston RL (2016) Biological control of weeds in the 22 Pacific island countries and territories: current status and future prospects. In: Daehler CC, van Kleunen M, Pyšek P, Richardson DM (Eds) Proceedings of 13th International EMAPi conference, Waikoloa, Hawaii. NeoBiota 30: 167–192. doi: 10.3897/neobiota.30.7113 Biological co NeoBiota 30: 167–192 (2016) doi: 10.3897/neobiota.30.7113 http://neobiota.pensoft.net Biological co NeoBiota 30: 167–192 (2016) doi: 10.3897/neobiota.30.7113 http://neobiota.pensoft.net Biological co NeoBiota 30: 167–192 (2016) doi: 10.3897/neobiota.30.7113 http://neobiota.pensoft.net n the 22 Pacific isla REVIEW ARTICLE Copyright Michael D. Day, Rachel L. Winston. 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. Introduction Introduced invasive weeds are of increasing concern and importance in the Pacific region, which is reflected by the growing number of publications and websites docu­ menting their distribution and impacts (e.g. Swarbrick 1997, Waterhouse 1997, Mey­ er 2000, Shine et al. 2003, PIER 2013). Weeds decrease food security and income by smothering crops, infesting plantations, and overgrowing grazing lands (Waterhouse and Norris 1987, Orapa 2001, Day et al. 2012). Weeds also affect ecosystem processes through impacts such as degrading soil and reducing water quality and quantity, and are second only to land clearing as a major threat to biodiversity (Meyer 2000, Sherley and Lowe 2000, Dovey et al. 2004). Since 1985, at least six workshops have been held in the Pacific region to prioritise weeds for improved management (e.g. Waterhouse and Norris 1987, Sherley 2000, Shine et al. 2003, Dodd and Hayes 2009, Day 2013). Biological control is a long-term, self-sustaining and feasible option for managing many weeds (Dovey et al. 2004, Julien et al. 2007). Biocontrol of weeds is particu­ larly beneficial and applicable to many Pacific island countries and territories (PICTs) where the capacity to tackle major weed problems is often restricted due to limited infrastructure, resources, and skills (Dovey et al. 2004). The earliest case of the delib­ erate introduction of biocontrol agents from their native range to control a weed was in 1902 when 23 insect species were imported into Hawaii from Mexico to control Lantana camara (Swezey 1923). One agent, the seed-feeding fly Ophiomyia lantanae, which successfully established in Hawaii, was subsequently introduced into Fiji and New Caledonia in 1911 (Guiterrez and Forno 1989), becoming the first weed biocon­ trol agent released in the PICTs. Introduced invasive weeds are of increasing concern and importance in the Pacific region, which is reflected by the growing number of publications and websites docu­ menting their distribution and impacts (e.g. Swarbrick 1997, Waterhouse 1997, Mey­ er 2000, Shine et al. 2003, PIER 2013). Weeds decrease food security and income by smothering crops, infesting plantations, and overgrowing grazing lands (Waterhouse and Norris 1987, Orapa 2001, Day et al. 2012). Weeds also affect ecosystem processes through impacts such as degrading soil and reducing water quality and quantity, and are second only to land clearing as a major threat to biodiversity (Meyer 2000, Sherley and Lowe 2000, Dovey et al. 2004). Introduction Since 1985, at least six workshops have been held in the Pacific region to prioritise weeds for improved management (e.g. Waterhouse and Norris 1987, Sherley 2000, Shine et al. 2003, Dodd and Hayes 2009, Day 2013). y y y Biological control is a long-term, self-sustaining and feasible option for managing many weeds (Dovey et al. 2004, Julien et al. 2007). Biocontrol of weeds is particu­ larly beneficial and applicable to many Pacific island countries and territories (PICTs) where the capacity to tackle major weed problems is often restricted due to limited infrastructure, resources, and skills (Dovey et al. 2004). The earliest case of the delib­ erate introduction of biocontrol agents from their native range to control a weed was in 1902 when 23 insect species were imported into Hawaii from Mexico to control Lantana camara (Swezey 1923). One agent, the seed-feeding fly Ophiomyia lantanae, which successfully established in Hawaii, was subsequently introduced into Fiji and New Caledonia in 1911 (Guiterrez and Forno 1989), becoming the first weed biocon­ trol agent released in the PICTs. Over 60 weed biocontrol agents have since been introduced deliberately into 17 of the 22 PICTs, not including Australia, New Zealand, or Hawaii (Winston et al. 2014). However, for most biocontrol agents, the number of PICTs in which they have been introduced or naturally spread is only a fraction of the number of PICTs where the target weeds occur. Consequently, there is great potential for further introductions within the PICTs. In addition, there are many more weeds present for which biocon­ trol has not been attempted in the PICTs. Effective biocontrol agents for some of these are available elsewhere and could be introduced. One of the limiting factors for weed biocontrol in many PICTs is the knowledge of what agents are available and effective. Numerous workshops involving the PICTs have been conducted, with the last being held in Auckland in 2009 (Dodd and Hayes 2009) where potential biocontrol agents were discussed. These workshops have often resulted in new biocontrol programs being implemented, with new or existing agents being introduced into one or more countries (Winston et al. 2014).hf This paper reviews the current status of biocontrol efforts against introduced weeds in the PICTs and identifies existing biocontrol agents that could be moved around the Pacific as well as additional effective biocontrol agents that could be introduced into the region. Keywordsi Host specificity, establishment, biocontrol agents Copyright Michael D. Day, Rachel L. Winston. 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. 168 Michael D. Day & Rachel L. Winston / NeoBiota 30: 167–192 (2016) Materials and methods The number of weed biocontrol agents introduced into the 22 PICTs, their estab­ lishment status, and their current impact were extracted from Winston et al. (2014) and supplemented by recent publications and personal communications with local researchers to provide an updated account through to 2015. The assessment did not include Australia, Norfolk Island (a territory of Australia), New Zealand, Hawaii, and Easter Island (a territory of Chile).f From the compiled dataset, we determined the weed biocontrol effort of each country, including the number of weeds targeted and the number of agents deliber­ ately introduced. We also analysed the dataset by target weed to determine how many biocontrol agents have been introduced into the region, how many have established, and their overall level of impact against their target weeds. The level of impact was obtained from Winston et al. (2014) or from the perception of local researchers and took into consideration varying habitats and climates, with the understanding that a weed may not be under the same level of control in all areas where it exists. The two analyses allowed us to ascertain which weeds were most amenable to biocontrol, and which biocontrol agents were the most widespread, damaging, and effective against their target weed. g Numerous sources were utilized to determine the distribution of weeds in the Pa­ cific, including workshop reports, websites, and personal communications with local land managers (Swarbrick 1997, Waterhouse 1997, Meyer 2000, Shine et al. 2003, Dodd and Hayes 2009, PIER 2013, Endemia 2015). Some of the weed biocontrol prioritisation workshops utilized herein asked participants to list the top 10 weeds in their country. In these circumstances, not all weeds present in a country were captured. The weed lists were then collated into a comprehensive compilation of weeds occurring in each country and cross-checked against weed species that have already been targeted for biocontrol worldwide (Winston et al. 2014), as well as against weed species being evaluated as potential new candidates now or in the near future (Q. Paynter, Landcare Research pers. comm. 2015, T. Johnson, US Department of Agriculture, pers. comm. 2015). Weed species not targeted for weed biocontrol were deleted from the dataset. After combining the two datasets, we determined which biocontrol agents could be introduced into particular countries where the target weed occurs but no biocon­ trol agents have established to date. Introduction This information provides a platform for PICTs to identify the best and most appropriate weed biocontrol opportunities to pursue, and should be considered Biological control of weeds in the 22 Pacific island countries and territories... 169 against other factors such as weed importance and available resources in each country. Australia, Norfolk Island (a territory of Australia), New Zealand, and Hawaii are not included in this paper as they already have well-established biocontrol programmes, and extensive reviews on their programmes have already been conducted (Conant et al. 2013, Fowler et al. 2000, 2010, Funasaki 1988, Julien et al. 2012, Smith 2002, Trujillo 2005). Materials and methods In doing so, we only considered those biocontrol agents that had been deliberately released into at least one country. This excluded spe­ Michael D. Day & Rachel L. Winston / NeoBiota 30: 167–192 (2016) 170 cies that had found their way into countries naturally but had never been deliberately introduced into any country. The rationale behind excluding these species is that they are not bona fide biocontrol agents, nor have they been subjected to detailed host specificity testing; consequently, there is a risk of non-target impacts if introduced into a new region. There are no native species in the Pacific region that have been used as weed biocontrol agents. Results were separated into three lists based on whether 1) the agent is already es­ tablished in at least one of the PICTs and is having at least a medium impact (weed is partially or fully controlled in most areas) on the target weed, 2) the agent is not yet in any PICTs but has at least a medium impact on the target weed elsewhere, and 3) the agent has only a slight impact (may cause damage but does not reduce weed popula­ tions) on the target weed either in any of the PICTs or elsewhere. A fourth list docu­ ments the agents that have been recently released and are still being evaluated, and any new target weeds for which agent exploration or host specificity testing of new agents are currently being conducted. As much of the data on weed presence or importance by country is not well defined, no attempt was made to suggest specific actions. ii Our analysis excluded agents that did not establish in any country in which they were introduced, agents that had established in at least one country but were consid­ ered to have no impact against the target weed, and agents that have caused significant impacts to non-target species. We determined that these agents were unlikely to suc­ ceed in terms of achieving establishment and causing a significant impact to the target weed and/or had great potential to damage non-target species in a new country (Julien et al. 2007, Paynter et al. 2015). Results Seventeen of the 22 PICTs have deliberately introduced at least one biocontrol agent (Table 1). Fiji (30 biocontrol agents introduced against 11 weed species) and Papua New Guinea (19 agents released against 12 weed species) have been the most active. Guam (16 agents against 4 weed species), Federated States of Micronesia (13 agents against 3 weed species), and Palau (11 agents against 4 weed species) have also been actively involved in weed biocontrol. Five countries, namely Kiribati, Pitcairn Islands, Tokelau, Tuvalu, and Wallis and Futuna, have not deliberately introduced any weed biocontrol agents to date. These countries mainly consist of small, low-lying atolls, and weeds may not be at sufficient densities to warrant biocontrol. fi Since 1911, there has been a steady stream of biocontrol agents introduced into the PICTs (Fig. 1). A total of 62 biocontrol agents targeting 21 weed species have been deliberately released into at least one country in the PICTs (Table 2). Of these, 32 agents have established on 17 weed species. Two biocontrol agents, Neogalea sunia and Epiblema strenuana, did not establish when deliberately introduced into the region, but were later found to have spread into some PICTs of their own accord (Table 2). In addition, Acalitus adoratus and Maravalia cryptostegiae also self-introduced into Biological control of weeds in the 22 Pacific island countries and territories... 171 Table 1. The number of weed species targeted for biocontrol and the number of biocontrol agents that have been deliberately introduced (intentional) and agents that were not deliberately introduced but have been found (unintentional) in the PICTs. Table 1. The number of weed species targeted for biocontrol and the number of biocontrol agents that have been deliberately introduced (intentional) and agents that were not deliberately introduced but have been found (unintentional) in the PICTs. Intentional introductions Unintentional introductions Combined introductions Country No. of weed species No. of agents released No. of agents establ. No. of weed species No. of agents establ. No. of weed species No. of agents establ. Results American Samoa 2 2 2 0 0 2 2 Cook Islands 4 11 2 0 0 4 2 Federated States of Micronesia 3 13 10 2 2 3 12 Fiji 11 30 17 0 0 11 17 French Polynesia 2 3 3 0 0 2 3 Guam 4 16 9 2 4 4 13 Marshall Islands 1 1 1 0 0 1 1 Nauru 1 1 0 0 0 1 0 New Caledonia 4 7 6 3 4 5 10 Niue 2 4 3 1 1 3 4 Northern Mariana Islands 4 8 7 2 5 4 12 Palau 4 11 6 2 4 4 10 Papua New Guinea 12 19 12 3 6 13 18 Samoa 4 5 3 1 1 4 4 Solomon Islands 5 7 4 2 2 5 6 Tonga 3 6 5 2 2 4 7 Vanuatu 8 9 8 3 6 9 14 Figure 1. Cumulative number of deliberate biocontrol agent introductions in the PICTs since 1911. The values include those introductions where the agent failed to establish in any country. Intentional introductions Unintentional introductions Combined introductions Country No. of weed species No. of agents released No. of agents establ. No. of weed species No. of agents establ. No. of weed species No. of agents establ. American Samoa 2 2 2 0 0 2 2 Cook Islands 4 11 2 0 0 4 2 Federated States of Micronesia 3 13 10 2 2 3 12 Fiji 11 30 17 0 0 11 17 French Polynesia 2 3 3 0 0 2 3 Guam 4 16 9 2 4 4 13 Marshall Islands 1 1 1 0 0 1 1 Nauru 1 1 0 0 0 1 0 New Caledonia 4 7 6 3 4 5 10 Niue 2 4 3 1 1 3 4 Northern Mariana Islands 4 8 7 2 5 4 12 Palau 4 11 6 2 4 4 10 Papua New Guinea 12 19 12 3 6 13 18 Samoa 4 5 3 1 1 4 4 Solomon Islands 5 7 4 2 2 5 6 Tonga 3 6 5 2 2 4 7 Vanuatu 8 9 8 3 6 9 14 Figure 1. Cumulative number of deliberate biocontrol agent introductions in the PICTs since 1911. The values include those introductions where the agent failed to establish in any country. Michael D. Day & Rachel L. Results Winston / NeoBiota 30: 167–192 (2016) 172 Table 2. Status of weed biocontrol agents deliberately released (intentional) and/or spread of their own accord (unintentional) into the 22 PICTs and the potential countries in which they could be introduced. Countries: AS=American Samoa, CI=Cook Islands, FSM=Federated States of Micronesia, Fi=Fiji, FP=French Poly­ nesia, Gu=Guam, Ki=Kiribati, MI=Marshall Islands, Na=Nauru, NC=New Caledonia, Ni=Niue, NMI=Northern Mariana Islands, Pa=Palau, PNG=Papua New Guinea, PI=Pitcairn Islands, Sa=Samoa, SI=Solomon Islands, Tk=Tokelau, To=Tonga, Tu=Tuvalu, Va=Vanuatu, WF=Wallis & Futuna. Status: I=intentionally intro­ duced, U=unintentionally introduced, E=established, F=failed to establish. Impact: H=high, M=moderate, N=none, S=slight, V=variable, ?=unknown. * Potential countries where agents could be introduced (based on weed occurrence in each country, not weed density). Weed family Weed species Biocontrol agent family Biocontrol agent species AS CI FSM Fi FP Gu Ki MI Na NC Ni NMI Pa PNG PI Sa SI Tk To Tu Va WF Apocynaceae Cryptostegia grandiflora R. Br. Chaconiaceae Maravalia cryptostegiae (Cummins) Ono UE? Araceae Pistia stratiotes L. Curculionidae Neohydronomus affinis Hustache * * * * * * * IEV * IEV Asteraceae Chromolaena odorata (L.) R. M. King & H. Rob. Eriophyidae Acalitus adoratus Keifer UES UES UES UES UES Brentidae Apion brunneonigrum Béguin-Billecocq IF Agromyzidae Calycomyza eupatorivora Spencer IF Tephritidae Cecidochares connexa Macquart IEH IEM * * IEH IES IEV Erebidae Pareuchaetes pseudoinsulata Rego Barros IEV IEM IEV IF IEV Pyralidae Phestinia costella Hampson IF Elephantopus mollis Kunth Tephritidae Tetraeuaresta obscuriventris (Loew) * * IEM * * * UE? * * * * UEN UEN * Mikania micrantha Kunth Phlaeothripidae Liothrips mikaniae (Priesner) IF Pucciniaceae Puccinia spegazzinii De Toni * * IE? * * * * * * * * IE? * * UE? * * * IE? * Parthenium hysterophorus L. Tortricidae Epiblema strenuana (Walker) * * UE? Chrysomelidae Zygogramma bicolorata Pallister I? Xanthium strumarium L. Tortricidae Epiblema strenuana (Walker) * * * * * IF* * Tephritidae Euaresta aequalis Loew IF of weed biocontrol agents deliberately released (intentional) and/or spread of their own accord (unintentional) into the 22 PICTs and the potential h they could be introduced. Countries: AS=American Samoa, CI=Cook Islands, FSM=Federated States of Micronesia, Fi=Fiji, FP=French Poly­ , Ki=Kiribati, MI=Marshall Islands, Na=Nauru, NC=New Caledonia, Ni=Niue, NMI=Northern Mariana Islands, Pa=Palau, PNG=Papua New irn Islands, Sa=Samoa, SI=Solomon Islands, Tk=Tokelau, To=Tonga, Tu=Tuvalu, Va=Vanuatu, WF=Wallis & Futuna. Status: I=intentionally intro­ ntionally introduced, E=established, F=failed to establish. Impact: H=high, M=moderate, N=none, S=slight, V=variable, ?=unknown. Results * Potential gents could be introduced (based on weed occurrence in each country, not weed density). Table 2. Status of weed biocontrol agents deliberately released (intentional) and/or spread of their own accord (unintentional) into the 22 PICTs and the potential countries in which they could be introduced. Countries: AS=American Samoa, CI=Cook Islands, FSM=Federated States of Micronesia, Fi=Fiji, FP=French Poly­ nesia, Gu=Guam, Ki=Kiribati, MI=Marshall Islands, Na=Nauru, NC=New Caledonia, Ni=Niue, NMI=Northern Mariana Islands, Pa=Palau, PNG=Papua New Guinea, PI=Pitcairn Islands, Sa=Samoa, SI=Solomon Islands, Tk=Tokelau, To=Tonga, Tu=Tuvalu, Va=Vanuatu, WF=Wallis & Futuna. Status: I=intentionally intro­ duced, U=unintentionally introduced, E=established, F=failed to establish. Impact: H=high, M=moderate, N=none, S=slight, V=variable, ?=unknown. * Potential countries where agents could be introduced (based on weed occurrence in each country, not weed density). Biological control of weeds in the 22 Pacific island countries and territories... 173 Weed family Weed species Biocontrol agent family Biocontrol agent species AS CI FSM Fi FP Gu Ki MI Na NC Ni NMI Pa PNG PI Sa SI Tk To Tu Va WF Cerambycidae Nupserha vexator (Pascoe) IF Pucciniaceae Puccinia xanthii Schweinitz I? Cactaceae Acanthocereus tetragonus (L.) Hummelinck Pseudococcidae Hypogeococcus festerianus (Lizer y Trelles) IF Opuntia spp. Dactylopiidae Dactylopius sp. nr confusus (Cockerell) IEH Opuntia ficus- indica (L.) Mill. Pyralidae Cactoblastis cactorum (Berg) * * Opuntia monacantha (Willd.) Haw. Pyralidae Cactoblastis cactorum (Berg) * * * * * * * * * * * * * Opuntia stricta (Haw.) Haw. Pyralidae Cactoblastis cactorum (Berg) IEH * * Cucurbitaceae Coccinia grandis (L.) Voigt Curculionidae Acythopeus burkhartorum O’Brien & Pakaluk IF IF Curculionidae Acythopeus cocciniae O’Brien & Pakaluk * * IEH * IEM * * * * * * Sesiidae Melittia oedipus Oberthür * * IEH * IE? * * * * * * Cyperaceae Cyperus rotundus L. Curculionidae Athesapeuta cyperi Marshall IF IF IEN Tortricidae Bactra minima Meyrick IF IF IEN Tortricidae Bactra venosana (Zeller) IF IEN UE? Fabaceae Mimosa diplotricha C. Wright Psyllidae Heteropsylla spinulosa Muddiman, Hodkinson & Hollis IEH IEH IEH IEH * IE? * IEH IE? IEH IEV IEH IEH IEH IES * Saturniidae Psigida walkeri (Grote) IF Coreidae Scamurius sp. IF Mimosa pigra L. Chrysomelidae Acanthoscelides puniceus Johnson I? Chrysomelidae Acanthoscelides quadridentatus (Schaeffer) I? Malvaceae Sida acuta Burm. f. Chrysomelidae Calligrapha pantherina Stål * * * IEH * * * * * UE? * * * IEH I? * * IEH Sida rhombifolia L. Results Chrysomelidae Calligrapha pantherina Stål * * * IEH * * * * * * * * IEH * I? * * * * IEH * Michael D. Day & Rachel L. Winston / NeoBiota 30: 167–192 (2016) 174 Weed family Weed species Biocontrol agent family Biocontrol agent species AS CI FSM Fi FP Gu Ki MI Na NC Ni NMI Pa PNG PI Sa SI Tk To Tu Va WF Melastomataceae Clidemia hirta (L.) D. Don Crambidae Ategumia matutinalis (Guenée) IF Phlaeothripidae Liothrips urichi Karny IEM * IEH IEV * * IF* * Miconia calvescens DC. Glomerellaceae Colletotrichum gloeosporioides (Penz.) Penz. & Sacc. f. sp. miconiae Killgore & L. Sugiyama IEV * * Pontederiaceae Eichhornia crassipes (Mart.) Solms Erirhinidae Neochetina bruchi Hustache * * * * * * * * * * * IEH * * IE? Erirhinidae Neochetina eichhorniae Warner * * * IEH * * * I? * * * IEH * IES IEH Crambidae Niphograpta albiguttalis (Warren) IF Crambidae Xubida infusella (Walker) I? Salviniaceae Salvinia molesta D.S. Mitch. Erirhinidae Cyrtobagous salviniae Calder & Sands * IEH * * * * IEH Erirhinidae Cyrtobagous singularis Hustache IEN Pauliniidae Paulinia acuminata (De Geer) IEN Crambidae Samea multiplicalis (Guenée) IEN Verbenaceae Lantana camara L. sens. lat. Agromyzidae Calycomyza lantanae (Frick) UEM IE? UE? UE? UES UES UES Chrysomelidae Charidotis pygmaea Klug IF Tortricidae Crocidosema lantana Busck IEM UEM IE? UEM UEM UES Noctuidae Diastema tigris Guenée IF IF Erebidae Hypena laceratalis Walker IEN IES IEN UES UEN UES UES Pterophoridae Lantanophaga pusillidactyla (Walker) IEM UEM UEM IES UES Tingidae Leptobyrsa decora Drake I? IF IF IF I? Noctuidae Neogalea sunia (Guenée) IF UES Chrysomelidae Octotoma championi Baly IF Chrysomelidae Octotoma scabripennis Guérin-Méneville * IF* * IF* * IF* * * * IES IF* * * * * * I? * * * * 175 Biological control of weeds in the 22 Pacific island countries and territories... Weed family Weed species Biocontrol agent family Biocontrol agent species AS CI FSM Fi FP Gu Ki MI Na NC Ni NMI Pa PNG PI Sa SI Tk To Tu Va WF Agromyzidae Ophiomyia lantanae (Froggatt) I? IE? IES IE? IEM IEM UEM UEM UE? UE? UE? UE? Cerambycidae Plagiohammus spinipennis (Thomson) IF IF Crambidae Pseudopyrausta santatalis (Barnes & McDunnough) IF IF Crambidae Salbia haemorrhoidalis Guenée IES IES IF IF Lycaenidae Strymon bazochii (Godart) I? Results Tingidae Teleonemia elata Drake IF Tingidae Teleonemia scrupulosa Stål * * IEV IEV IE? IEV * * * IEV IEH IEV IEV IEH * IEH IEH IES * IES * Lycaenidae Tmolus echion (L.) IF Chrysomelidae Uroplata fulvopustulata Baly IF Chrysomelidae Uroplata girardi Pic * IEH IEM IEV * IEM * * * IEM IEM IEV IEM IES * IEM IEH IEM * IES * gophyllaceae Tribulus cistoides L. Curculionidae Microlarinus lareynii (Jacquelin du Val) IF Microlarinus lypriformis (Wollaston) * * * * * * * IEH Tribulus terrestris L. Curculionidae Microlarinus lypriformis (Wollaston) * * Michael D. Day & Rachel L. Winston / NeoBiota 30: 167–192 (2016) 176 some PICTs. In total, 36 weed biocontrol agents are now confirmed as present in the PICTs, attacking 19 weed species. The overall impact of these biocontrol agents ranges from no damage to high impact on the target weed, depending on country and region (Tables 2, 3). Of the weed species on which at least one biocontrol agent has established, seven are deemed to be under complete control overall, due to the high impact of the agent(s) (Ta­ ble 3). A further six weed species are deemed to be under partial to full control. The im­ pacts of biocontrol agents on two weed species have been variable. For four weed species where biocontrol agents have only recently established, the establishment and impacts of biocontrol agents are still being evaluated. There are three weed species for which agents have either not established, or there is little, no, or unknown impact of biocontrol agents.h The most widespread and damaging biocontrol agent in the PICTs is the psyl­ lid Heteropsylla spinulosa, which was introduced and has established in 13 of the 16 countries where its target weed Mimosa diplotricha occurs. In most areas within most countries, M. diplotricha is under control (Tables 2, 3). However, in high rainfall areas, control is not always achieved because heavy rain can wash the psyllids from plants. Sida acuta and S. rhombifolia are deemed under control in three of the four coun­ tries where the leaf-feeding beetle Calligrapha pantherina was intentionally introduced and established. The establishment of C. pantherina in the fourth country, Samoa, is not known. Calligrapha pantherina has recently been reported in New Caledonia, although its mode of entry and impact on the Sida spp. are unknown. Results Other weeds considered under control by biocontrol agents in the PICTs include Salvinia molesta, Tribulus cistoides, Opuntia stricta, and unspecified Opuntia spp. (Tables 2, 3). i Eichhornia crassipes and Pistia stratiotes are generally under a high degree of control in each of the countries where their respective biocontrol agents have been released and established (Tables 2, 3). Control of E. crassipes is generally higher if both Neochetina eichhorniae and N. bruchi are present. Control of both aquatic weeds appears to be incomplete in shaded locations. Cecidochares connexa has established and is aiding the control of Chromolaena odo­ rata in all five countries in which it has been introduced (Tables 2, 3). However, C. connexa appears to be less effective at altitudes greater than 1000 m above sea level or in areas where rainfall is high, such as West New Britain, Papua New Guinea. Of the two agents introduced to control Clidemia hirta, only Liothrips urichi estab­ lished. This agent appears to be effective at controlling C. hirta in only sunny areas of the three countries in which it has established (Tables 2, 3); there is little impact where C. hirta is growing in shaded areas. Three agents have been released against Coccinia grandis, but only two have estab­ lished. Melittia oedipus has been released in Guam and the Northern Mariana Islands, and is having a high degree of impact in both countries. Acythopeus cocciniae is having a high degree of impact in Guam, while its establishment in the Northern Mariana Islands has not been confirmed (Tables 2, 3). i Twenty biocontrol agents have been intentionally introduced against L. camara in the PICTs. Of these, nine agents have established in at least one country (Table 2). Biological control of weeds in the 22 Pacific island countries and territories... 177 Table 3. Summary of the biocontrol effort against each target weed species, including the number of PICTs where biocontrol agents have established without being deliberately released. For weeds where multiple agents have been released, numbers have been pooled. Weed family Weed species No. countries weed occurs No. agents established in the Pacific No. Results countries all agents established Overall impact on weed** Apocynaceae Cryptostegia grandiflora 8 1 1 unknown Araceae Pistia stratiotes 9 1 2 medium to high Asteraceae Chromolaena odorata 7 3 5 medium to high Elephantopus mollis 14 1 4 variable Mikania micrantha 20 1 4 still evaluating Parthenium hysterophorus 3 1 1 still evaluating Xanthium strumarium 7 0* 0 still evaluating Cactaceae Acanthocereus tetragonus 1 0 0 none Opuntia spp. 1 1 1 high Opuntia stricta 3 1 1 high Cucurbitaceae Coccinia grandis 11 2 2 medium to high Cyperaceae Cyperus rotundus 21 3 2 none Fabaceae Mimosa diplotricha 16 1 13 high Mimosa pigra 1 0* 0 still evaluating Malvaceae Sida acuta 18 1 4 high Sida rhombifolia 22 1 3 high Melastomataceae Clidemia hirta 9 1 3 low to high Miconia calvescens 3 1 1 variable Pontederiaceae Eichhornia crassipes 15 2 4 medium to high Salviniaceae Salvinia molesta 7 4 2 high Verbenaceae Lantana camara 21 10 15 slight to high Zygophyllaceae Tribulus cistoides 8 1 1 high * Biocontrol agents have recently been released, but establishment is not confirmed ** Rating is based on the overall level of control as per Winston et al. (2014) * Biocontrol agents have recently been released, but establishment is not confirmed ** Rating is based on the overall level of control as per Winston et al. (2014) Uroplata girardi and Teleonemia scrupulosa have been released and have established in 13 countries; both reportedly have a moderate to high overall impact in most coun­ tries where they have established. Crocidosema lantana, Lantanophaga pusillidactyla, and Ophyiomyia lantanae have a moderate impact in some countries but only a slight impact in other countries. The remaining agents have little or no impact on L. camara. ph g g p Of the biocontrol agents that have established in the PICTs and are having a me­ dium to high impact on the target weed, many have not been released in all PICTs where their respective target weed has been recorded. For example, C. pantherina has proven very effective against S. acuta and S. rhombifolia in three countries, and could potentially be introduced into 14 and 18 additional countries, respectively. Likewise, 178 Michael D. Day & Rachel L. Winston / NeoBiota 30: 167–192 (2016) N. bruchi and N. eichhorniae could potentially be introduced against E. crassipes in 13 additional countries, while the biocontrol agents for C. Results grandis could be introduced into nine countries. Cactoblastis cactorum was introduced into New Caledonia to control O. stricta. However, the agent also attacks Opuntia monacantha, and so could be released in the 13 countries in which this weed occurs. Similarly, Microlarinus lypriformis was released against Tribulus cistoides, but could also be used against Tribulus terrestris in Fiji and Papua New Guinea. The countries in which established and effective agents within the PICTs could potentially be redistributed are listed in Table 2. Because biocontrol agents can spread naturally between islands, it is recommended that countries conduct surveys to determine what biocontrol agents are present prior to any introductions.hf There are also opportunities to introduce biocontrol agents that have proven effec­ tive outside the PICTs (Table 4), provided target weed densities are sufficiently high to warrant this. Additional agents attacking L. camara, O. stricta, and Parthenium hystero­ phorus could be introduced in the PICTs to supplement the biocontrol agents already established against these species. There are also effective agents for weeds that have not been targeted for biocontrol in the PICTs to date. These weed species include Arundo donax (present in 12 countries), Dolichandra unguis-cati (7 countries), and Melaleuca quinquenervia (7 countries) (Table 4). Because biocontrol agents may do poorly in one region and have spectacular suc­ cess elsewhere, agents having slight or variable impacts on their target weed(s) in at least one country within or outside the Pacific region are listed in Table 5. i Numerous weed species occurring in the PICTs are currently weed biocontrol tar­ gets elsewhere, but the agents have either been only recently released and not yet evalu­ ated or not yet released (Table 6). In addition, there are several previously targeted weeds (e.g. C. odorata, E. crassipes, and L. camara) for which new agents were recently released and are currently being evaluated for establishment and/or impact (Table 6). Should any of these agents prove to be specific and effective against their target weeds, they could also be considered for introduction in the PICTs in the future. Discussion Biological control of weeds has been practiced in the PICTs for over 100 years, with over 20 weed species targeted. In that time, 17 countries have deliberately introduced at least one biocontrol agent (Winston et al. 2014). In addition to agents deliberately released into the PICTs, four biocontrol agents have found their way into the Pacific region either through natural means or unintentionally on imported goods. For over half the weed species targeted, biocontrol agents are having a medium to high impact. Consequently, weed biocontrol to date has been very cost-effective and has provided relief to farmers and land managers trying to control those weeds, and has resulted in increased production and income (e.g. Julien and Orapa 2001, Day et al. 2013a, Day and Bule this edition). Biological control of weeds in the 22 Pacific island countries and territories... 179 Table 4. Weed biocontrol agents that have medium to high impacts in at least one country outside the PICTs and could be introduced into the region. Prior to introduction, additional host specificity testing may be needed. Countries: AS=American Samoa, CI=Cook Islands, FSM=Federated States of Micronesia, Fi=Fiji, FP=French Polynesia, Gu=Guam, Ki=Kiribati, MI=Marshall Islands, Na=Nauru, NC=New Caledonia, Ni=Niue, NMI=Northern Mariana Islands, Pa=Palau, PNG=Papua New Guinea, PI=Pitcairn Islands, Sa=Samoa, SI=Solomon Islands, Tk=Tokelau, To=Tonga, Tu=Tuvalu, Va=Vanuatu, WF=Wallis & Futuna. Weed family Weed species Biocontrol agent family Biocontrol agent species Possible countries for introduction# Asteraceae Ageratina adenophora (Spreng.) R. M. King & H. Rob. Mycosphaerellaceae Passalora ageratinae Crous & A.R. Wood FP Parthenium hysterophorus Chrysomelidae Zygogramma bicolorata* FP, NC, Va Curculionidae Listronotus setosipennis (Hustache) FP, NC, Va Xanthium strumarium Pucciniaceae Puccinia xanthii Schweinitz* CI, Fi, FP, Gu, NC, PNG, To Azollaceae Azolla filiculoides Lam. Erirhinidae Stenopelmus rufinasus Gyllenhal CI Basellaceae Anredera cordifolia (Ten.) Steenis Chrysomelidae Plectonycha correntina Lacordaire CI, Fi, FP, NC, Ni, PI Bignoniaceae Dolichandra unguis-cati (L.) L. G. Lohmann Buprestidae Hedwigiella jureceki (Obenberger) CI, FSM, FP, Gu, NC, Ni, Va Tingidae Carvalhotingis visenda Drake CI, FSM, FP, Gu, NC, Ni, Va Cactaceae Opuntia ficus-indica Dactylopiidae Dactylopius opuntiae (Cockerell) FP, NC Opuntia monacantha Dactylopiidae Dactylopius ceylonicus (Green) AS, CI, FSM, Fi, Gu, Na, NC, Ni, NMI, Pa, Sa, SI, To Opuntia stricta Dactylopiidae Dactylopius opuntiae (Cockerell) NC, Sa, SI Pereskia aculeata Mill. Chrysomelidae Phenrica guerini Bechyné FP, NC, Pa Fabaceae Acacia dealbata Link Curculionidae Melanterius maculatus Lea FP Acacia mearnsii De Wild. Discussion Curculionidae Anthonomus santacruzi Hustache CI, Fi, FP, NC, SI, To Tingidae Gargaphia decoris Drake CI, Fi, FP, NC, SI, To Verbenaceae Lantana camara Agromyzidae Ophiomyia camarae Spencer AS, CI, FSM, Fi, FP, Gu, Ki, MI, Na, NC, Ni, NMI, Pa, PNG, PI, Sa, SI, To, Tu, Va, WF Eriophyidae Aceria lantanae (Cook) AS, CI, FSM, Fi, FP, Gu, Ki, MI, Na, NC, Ni, NMI, Pa, PNG, PI, Sa, SI, To, Tu, Va, WF Miridae Falconia intermedia (Distant) AS, CI, FSM, Fi, FP, Gu, Ki, MI, Na, NC, Ni, NMI, Pa, PNG, PI, Sa, SI, To, Tu, Va, WF Zygophyllaceae Tribulus cistoides Curculionidae Microlarinus lareynii* CI, Fi, FP, Gu, Ki, MI, NC, PNG Tribulus terrestris Curculionidae Microlarinus lareynii Fi, PNG * Introduced previously but failed to establish # Based on weed occurrence in each country, not weed density Biological control of weeds in the 22 Pacific island countries and territories... 181 Table 5. Weed biocontrol agents that have slight, variable, or unknown impacts in at least one country within or outside the PICTs that could be investigated further to assess their suitability for introduction/redistribution in the region. Prior to introduction, additional host specificity testing may be needed. Coun­ tries: AS=American Samoa, CI=Cook Islands, FSM=Federated States of Micronesia, Fi=Fiji, FP=French Polynesia, Gu=Guam, Ki=Kiribati, MI=Marshall Islands, Na=Nauru, NC=New Caledonia, Ni=Niue, NMI=Northern Mariana Islands, Pa=Palau, PNG=Papua New Guinea, PI=Pitcairn Islands, Sa=Samoa, SI=Solomon Islands, Tk=Tokelau, To=Tonga, Tu=Tuvalu, Va=Vanuatu, WF=Wallis & Futuna. Weed family Weed species Biocontrol agent family Biocontrol agent species No. of countries in Pacific agent established Possible countries for introduction# Apocynaceae Cryptostegia grandiflora Crambidae Euclasta whalleyi Popescu-Gorj & Constantinescu Fi, FP, Gu, MI, NC, NMI, PNG, SI Chaconiaceae Maravalia cryptostegiae 1 Fi, FP, Gu, MI, NC, NMI, SI Asteraceae Ageratina adenophora Pterophoridae Oidaematophorus beneficus Yano & Heppner FP Tephritidae Procecidochares utilis Stone FP Chromolaena odorata Agromyzidae Calycomyza eupatorivora FSM, Gu, MI, NC, NMI, Pa, PNG Erebidae Pareuchaetes insulata (Walker) FSM, Gu, MI, NC, NMI, Pa, PNG Erebidae Pareuchaetes pseudoinsulata 5 MI, NC Cirsium vulgare (Savi) Ten. Curculionidae Larinus carlinae (Olivier) NC Curculionidae Rhinocyllus conicus (Frölich) NC Curculionidae Trichosirocalus horridus (Panzer) NC Syrphidae Cheilosia grossa (Fallén) NC Tephritidae Urophora stylata (Fabricius) NC Parthenium hysterophorus Bucculatricidae Bucculatrix parthenica Bradley FP, NC, Va Curculionidae Conotrachelus albocinereus Fiedler FP, NC, Va Curculionidae Smicronyx lutulentus Dietz FP, NC, Va Delphacidae Stobaera concinna (Stål) FP, NC, Va Pucciniaceae Puccinia abrupta Dietel & Holw. var. partheniicola (H.S. Discussion Cecidomyiidae Dasineura rubiformis Kolesik CI Curculionidae Melanterius maculatus Lea CI Acacia melanoxylon R. Br. Curculionidae Melanterius acaciae Lea CI Acacia pycnantha Benth. Curculionidae Melanterius maculatus Lea Gu Pteromalidae Trichilogaster signiventris (Girault) Gu Mimosa pigra Chrysomelidae Acanthoscelides spp. PNG Chrysomelidae Malacorhinus irregularis Jacoby PNG Curculionidae Chalcodermus serripes Fåhraeus PNG Geometridae Macaria pallidata (Warren) PNG Gracillariidae Neurostrota gunniella (Busck) PNG Sesiidae Carmenta mimosa Eichlin & Passoa PNG Michael D. Day & Rachel L. Winston / NeoBiota 30: 167–192 (2016) 180 Weed family Weed species Biocontrol agent family Biocontrol agent species Possible countries for introduction# Paraserianthes lophantha (Willd.) Nielsen Curculionidae Melanterius servulus Pascoe CI Ulex europaeus L. Tetranychidae Tetranychus lintearius Dufour PNG Vachellia nilotica subsp. indica (Benth.) Kyal. & Boatwr Geometridae Chiasmia assimilis (Warren) FP, NC, SI, WF Hydrocharitaceae Hydrilla verticillata (L. f.) Royle Ephydridae Hydrellia pakistanae Deonier Fi, Gu, NC, PNG Lamiaceae Marrubium vulgare L. Pterophoridae Wheeleria spilodactylus (Curtis) NC Sesiidae Chamaesphecia mysiniformis Rambur NC Myrtaceae Melaleuca quinquenervia (Cav.) S. T. Blake Cecidomyiidae Lophodiplosis trifida Gagné FSM, Fi, FP, Gu, NC, Pa, PNG Curculionidae Oxyops vitiosa Pascoe FSM, Fi, FP, Gu, NC, Pa, PNG Psyllidae Boreioglycaspis melaleucae Moore FSM, Fi, FP, Gu, NC, Pa, PNG Pucciniaceae Puccinia psidii G. Winter FSM, Fi, FP, Gu, NC, Pa, PNG Passifloraceae Passiflora tarminiana Coppens & V. E. Barney Mycosphaerellaceae Septoria passiflorae Pallister Gu Poaceae Arundo donax L. Eurytomidae Tetramesa romana Walker CI, FSM, Fi, FP, Gu, Na, NC, Pa, PNG, Sa, To, WF Polygonaceae Rumex crispus L. Sesiidae Pyropteron doryliformis (Ochsenheimer) Fi, FP, NC, PNG Solanaceae Solanum mauritianum Scop. Discussion Jacks.) Parmelee FP, NC, Va Pucciniaceae Puccinia xanthii Schwein. var. parthenii- hysterophorae Seier, H.C. Evans & Á. Romero FP, NC, Va Sesiidae Carmenta sp. nr ithacae (Beutenmüller) FP, NC, Va Tortricideae Platphalonidia mystica (Razowski & Becker) FP, NC, Va Michael D. Day & Rachel L. Winston / NeoBiota 30: 167–192 (2016) 182 Weed family Weed species Biocontrol agent family Biocontrol agent species No. of countries in Pacific agent established Possible countries for introduction# Pluchea carolinensis (Jacq.) G. Don Tephritidae Acinia picturata (Snow) CI, FP, Gu, Ki, MI, Na, NC, NMI, Pa, To, Va, WF Xanthium strumarium Cerambycidae Nupserha vexator CI, Fi, FP, Gu, NC, PNG, To Bignoniaceae Dolichandra unguis-cati Chrysomelidae Charidotis auroguttata Boheman CI, FSM, FP, Gu, NC, Ni, Va Tingidae Carvalhotingis hollandi Drake CI, FSM, FP, Gu, NC, Ni, Va Cactaceae Opuntia ficus-indica Cerambycidae Lagocheirus funestus Thomson FP, NC Dryophthoridae Metamasius spinolae (Gyllenhal) FP, NC Nectriaceae Fusarium oxysporum Schlecktendahl FP, NC Opuntia monacantha Dactylopiidae Dactylopius opuntiae AS, CI, FSM, Fi, Gu, Na, NC, Ni, NMI, Pa, Sa, SI, To Opuntia stricta Cerambycidae Moneilema blapsides (Newman) subsp. ulkei Horn NC, Sa, SI Convolvulaceae Convolvulus arvensis L. Eriophyidae Aceria malherbae Nuzzaci Pa Noctuidae Tyta luctuosa (Denis & Schiffermüller) Pa Fabaceae Acacia podalyriifolia A. Cunn. ex G. Don Curculionidae Melanterius maculatus NC Caesalpinia decapetala (Roth) Alston Chrysomelidae Sulcobruchus subsuturalis (Pic) Fi, FP, NC Leucaena leucocephala (Lam.) de Wit Chrysomelidae Acanthoscelides macrophthalmus (Schaeffer) AS, CI, FSM, Fi, FP, Gu, Ki, MI, Na, NC, Ni, NMI, Pa, PNG, PI, Sa, SI, To, Tu, Va, WF Mimosa pigra Brentidae Coelocephalapion pigrae Kissinger PNG Cerambycidae Rhytiphora piperitia Hope PNG Chrysomelidae Chlamisus mimosae Karren PNG Geometridae Leuciris fimbriaria (Stoll) PNG Parkinsonia aculeata Chrysomelidae Penthobruchus germaini (Pic) FSM, FP, Gu, NC, SI Prosopis juliflora (Sw.) DC. Chrysomelidae Algarobius prosopis (Le Conte) FP, PNG Ulex europaeus Brentidae Exapion ulicis (Forster) PNG Oecophoridae Agonopterix umbellana (Fabricius) PNG Biological control of weeds in the 22 Pacific island countries and territories... 183 Weed family Weed species Biocontrol agent family Biocontrol agent species No. of countries in Pacific agent established Possible countries for introduction# Pyralidae Pempelia genistella (Duponchel) PNG Tetranychidae Tetranychus linterarius Dufour PNG Thripidae Sericothrips staphylinus Haliday PNG Tortricidae Cydia succedana (Denis & Schiffermüller) PNG Vachellia nilotica subsp. Discussion indica Chrysomelidae Bruchidius sahlbergi Schilsky FP, NC, SI, WF Melastomataceae Clidemia hirta Buprestidae Lius poseidon Napp AS, FSM, Fi, Pa, PNG, Sa, SI, Va, WF Crambidae Ategumia matutinalis (Guenée) AS, FSM, Fi, Pa, PNG, Sa, SI, Va, WF Erebidae Antiblemma acclinalis Hübner AS, FSM, Fi, Pa, PNG, Sa, SI, Va, WF Glomerellaceae Colletotrichum clidemiae B. Weir & P.R. Johnst. AS, FSM, Fi, Pa, PNG, Sa, SI, Va, WF Momphidae Mompha trithalama Meyrick AS, FSM, Fi, Pa, PNG, Sa, SI, Va, WF Poaceae Arundo donax Diaspididae Rhizaspidiotus donacis Leonardi CI, FSM, Fi, FP, Gu, Na, NC, Pa, PNG, Sa, To, WF Polygonaceae Emex australis Brentidae Perapion antiquum (Gyllenhal) NC Pontederiaceae Eichhornia crassipes Crambidae Niphograpta albiguttalis AS, CI, FSM, Fi, FP, Gu, MI, Na, NC, NMI, Pa, PNG, Sa, SI, Va Crambidae Xubida infusella AS, CI, FSM, Fi, FP, Gu, MI, Na, NC, NMI, Pa, PNG, Sa, SI, Va Galumnidae Orthogalumna terebrantis Wallwork AS, CI, FSM, Fi, FP, Gu, MI, Na, NC, NMI, Pa, PNG, Sa, SI, Va Miridae Eccritotarsus catarinensis (Carvalho) AS, CI, FSM, Fi, FP, Gu, MI, Na, NC, NMI, Pa, PNG, Sa, SI, Va Salviniaceae Salvinia molesta Crambidae Samea multiplicalis CI, Fi, FP, Gu, NC, NMI, PNG Pauliniidae Paulinia acuminata CI, Fi, FP, Gu, NC, NMI, PNG Michael D. Day & Rachel L. Winston / NeoBiota 30: 167–192 (2016) 184 Weed family Weed species Biocontrol agent family Biocontrol agent species No. of countries in Pacific agent established Possible countries for introduction# Scrophulariaceae Buddleja davidii Franch. Curculionidae Cleopus japonicus Wingelmüller Fi, NC, PNG Verbenaceae Lantana camara Agromyzidae Calycomyza lantanae 7 AS, CI, FP, Ki, MI, Na, NC, Ni, NMI, PI, Sa, To, Tu, WF Agromyzidae Ophiomyia lantanae 11 AS, CI, Ki, MI, Na, Ni, PI, SI, Tu, WF Brentidae Coelocephalapion camarae Kissinger AS, CI, FSM, Fi, FP, Gu, Ki, MI, Na, NC, Ni, NMI, Pa, PNG, PI, Sa, SI, To, Tu, Va, WF Mycosphaerellaceae Passalora lantanae (Chupp) U. Braun & Crous var. lantanae AS, CI, FSM, Fi, FP, Gu, Ki, MI, Na, NC, Ni, NMI, Pa, PNG, PI, Sa, SI, To, Tu, Va, WF Mycosphaerellaceae Septoria sp. Discussion AS, CI, FSM, Fi, FP, Gu, Ki, MI, Na, NC, Ni, NMI, Pa, PNG, PI, Sa, SI, To, Tu, Va, WF Cerambycidae Plagiohammus spinipennis AS, CI, FSM, Fi, FP, Gu, Ki, MI, Na, NC, Ni, NMI, Pa, PNG, PI, Sa, SI, To, Tu, Va, WF Chrysomelidae Octotoma championi AS, CI, FSM, Fi, FP, Gu, Ki, MI, Na, NC, Ni, NMI, Pa, PNG, PI, Sa, SI, To, Tu, Va, WF Chrysomelidae Uroplata fulvopustulata AS, CI, FSM, Fi, FP, Gu, Ki, MI, Na, NC, Ni, NMI, Pa, PNG, PI, Sa, SI, To, Tu, Va, WF Crambidae Salbia haemorrhoidalis 2 AS, CI, FP, Gu, Ki, MI, Na, NC, Ni, NMI, Pa, PNG, PI, Sa, SI, To, Tu, Va, WF Erebidae Hypena laceratalis 7 AS, CI, FP, Ki, MI, Na, Ni, Pa, PI, Sa, SI, To, Tu, WF Gracillariidae Cremastobombycia lantanella Busck AS, CI, FSM, Fi, FP, Gu, Ki, MI, Na, NC, Ni, NMI, Pa, PNG, PI, Sa, SI, To, Tu, Va, WF 185 Biological control of weeds in the 22 Pacific island countries and territories... Weed family Weed species Biocontrol agent family Biocontrol agent species No. of countries in Pacific agent established Possible countries for introduction# Noctuidae Neogalea sunia 1 AS, CI, FSM, Fi, FP, Gu, Ki, MI, Na, Ni, NMI, Pa, PNG, PI, Sa, SI, To, Tu, Va, WF Pterophoridae Lantanophaga pusillidactyla 5 AS, CI, Fi, FP, Ki, MI, Na, NC, Ni, PI, Sa, SI, To, Tu, Va, WF Uropyxidaceae Prospodium tuberculatum (Spegazzini) Arthur AS, CI, FSM, Fi, FP, Gu, Ki, MI, Na, NC, Ni, NMI, Pa, PNG, PI, Sa, SI, To, Tu, Va, WF Tephritidae Eutreta xanthochaeta Aldrich AS, CI, FSM, Fi, FP, Gu, Ki, MI, Na, NC, Ni, NMI, Pa, PNG, PI, Sa, SI, To, Tu, Va, WF Tingidae Leptobyrsa decora AS, CI, FSM, Fi, FP, Gu, Ki, MI, Na, NC, Ni, NMI, Pa, PNG, PI, Sa, SI, To, Tu, Va, WF Tortricideae Crocidosema lantana 6 AS, CI, Fi, FP, Ki, Na, NC, Ni, PNG, PI, Sa, SI, To, Tu, WF Lantana montevidensis (Spreng.) Briq. Agromyzidae Calycomyza lantanae 7 FP, NC, WF Erebidae Hypena laceratalis 7 FP, SI, WF Pterophoridae Lantanophaga pusillidactyla 5 Fi, FP, NC, SI, WF Based on weed occurrence in each country, not weed density Weed family Michael D. Day & Rachel L. Winston / NeoBiota 30: 167–192 (2016) 186 Table 6. Weed species currently under evaluation outside the PICTs. Discussion Crambidae Neomusotima conspurcatalis (Warren) FSM, Fi, Gu, NMI, Pa, PNG, SI Eriophyidae Floracarus perrepae Knihinicki & Boczek FSM, Fi, Gu, NMI, Pa, PNG, SI Myrtaceae Psidium cattleianum Sabine Eriococcidae Tectococcus ovatus Hempel CI, FSM, Fi, FP, NC, Pa, PNG, PI, Sa, SI Passifloraceae Passiflora rubra L.*     AS, CI Pontederiaceae Eichhornia crassipes Acrididae Cornops aquaticum (Brüner) AS, CI, FSM, Fi, FP, Gu, MI, Na, NC, NMI, Pa, PNG, Sa, SI, Va Delphacidae Megamelus scutellaris Berg AS, CI, FSM, Fi, FP, Gu, MI, Na, NC, NMI, Pa, PNG, Sa, SI, Va Sapindaceae Cardiospermum grandiflorum Sw. Curculionidae Cissoanthonomus tuberculipennis Hustache CI, FP Verbenaceae Lantana camara Chrysomelidae Longitarsus bethae Savini & Escalona AS, CI, FSM, Fi, FP, Gu, Ki, MI, Na, NC, Ni, NMI, Pa, PNG, PI, Sa, SI, To, Tu, Va, WF Zingiberaceae Hedychium gardnerianum Sheppard ex Ker Gawl.*     CI, FSM, Fi, FP, NC # Based on weed occurrence in each country, not weed density * Field exploration and host specificity being conducted Weed family Weed species Biocontrol agent family Biocontrol agent species Possible countries for introduction# Lygodiaceae Lygodium microphyllum (Cav.) R. Br. Crambidae Neomusotima conspurcatalis (Warren) FSM, Fi, Gu, NMI, Pa, PNG, SI Eriophyidae Floracarus perrepae Knihinicki & Boczek FSM, Fi, Gu, NMI, Pa, PNG, SI Myrtaceae Psidium cattleianum Sabine Eriococcidae Tectococcus ovatus Hempel CI, FSM, Fi, FP, NC, Pa, PNG, PI, Sa, SI Passifloraceae Passiflora rubra L.*     AS, CI Pontederiaceae Eichhornia crassipes Acrididae Cornops aquaticum (Brüner) AS, CI, FSM, Fi, FP, Gu, MI, Na, NC, NMI, Pa, PNG, Sa, SI, Va Delphacidae Megamelus scutellaris Berg AS, CI, FSM, Fi, FP, Gu, MI, Na, NC, NMI, Pa, PNG, Sa, SI, Va Sapindaceae Cardiospermum grandiflorum Sw. Curculionidae Cissoanthonomus tuberculipennis Hustache CI, FP Verbenaceae Lantana camara Chrysomelidae Longitarsus bethae Savini & Escalona AS, CI, FSM, Fi, FP, Gu, Ki, MI, Na, NC, Ni, NMI, Pa, PNG, PI, Sa, SI, To, Tu, Va, WF Zingiberaceae Hedychium gardnerianum Sheppard ex Ker Gawl.*     CI, FSM, Fi, FP, NC # Based on weed occurrence in each country, not weed density * Field exploration and host specificity being conducted Weed family Weed species Biocontrol agent family Biocontrol agent species Possible countries for introduction# Lygodiaceae Lygodium microphyllum (Cav.) R. Br. Discussion Agents have either not been released to date, or have been released and not yet evaluated. Bio­ control agents could potentially be introduced against these weeds in the PICTs in the future. Countries: AS=American Samoa, CI=Cook Islands, FSM=Federated States of Micronesia, Fi=Fiji, FP=French Polynesia, Gu=Guam, Ki=Kiribati, MI=Marshall Islands, Na=Nauru, NC=New Caledonia, Ni=Niue, NMI=Northern Mariana Islands, Pa=Palau, PNG=Papua New Guinea, PI=Pitcairn Islands, Sa=Samoa, SI=Solomon Islands, Tk=Tokelau, To=Tonga, Tu=Tuvalu, Va=Vanuatu, WF=Wallis & Futuna. Weed family Weed species Biocontrol agent family Biocontrol agent species Possible countries for introduction# Asteraceae Ageratina adenophora Pucciniosiraceae Baeodromus eupatorii (Arthur) Arthur FP Chromolaena odorata Curculionidae Lixus aemulus Petri FSM, Gu, MI, NC, NMI, Pa, PNG Tortricidae Dichrorampha odorata Brown & Zachariades FSM, Gu, MI, NC, NMI, Pa, PNG Bignoniaceae Spathodea campanulata P. Beauv.*   AS, CI, FSM, Fi, FP, Gu, Ki, MI, Na, NC, Ni, NMI, Pa, PNG, PI, Sa, SI, To, Va, WF Tecoma stans (L.) Juss. ex Kunth var. stans Coccinellidae Mada polluta (Mulsant) AS, CI, FSM, Fi, FP, Gu, Ki, MI, Na, NC, Ni, NMI, Pa, PNG, Sa, SI, To, WF Dolichandra unguis-cati Pyralidae Hypocosmia pyrochroma Jones CI, FSM, FP, Gu, NC, Ni, Va Cactaceae Pereskia aculeata Coreidae Catorhintha schaffneri Brailovsky & Garcia FP, NC, Pa Commelinaceae Tradescantia fluminensis Vell. Chrysomelidae Lema basicostata Monros FP, Na Chrysomelidae Neolema abbreviata Lacordaire FP, Na Chrysomelidae Neolema ogloblini (Monros) FP, Na Dioscoreaceae Dioscorea bulbifera L. Chrysomelidae Lilioceris cheni Gressitt & Kimoto AS, FSM, Fi, FP, Gu, MI, Ni, NMI, Pa, PNG, Sa, SI, To, Va, WF Fabaceae Falcataria moluccana (Miq.) Barneby & J.W. Grimes*     AS, CI, FSM, Fi, FP, Gu, NC, Ni, Pa, PNG, Sa, SI, To, WF Mimosa pigra Chrysomelidae Nesaecrepida infuscata (Schaeffer) PNG Raveneliaceae Diabole cubensis (Arthur & J.R. Johnst.) Arthur PNG Parkinsonia aculeata L. Geometridae Eueupithecia cisplatensis Prout FSM, FP, Gu, NC, SI Lamiaceae Clerodendrum chinensis (Osbeck) Mabb. Chrysomelidae Phyllocharis undulata (L.) AS, CI, FSM, Fi, FP, Gu, Ni, NMI, PNG, Sa, SI, To, Va 187 Biological control of weeds in the 22 Pacific island countries and territories... Weed family Weed species Biocontrol agent family Biocontrol agent species Possible countries for introduction# Lygodiaceae Lygodium microphyllum (Cav.) R. Br. Discussion Crambidae Neomusotima conspurcatalis (Warren) FSM, Fi, Gu, NMI, Pa, PNG, SI Eriophyidae Floracarus perrepae Knihinicki & Boczek FSM, Fi, Gu, NMI, Pa, PNG, SI Myrtaceae Psidium cattleianum Sabine Eriococcidae Tectococcus ovatus Hempel CI, FSM, Fi, FP, NC, Pa, PNG, PI, Sa, SI Passifloraceae Passiflora rubra L.*     AS, CI Pontederiaceae Eichhornia crassipes Acrididae Cornops aquaticum (Brüner) AS, CI, FSM, Fi, FP, Gu, MI, Na, NC, NMI, Pa, PNG, Sa, SI, Va Delphacidae Megamelus scutellaris Berg AS, CI, FSM, Fi, FP, Gu, MI, Na, NC, NMI, Pa, PNG, Sa, SI, Va Sapindaceae Cardiospermum grandiflorum Sw. Curculionidae Cissoanthonomus tuberculipennis Hustache CI, FP Verbenaceae Lantana camara Chrysomelidae Longitarsus bethae Savini & Escalona AS, CI, FSM, Fi, FP, Gu, Ki, MI, Na, NC, Ni, NMI, Pa, PNG, PI, Sa, SI, To, Tu, Va, WF Zingiberaceae Hedychium gardnerianum Sheppard ex Ker Gawl.*     CI, FSM, Fi, FP, NC # Based on weed occurrence in each country not weed density 188 Michael D. Day & Rachel L. Winston / NeoBiota 30: 167–192 (2016) However, many biocontrol agents that have established in the PICTS are only found in a fraction of the countries in which their respective target weed occurs. This could be because weed densities in countries where agents are not present are not high enough to warrant biocontrol, or because human population base, infrastructure, expertise, experience and funding to implement biocontrol programmes are limited (Dovey et al. 2004). Both the Secretariat of the Pacific Community (SPC) and the South Pacific Re­ gional Environmental Program (SPREP) have a responsibility in helping member countries in agricultural and environmental issues respectively, and could therefore assist in coordination of biocontrol programmes, while Australia, the USA and New Zealand could help in a technical capacity, especially regarding the additional testing of biocontrol agents (Dovey et al. 2004). g Another constraint to successfully implementing biocontrol in the PICTs is due to the nature of the Pacific. The Pacific region covers 30 million km2, of which only 2% is landmass and is spread over 7,500 islands (Shine et al. 2003). Therefore, releasing biocontrol agents into all countries and on all islands where target weeds occur can be challenging and expensive (Dovey et al. 2004, Day et al. 2013a, c). This contrasts greatly with Asia or Africa where biocontrol agents have readily spread within and to other countries, as weed populations are often contiguous (Winston et al. 2014). Discussion To help overcome these logistical difficulties, many biocontrol programs in the Pacific region have been funded by donor organisations from Australia, Europe, the USA and New Zealand and/or have involved the assistance of the SPC. Within these programs, substantial funds are frequently allocated to conducting weed and biocontrol agent distribution surveys in order to identify locations where a target weed is present but no agents have established. Such surveys have been con­ ducted recently in Papua New Guinea and Vanuatu, with funding from the Australian Government. Program funds are also frequently spent on increasing capacity, such as improving infrastructure and training staff, as well as releasing biocontrol agents.f f A cost-effective solution to weed biocontrol research in the PICTs is to redistribute effective agents already established in the region (Dovey et al. 2004, Julien et al. 2007, Paynter et al. 2015). In general, redistribution of agents within the Pacific requires little to no extra host specificity testing because plant assemblages are often similar be­ tween countries, and many agents have been established long enough to both identify the most highly effective agents and to detect any non-target impacts. Utilising tried and proven agents overcomes the considerable cost of host specificity testing of new agents, and reduces the likelihood of agents not establishing or having minimal impact on the target weeds (Julien et al. 2007, Paynter et al. 2015).i Countries wishing to introduce any biocontrol agent from within the Pacific re­ gion should conduct surveys to determine what agents are already present in their country. There are many examples of agents previously not reported, being found in countries following the conduct of dedicated or even opportunistic surveys (Winston et al. 2014). Regardless of the mode of entry into a country, once established within the region, biocontrol agents can spread naturally to new islands and/or countries. Cal­ Biological control of weeds in the 22 Pacific island countries and territories... 189 ligrapha pantherina was released onto only 14 islands in Vanuatu and is now present on 21 islands (Day and Bule this edition). Within the PICTs, Calycomyza lantanae was deliberately released into only Fiji for the control of L. camara, but it is now found in seven countries in the PICTs. Incidentally, although C. lantanae has only ever been deliberately released into three countries (Australia, Fiji and South Africa), it is now found in 28 countries worldwide (Day et al. Discussion 2003, Winston et al. 2014). In addition to redistributing agents already established within the PICTS, there are many more biocontrol agents released outside the PICTs that cause medium to high impacts on their target weed(s) and could be considered for introduction into the PICTs (Winston et al. 2014). However, such agents may not have the same efficacy in the PICTs, so climate-matching and other suitability studies may need to be con­ ducted prior to their consideration. More importantly, because host specificity testing of these agents may have occurred in regions with very different plant assemblages, PICTs wishing to import particular agents from outside the region should determine if additional host specificity testing is required prior to the agents’ importation. pi g q p g p Under an Australian Government funded programme, Puccinia spegazzinii was tested against an additional 17 local plant species by CABI prior to its introduction into PNG and Fiji. This was despite the agent being tested against 170 species on behalf of India and China prior to its introduction into those countries (Day et al. 2013b). Conversely, both Neochetina spp. and C. pantherina were introduced into the PICTs without any additional testing following their testing and subsequent release in Australia (Julien et al. 2007). J Biocontrol is seen as the most cost-effective, environmentally friendly, and sustain­ able option to manage many weeds in the Pacific and elsewhere. Utilising tried and proven agents that are both host specific and effective against the target weed species in other countries maximises the chance of success in new countries while minimising the risks of non-target impacts (Dovey et al. 2004, Julien et al. 2007, Paynter et al. 2015). With over 60 agents already deliberately released against more than 20 weed species, biocontrol of weeds in the PICTs is not a new concept. However, as many of these agents are found in only a few countries, there is great potential to manage the target weeds in other countries in the Pacific through their redistribution. In addition, highly damaging and host specific agents established outside the Pacific could be introduced to control those weed species not yet targeted.h g Through coordinated responses, possibly involving the SPC and the SPREP, as well as Australia, the USA and New Zealand, the impacts of weeds in the Pacific region can be reduced through biocontrol, and food security for its inhabitants increased. Acknowledgements The authors wish to thank researchers within National Agricultural Research Institute and National Agricultural Quarantine and Inspection Authority, Papua New Guinea, Biosecurity Vanuatu, and the Secretariat of the Pacific Community for their input into Michael D. Day & Rachel L. Winston / NeoBiota 30: 167–192 (2016) 190 projects over the past few decades. The USDA Forest Service assisted with funding the production of the weed biocontrol catalogue, while Landcare Research New Zealand Ltd funded attendance at the EMAPI Conference held in Hawaii. 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https://openalex.org/W2944092791
https://europepmc.org/articles/pmc6572060?pdf=render
English
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Prevalence and Risk Factors for Positive Nasal Methicillin-Resistant Staphylococcus aureus Carriage among Orthopedic Patients in Korea
Journal of clinical medicine
2,019
cc-by
6,607
Received: 14 April 2019; Accepted: 7 May 2019; Published: 8 May 2019 Abstract: Methicillin-resistant Staphylococcus aureus (MRSA) causes purulent skin and soft tissue infections as well as other life-threatening diseases. Recent guidelines recommend screening for MRSA at the time of admission. However, few studies have been conducted to determine the prevalence and risk factors for MRSA colonization. A prospective data collection and retrospective analysis was performed. MRSA screening tests were performed using nasal swabs in patients enrolled between January 2017 and July 2018. Demographic data, socio-economic data, medical comorbidities, and other risk factors for MRSA carriage were evaluated among 1577 patients enrolled in the study. The prevalence of MRSA nasal carriage was 7.2%. Univariate regression analysis showed that colonization with MRSA at the time of hospital admission was significantly related to patient age, body mass index, smoking, alcohol, trauma, recent antibiotic use, and route of hospital admission. Multiple logistic regression analysis for the risk factors for positive MRSA nasal carriage showed that being under- or overweight, trauma diagnosis, antibiotic use one month prior to admission, and admission through an emergency department were related to MRSA colonization. This study highlights the importance of a preoperative screening test for patients scheduled to undergo surgery involving implant insertion, particularly those at risk for MRSA. Keywords: Methicillin-resistant Staphylococcus aureus (MRSA); nasal screening test; infection; prevalence; risk factors Sung-Woo Choi 1,†,*, Jae Chul Lee 1 , Jahyung Kim 1, Ji Eun Kim 2, Min Jung Baek 3,†, Se Yoon Park 4, Suyeon Park 5 and Byung-Joon Shin 1,* 1 Department of Orthopedic Surgery, Soonchunhyang University Hospital, College of Medicine, Seoul 04401, Korea; jlee@schmc.ac.kr (J.C.L.); t0152@schmc.ac.kr (J.K.) 1 Department of Orthopedic Surgery, Soonchunhyang University Hospital, College of Medicine, Seoul 04401, Korea; jlee@schmc.ac.kr (J.C.L.); t0152@schmc.ac.kr (J.K.) Department of Laboratory Medicine, Soonchunhyang University College of Medicine, Seoul 04401, Korea; jkim@schmc.ac.kr 2 Department of Laboratory Medicine, Soonchunhyang University College of Medicine, Seoul 04401, Kore jkim@schmc.ac.kr 3 Department of Obstetrics and Gynecology, Bundang CHA Hospital, Seongnam 13496, Korea; goodgood75@naver.com Department of Obstetrics and Gynecology, Bundang CHA Hospital, Seongnam 13496, Korea; goodgood75@naver.com 4 Division of Infectious Diseases, Department of Internal Medicine, Soonchunhyang University Hospital College of Medicine, Seoul 04401, Korea; sypark@schmc.ac.kr g yp 5 Department of Biostatistics, Soonchunhyang University College of Medicine, Seoul 04401, Korea; sue3517@schmc.ac.kr * Correspondence: swchoi@schmc.ac.kr (S.-W.C.); schsbj@schmc.ac.kr (B.-J.S.); Tel.: +82-2-709-9259 (S.-W.C.) +82-2-709-9250 (B.-J.S.) † These authors contributed equally to this paper. Received: 14 April 2019; Accepted: 7 May 2019; Published: 8 May 2019 Journal of Clinical Medicine Journal of Clinical Medicine Journal of Clinical Medicine Journal of Clinical Medicine Prevalence and Risk Factors for Positive Nasal Methicillin-Resistant Staphylococcus aureus Carriage among Orthopedic Patients in Korea Sung-Woo Choi 1,†,*, Jae Chul Lee 1 , Jahyung Kim 1, Ji Eun Kim 2, Min Jung Baek 3,†, Se Yoon Park 4, Suyeon Park 5 and Byung-Joon Shin 1,* 1. Introduction The emergence of methicillin-resistant Staphylococcus aureus (MRSA) since the early 1960s and its spread throughout hospitals and communities decades later have complicated antibiotic therapy [1–5]. The rate of methicillin resistance is higher in the orthopedics department, where artificial implantation is more common than in other medical specialties [6]. If the infection is caused by MRSA, either due to J. Clin. Med. 2019, 8, 631; doi:10.3390/jcm8050631 www.mdpi.com/journal/jcm www.mdpi.com/journal/jcm J. Clin. Med. 2019, 8, 631 2 of 11 antibiotic resistance or biofilm formation, the treatment of an orthopedic infection becomes difficult and may lead to a higher economic burden [7]. Therefore, early detection and prevention of MRSA are important, particularly among orthopedic patients. The recent guidelines published by the Society for Health Care Epidemiology for America recommend surveillance of cultures at the time of hospital admission for patients scheduled to undergo high-risk surgeries, including some orthopedic and cardiothoracic procedures [8,9]. The Korea Center for Disease Control and Prevention suggests a screening test only for patients who are admitted to high-risk departments such as the intensive care unit, the hemato-oncology department, and the organ transplantation department; patients with a prior diagnosis of MRSA; and patients transferred from a nursing facility [10]. The recommended screening method for MRSA is nasal sampling because the anterior nasal cavity is one of the preferred carrier sites of this bacterium and because the frequency of skin colonization depends on nasal carriage [11,12]. In addition, Yano et al. [13] reported that patients with positive preoperative nasal cultures for MRSA had a higher occurrence of surgical site infection with MRSA after orthopedic surgery. However, a limited number of studies have been conducted to determine the prevalence and risk factors for colonization at the time of admission in orthopedic patients [14–18], especially in Korea. The prevalence of MRSA is extremely high in Korea. The Asian Network for Surveillance of Resistant Pathogens (ANSORP) study, which included seven hospitals in Korea, showed an average MRSA prevalence of 77.6% for nosocomial S. aureus isolates [19]. The recent report of the Regional Resistance Surveillance (RRS) program showed that 73% of the clinical S. aureus isolates from two hospitals in Korea were MRSA [20]. Korea has the highest MRSA prevalence among the 12 surveillance countries in the RRS program [20,21]. The present study primarily aimed to assess the prevalence of MRSA carriage in the orthopedic department. 1. Introduction The secondary objective was to identify potential risk factors for MRSA colonization with respect to the demographic and medical data. 2.2. Sampling The patients, who were admitted to the hospital during the study period and satisfied the inclusion criteria, had nasal swabs collected from them. All sampling was performed by well-trained orthopedic nurses. Nasal swabs were obtained within a day of admission. A sterile transport swab (COPAN, Brescia, Italy) was rotated in the anterior nasal cavity of the patients by the nurses. The swab was transported at room temperature and each swab was processed as described here within four hours of collection. Direct culture was performed onto chromID MRSA agar (bioMérieux, Nürtingen, Germany) and was examined at 24 and 48 hours after incubation. A positive culture was defined as growth with morphological features comparable to MRSA. This was confirmed by the coagulase test using a commercial latex agglutination kit (Pastorex Staph Plus, Bio Rad Laboratories, Hemel Hempstead, UK). 2.1. Inclusion and Exclusion Criteria The patients admitted to the orthopedic surgery department of Soonchunhyang University Hospital, Korea were enrolled in the study based on the following inclusion criteria: patients aged 18 to 90 years were included in the study and patients with a current infection were excluded. 2. Material and Methods The study involved prospective data collection and retrospective data analysis and was conducted between January 2017 and July 2018 at the orthopedic surgery department of Soonchunhyang University Hospital and approved by the Institutional Review Board (IRB No.: SCHUH 2018-11-021). 2.3. Data Collection Data collection was coordinated by an orthopedic surgeon who completed standardized forms for each included patient. Overall data was recorded for the following variables: demographic 3 of 11 J. Clin. Med. 2019, 8, 631 characteristics, socio-economic characteristics, medical comorbidities, and other risk factors for MRSA carriage. Sex, age, body mass index (BMI), history of smoking and alcohol consumption, diagnosis of trauma or disease, and body part involved were analyzed as demographic data. BMI was divided into four groups (underweight (<18.5 kg/m2), normal weight (18.5–25 kg/m2), overweight (25–30 kg/m2), and obese (>30 kg/m2)) [22]. Smoking history was classified into three groups (never-smoker, ex-smoker, and current-smoker). An ex-smoker was defined as someone who had smoked more than 100 cigarettes in their lifetime but had not smoked in the last 28 days [23]. A current-smoker was defined as an adult who had smoked more than 100 cigarettes in his or her lifetime and was still smoking. History of alcohol consumption was divided into three groups (never-drinker, former-drinker, and current-drinker). A current-drinker was a person who consumed up to 12 drinks per year, while a former-drinker was a patient who had stopped drinking for >1 year [24]. The body part involved was divided into five categories (spine, knee and shoulder, hand and elbow, hip, and foot). Using the ICD-10-CM (International Classification of Diseases, Tenth Revision, Clinical Modification) codes, patients were divided into two groups (coded as trauma (S) and disease (M)). Education status was collected as socio-economic data [25–27]. Medical comorbidities included hypertension, diabetes mellitus, cardiovascular diseases, hepatic diseases, and dialysis. Other risk factors for MRSA carriage were recent hospitalization, recent antibiotics use, presence of a ureteral catheter, and type of hospital admission (i.e., whether emergency or outpatient department) (Table 1). Recent hospitalization and recent antibiotic use were defined as stationary hospitalization within 12 months prior to admission and antimicrobial therapy within one month before the screening test, respectively [28]. Table 1. Overview of collected patient data. Table 1. Overview of collected patient data. 2.3. Data Collection Patient Data Type of Acquired Information Demographic Gender Age Body mass index (BMI) Smoking (current-smoker/ex-smoker/never-smoker) Regular consumption of alcohol (current-drinker/former-drinker/never-drinker) Socio-economic Status of education (under high school/high school graduate/denied or unanswered) Medical comorbidities Hypertension, diabetes mellitus, cardiovascular diseases, hepatic disease, dialysis Other risk factors Stationary hospitalization in last 12 months Antimicrobial therapy in the past month Presence of urethral catheter Route of admission (ER/OPD) Trauma/disease Body parts involved (spine/knee and shoulder/hand and elbow/hip/foot) ER = Emergency Room, OPD = Outpatient department. Table 1. Overview of collected patient data. Table 1. Overview of collected patient data. Patient Data Type of Acquired Information Demographic Gender Age Body mass index (BMI) Smoking (current-smoker/ex-smoker/never-smoker) Regular consumption of alcohol (current-drinker/former-drinker/never-drinker) Socio-economic Status of education (under high school/high school graduate/denied or unanswered) Medical comorbidities Hypertension, diabetes mellitus, cardiovascular diseases, hepatic disease, dialysis Other risk factors Stationary hospitalization in last 12 months Antimicrobial therapy in the past month Presence of urethral catheter Route of admission (ER/OPD) Trauma/disease Body parts involved (spine/knee and shoulder/hand and elbow/hip/foot) ER = Emergency Room, OPD = Outpatient department. 2.4. Statistical Analysis Statistical analysis was performed using parametric or nonparametric tests, where appropriate. Logistic regression analysis was used to identify independent predictors of colonization with MRSA at the time of admission to the hospital as well as clinical conditions associated with the development of symptomatic MRSA infection. The association between the risk factors and MRSA colonization was analyzed using the chi-square test or Fisher’s exact test, as appropriate. Data were analyzed using SPSS Statistics (Statistical Package for the Social Sciences, version 25.0; 2017, IBM Corp. Armonk, NY, USA) and two-tailed p-values of ≤0.05 were considered as statistically significant. 4 of 11 J. Clin. Med. 2019, 8, 631 3. Results 3.1. Overall Result 3.1. Overall Result A total of 1577 patients were enrolled during the study period. Of these, 617 (39.1%) were women and 960 (60.9%) were men. The average patient age was 59.2 years; particularly, 288 patients (18.3%) were aged under 40 years, 776 (49.2%) were aged between 40 and 70 years, and 513 (32.5%) were aged older than 70 years. A total of 1332 patients (84.5%) were tested in the ward after admission, while 245 (15.5%) were tested in the outpatient department. Ultimately, 114 patients were found to be colonized with MRSA; thus, the prevalence of nasal MRSA carriage was 7.2%. Of the 114 colonized samples, 99 (86.8%) samples were collected after admission and 15 (13.2%) samples were collected in the outpatient department. 3.2.1. Univariate Logistic Regression Analysis Selected variables of interest were subjected to analysis (Tables 2 and 3). The results of the univariate logistic regression analysis showed that the colonization with MRSA at the time of hospital admission was significantly related to patient age, BMI, smoking, alcohol consumption, body part involved, diagnosis of trauma, type of hospital admission, and recent antibiotic use (Table 4). Patients aged 40 to 70 years and those older than 70 years had 0.237 (95% Confidence Interval (CI): 0.151–0.374; p < 0.001) and 0.292 (95% CI: 0.180–0.474; p < 0.001) times the risk of being a carrier of MRSA as compared to those aged under 40 years, respectively. Additionally, patients whose BMI was under 18.5 kg/m2, 25 to 30 kg/m2, and higher than 30 kg/m2 had 3.432 (95% CI: 1.308–9.000; p = 0.012), 0.290 (95% CI: 0.153–0.550; p < 0.01), 0.637 (95% CI: 0.238–1.702; p = 0.369) times the risk of being an MRSA carrier than those with a normal BMI. Current-smokers had an odds ratio of 4.204 (95% CI: 2.801–6.309; p < 0.01) and current-drinkers had an odds ratio of 4.204 (95% CI: 3.250–7.223; p < 0.001). Patients diagnosed with trauma had 3.401 (95% CI: 1.593–7.261; p < 0.01) times the risk of being an MRSA carrier than those diagnosed with disease. Furthermore, those who were recently treated with antibiotics tended to have an increased chance of being an MRSA carrier, with an odds ratio of 4.199 (95% CI: 2.831–6.228; p < 0.01). Lastly, patients admitted through the emergency department had 6.915 (95% CI: 2.863–16.702; p < 0.01) times the risk of being an MRSA carrier than those admitted through the outpatient department. Meanwhile, there was no strong association between MRSA carriage and other medical comorbidities, recent hospitalization, the presence of a urinary catheter, and socio-economic background. Table 2. Descriptive logistic regression analysis from data obtained at admission (demographics, socio-economic status). Number of Patients MRSA (−) (1463 Patients) MRSA (+) (114 Patients) p-Value a N = 1577 (%) N (%) N (%) Gender 0.359 Male 617 (39.1%) 577 (93.5%) 40 (6.5%) Female 960 (60.9%) 886 (92.3%) 74 (7.7%) Age group <0.01 <40 288 (18.3%) 239 (82.9%) 49 (17.1%) 40–70 776 (49.2%) 740 (95.4%) 36 (4.6%) ≥70 513 (32.7%) 484 (94.3%) 29 (5.7%) BMI group <0.01 Underweight (<18.5) 159 (10.1%) 116 (73.0%) 43 (27.0%) Table 2. Descriptive logistic regression analysis from data obtained at admission (demographics, socio-economic status). 5 of 11 J. 3.2.1. Univariate Logistic Regression Analysis Clin. Med. 2019, 8, 631 Table 2. Cont. Number of Patients MRSA (−) (1463 Patients) MRSA (+) (114 Patients) p-Value a N = 1577 (%) N (%) N (%) Normal (18.5–25) 735 (46.6%) 686 (93.3%) 49 (6.7%) Overweight (25–30) 566 (35.9%) 549 (97.0%) 17 (3.0%) Obese (≥30) 117 (7.4%) 112 (95.7%) 5 (4.3%) Smoking <0.01 Never-smoker 1294 (82.1%) 1226 (94.7%) 68 (5.3%) Current-smoker 238 (15.1%) 193 (81.1%) 45 (18.9%) Ex-smoker 45 (2.9%) 44 (97.8%) 1 (2.2%) Alcohol <0.01 Never-drinker 1292 (81.9%) 1228 (95.0%) 64 (5.0%) Current-drinker 248 (15.7%) 198 (79.8%) 50 (20.2%) Former-drinker 37 (2.3%) 37 (100.0%) 0 (0.0%) Status of education 0.372 Under high school 89 (5.6%) 82 (92.1%) 7 (7.9%) High school graduate 420 (26.6%) 374 (89.0%) 46 (11.0%) Denied, Unanswered 1068 (67.7%) 1007 (94.3%) 61 (5.7%) x% = Column percentage; N (xx%) = Number of patients (row percentage). OR = Odds ratio; CI = Confidence Interval; a = chi-square test. MRSA = Methicillin-resistant Staphylococcus aureus. = Column percentage; N (xx%) = Number of patients (row percentage). OR = Odds ratio; CI = Confidence erval; a = chi-square test. MRSA = Methicillin-resistant Staphylococcus aureus. Table 3. Descriptive logistic regression analysis from data obtained at admission (medical comorbidities, other risk factors for MRSA). able 3. Descriptive logistic regression analysis from data obtained at admission (medical comorbiditie ther risk factors for MRSA). Number of Patients MRSA (−) (1463 Patient) MRSA (+) (114 Patient) p-Value a N = 1577 (%) N (%) N (%) Hypertension 0.059 No 919 (58.3%) 843 (91.7%) 76 (8.3%) Yes 658 (41.7%) 620 (94.2%) 38 (5.8%) Cardiovascular disease 0.510 No 1486 (94.2%) 1377 (92.7%) 109 (7.3%) Yes 91 (5.8%) 86 (94.5%) 5 (5.5%) Diabetes Mellitus 0.697 No 1236 (78.4%) 1145 (92.6%) 91 (7.4%) Yes 341 (21.6%) 318 (93.3%) 23 (6.7%) Hepatic disease 0.516 No 1502 (95.2%) 1392 (92.7%) 110 (7.3%) Yes 75 (4.8%) 71 (94.7%) 4 (5.3%) Dialysis 0.405 * No 1565 (99.2%) 1451 (92.7%) 114 (7.3%) Yes 12 (0.8%) 12 (100.0%) 0 (0.0%) Recent hospitalization 0.951 No 1172 (74.3%) 1087 (92.7%) 85 (7.3%) Yes 405 (25.7%) 376 (92.8%) 29 (7.2%) Recent antibiotics <0.01 No 1105 (70.1%) 1061 (96.0%) 44 (4.0%) Yes 472 (29.9%) 402 (85.2%) 70 (14.8%) 6 of 11 J. Clin. Med. 2019, 8, 631 Table 3. Cont. 4. Discussion According to this study, the overall prevalence of nasal MRSA carriage is 7.2%. There is a huge difference in the epidemiology of MRSA worldwide. For example, the prevalence of community-associated MRSA infection in Japan, Germany, Turkey, Taiwan, and Malta was found to be 0.94% [29], 1.2% [17], 1.2% [30], 3.8% [31], and 8.81% [6] respectively. These differences can be attributed to variations in microbiological methods (sampling technique, culture sites, and method of MRSA identification), local infection control standards, and the local prevalence of MRSA. Gi et al. [32] reported that the methicillin resistance rate of staphylococcal isolates in Korea is slightly higher than that in other countries. Another study showed that the prevalence of MRSA is 73% among the nosocomial S. aureus isolates in Korea, which is higher than that in other Asian countries [21]. Therefore, we considered it worthwhile to study the prevalence and risk factors for MRSA colonization in the orthopedic field, where aseptic surgery is essential. Several studies have evaluated the risk factors associated with MRSA carriage [33–38]. Of the many known risk factors, the present study identified being underweight, having a diagnosis of trauma, recent antibiotic use, and admission through the emergency department as significant risk factors for MRSA carriage. Contrary to some prior studies, the present study did not reveal age as a significant risk factor [35,36]. The majority of studies have reported that the elderly patients have a higher tendency of being an MRSA carrier. An increased burden of infection in the elderly is linked to age-related decline in immune function, malnutrition, and anatomical and physiological changes [35]. However, some studies have reported that MRSA colonization is not influenced by age [39], and others found a higher carriage rate in younger patients [36]. Therefore, the association between age and MRSA colonization remains controversial. Our results showed that being underweight is a risk factor for MRSA colonization and being overweight decreases the colonization rate. However, obesity did not turn out to be a significant risk factor for MRSA colonization in this study. Neidhart et al. [17] reported a reduced risk for S. aureus carriage in obese (BMI ≥30.0 kg/m2) compared to overweight patients (BMI of 25.0 to 30 kg/m2). However, other studies reported the opposite result. Olsen et al. [40] found a significant positive correlation between BMI and MRSA carriage only in women, particularly among those aged 30–43 years. Campbell et al. 3.2.2. Multiple Logistic Regression Analysis Table 4 shows the results of the multiple logistic regression analysis for the risk factors found statistically significant in the univariate logistic regression analysis. Being underweight, having trauma, recent antibiotic use, and admission through the emergency room were found to be risk factors. Patients with a BMI lower than 18.5 kg/m2 had a 2.026 times higher risk of nasal MRSA carriage than patients with normal BMI. Patients diagnosed with trauma had a 1.795 times higher rate of nasal MRSA carriage than those diagnosed with disease. In addition, patients with recent antibiotics use had a 1.946 times higher risk of MRSA carriage than patients without antibiotic use. Furthermore, patients admitted through the emergency department had a 3.998 times higher MRSA carriage rate than those admitted through the outpatient department. 3.2.1. Univariate Logistic Regression Analysis Number of Patients MRSA (−) (1463 Patient) MRSA (+) (114 Patient) p-Value a N = 1577 (%) N (%) N (%) Urinary catheter 0.985 No 1329 (84.3%) 1233 (92.8%) 96 (7.2%) Yes 248 (15.7%) 230 (92.7%) 18 (7.3%) Route of admission <0.01 ER 227 (14.4%) 174 (76.7%) 53 (23.3%) OPD 1350 (85.6%) 1289 (95.5%) 61 (4.5%) Body part involved 0.343 Spine 567 (36.0%) 543 (95.8%) 24 (4.2%) Knee, shoulder 531 (33.7%) 506 (95.3%) 25 (4.7%) Hand, elbow 64 (4.1%) 64 (100.0%) 0 (0.0%) Hip 127 (8.1%) 119 (93.7%) 8 (6.3%) Foot 288 (18.1%) 231 (80.2%) 57 (19.8%) Disease/Trauma <0.01 Trauma 560 (35.5%) 500 (89.3%) 60 (10.7%) Disease 1017 (64.5%) 963 (94.7%) 54 (5.3%) x% = Column percentage; N (xx%) = Number of patients (row percentage). OR = Odds ratio; CI = Confidence Interval. a = chi-square test or * Fisher’s exact test. x% = Column percentage; N (xx%) = Number of patients (row percentage). OR = Odds ratio; CI = Confidence Interval. a = chi-square test or * Fisher’s exact test. Table 4. Univariable and multivariable logistic regression analysis of the risk factors associated with MRSA colonization. Table 4. Univariable and multivariable logistic regression analysis of the risk factors associated with MRSA colonization. Variables Univariable Analysis OR (95% CI) p-Value Multivariable Analysis Adjusted OR (95% CI) p-Value Age <40 1 40–70 0.237 (0.151–0.374) <0.01 ** >70 0.292 (0.180–0.474) <0.01 ** BMI Normal weight 1 1 Underweight 3.432 (1.308–9.000) 0.012 ** 2.026 (1.115–3.680) 0.020** Overweight 0.290 (0.153–0.550) <0.01 ** 0.389 (0.219–0.689) <0.01 ** Obese 0.637 (0.238–1.702) 0.369 0.637 (0.246–1.650) 0.354 Smoking Never-smoker 1 Current-smoker 4.204 (2.801–6.309) <0.01 ** Ex-smoker 0.410 (0.056–3.019) 0.381 Alcohol Never-drinker 1 Current-drinker 4.204 (3.250–7.223) <0.01 ** Former-drinker N/A Hypertension 0.680 (0.454–1.017) 0.061 Code Disease 1 1 Trauma 3.401 (1.593–7.261) <0.01 ** 1.795 (0.984–3.275) 0.048 ** Recent antibiotics use 4.119 (2.831–6.228) <0.01 ** 1.946 (1.178–3.217) <0.01 ** Route of admission OPD 1 1 ER 6.915 (2.863–16.702) <0.01 ** 3.998 (2.003–7.979) 0.047 ** **: p < 0.05. J. Clin. Med. 2019, 8, 631 7 of 11 7 of 11 4. Discussion [41] reported an increased risk of S. aureus colonization in patients with both obesity and asthma. The analysis of skin and soft tissue staphylococcal infections showed that obesity is related to the presence of methicillin resistance. Therefore, further studies are needed to prove a definite relation between BMI and MRSA colonization. In this study, smoking was not found to be a statistically significant risk factor for MRSA carriage. The influence of smoking on the colonization of MRSA is still controversial. Some studies proposed that smokers have higher rates of MRSA colonization than nonsmokers, thus increasing their risk of serious and difficult-to-treat infections [42,43]. Ellisa et al. reported that cigarette smoke increases MRSA hydrophobicity, thus increasing MRSA adherence and invasion [43]. They also stated that cigarette smoking increases the MRSA expression of genes linked to cell surface changes. In contrast, the smoke itself seems to influence the S. aureus load as indicated by the reduced identification rate J. Clin. Med. 2019, 8, 631 8 of 11 in the upper respiratory tract [44]. A possible explanation for this finding is the inherent toxicity of smoke. The capability of smoke to inhibit the growth capacity of Gram-positive bacteria, particularly S. aureus, has already been demonstrated [45]. However, whether smoking affects MRSA colonization remains unclear. In our study, patients admitted through the emergency department and those diagnosed with trauma had 3.998 and 1.795 times higher risk of MRSA colonization, respectively. Most trauma patients tend to visit the emergency department, and these two factors were considered by us to be similar. Quach et al. [46] reported that a visit to the emergency department is associated with more than threefold increased risk of acute infection. Patients with morbid conditions are more likely to visit the emergency department than patients with less morbid conditions. In addition, admission through the emergency department may increase the chance of contact with these patients, which may be expected to increase the risk of acquiring an infection. Also, patterns of injury differ between different mechanisms of trauma [47] which may cause patients diagnosed with trauma to be more susceptible to MRSA. For example, certain mechanisms of trauma may result in skin injuries, which may serve as a portal for entry. Furthermore, overcrowding of the emergency department creates more opportunities for cross-transmission [48], and overworked medical staffmay not follow preventive procedures and take inadequate precautions [49,50]. 4. Discussion Our findings on the correlation between trauma, admission through the emergency department, and MRSA detection may be due to these factors. This study proves the relationship between antibiotic use prior to admission and MRSA colonization. Muller et al. [28] reported individual exposure to fluoroquinolones and collective exposure to penicillin to be associated with MRSA isolation after adjustment for colonization pressure and other potential confounders. Tacconelli et al. [51] performed a meta-analysis of over 76 studies included 24,230 patients and found that the development of MRSA is significantly related to the length of antibiotic exposure. According to the Society for Healthcare Epidemiology of America guidelines for preventing nosocomial transmission of MRSA and vancomycin-resistant enterococci, the use of antimicrobial agents in US hospitals is commonly excessive or unnecessary [9]. This widespread use of antibiotics may create conditions in which resistant bacteria experience a competitive advantage. There are some limitations to our study. First, only nasal swabs were performed by us as the screening test considering cost- and time-effectiveness. Some studies suggest that collecting samples from additional sites (throat, groin, and thorax) may contribute to a higher detection rate than using a single site [17]. The prevalence of MRSA colonization might have been higher if samples from additional sites were collected. However, the anterior nasal cavity is known to be the most common carrier site of MRSA [11,12]. Second, we focused mainly on the screening test but whether the result of the screening test actually contributed to the development of post-operative infection has not been investigated. Third, this is a single-institute and single-department study. Additional multi-center studies must be performed to determine the actual frequency and risk factors for MRSA in Korea. 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SHEA guideline for preventing nosocomial transmission of multidrug-resistant strains of Staphylococcus aureus and enterococcus. Infect. Control Hosp. Epidemiol. 2003, 24, 362–386. [CrossRef] 10. Infection Control Guidelines for Multidrug Resistant Microorganisms in Healthcare Facilities. Korea Center for Disease Control & Prevention 2012. 5. Conclusions The present study focused on the prevalence of MRSA colonization and associated risk factors among patients admitted to the orthopedic surgery department. Being underweight, having a trauma diagnosis, antibiotic use one month prior to admission, and admission through the emergency department are risk factors for MRSA infection. The relatively high prevalence of MRSA in this study highlights the importance of pre-operative screening tests for patients scheduled for surgery involving implant insertion, particularly those at risk for MRSA. Among high risk patients, elective surgery could be delayed until the confirmation of the MRSA screening test. Prophylactic treatment prior to surgery is recommended for patients with positive MRSA colonization. J. Clin. Med. 2019, 8, 631 9 of 11 Author Contributions: Conceptualization, S.-W.C.; methodology, S.-W.C.; validation, S.-W.C.; formal analysis, J.K.; investigation, J.K. and S.-W.C.; resources, S.-W.C.; data curation, J.K. and S.P.; writing—original draft preparation, J.K.; writing—review and editing, J.C.L. and J.E.K. and M.J.B. and S.Y.P.; visualization, J.C.L.; supervision, B.-J.S.; funding acquisition, S.-W.C. Author Contributions: Conceptualization, S.-W.C.; methodology, S.-W.C.; validation, S.-W.C.; formal analysis, J.K.; investigation, J.K. and S.-W.C.; resources, S.-W.C.; data curation, J.K. and S.P.; writing—original draft preparation, J.K.; writing—review and editing, J.C.L. and J.E.K. and M.J.B. and S.Y.P.; visualization, J.C.L.; supervision, B.-J.S.; funding acquisition, S.-W.C. Funding: This study was supported by the Soonchunhyang University research foundation and National Research Foundation of Korea (NRF) grant funded by the Korea government (MIST). (2019R1A2B5B01005924). Funding: This study was supported by the Soonchunhyang University research foundation and National Research Foundation of Korea (NRF) grant funded by the Korea government (MIST). (2019R1A2B5B01005924). Acknowledgments: The authors sincerely thank Soon-hyo Kwon and Jae Heon Kim for their assistance with patient data analysis and consolidation. Acknowledgments: The authors sincerely thank Soon-hyo Kwon and Jae Heon Kim for their assistance with patient data analysis and consolidation. Conflicts of Interest: The authors declare no conflict of interest. The funders had no role in the design of the study; in the collection, analyses, or interpretation of data; in the writing of the manuscript, or in the decision to publish the results Conflicts of Interest: The authors declare no conflict of interest. The funders had no role in the design of the study; in the collection, analyses, or interpretation of data; in the writing of the manuscript, or in the decision to publish the results References Available online: http://www.gidcc.or.kr/wp-content/uploads/2016/ 12/7/_3-%EB%8B%A4%EC%A0%9C%EB%82%B4%EC%84%B1%EA%B7%A0/_%EA%B4%80%EB%A6% AC%EC%A7%80%EC%B9%A8.pdf (accessed on 14 April 2019). 11. Kluytmans, J.; van Belkum, A.; Verbrugh, H. Nasal carriage of Staphylococcus aureus: Epidemiology, underlying mechanisms, and associated risks. Clin. Microbiol. Rev. 1997, 10, 505–520. [CrossRef] 12. Lucet, J.C. The importance of detecting methicillin-resistant Staphylococcus aureus in an intensive care setting. Ann. Fr. Anesth. Reanim. 2002, 21, 384–391. [CrossRef] 13. Yano, K.; Minoda, Y.; Sakawa, A.; Kuwano, Y.; Kondo, K.; Fukushima, W.; Tada, K. Positive nasal culture of methicillin-resistant Staphylococcus aureus (MRSA) is a risk factor for surgical site infection in orthopedics. Acta Orthop. 2009, 80, 486–490. [CrossRef] 14. Nelwan, E.J.; Sinto, R.; Subekti, D.; Adiwinata, R.; Waslia, L.; Loho, T.; Safari, D.; Widodo, D. Screening of methicillin-resistant Staphylococcus aureus nasal colonization among elective surgery patients in referral hospital in Indonesia. BMC Res. Notes 2018, 11, 56. [CrossRef] p 15. Price, C.S.; Williams, A.; Philips, G.; Dayton, M.; Smith, W.; Morgan, S. Staphylococcus aureus nasal colonization in preoperative orthopaedic outpatients. Clin. Orthop. Relat. Res. 2008, 466, 2842–2847. 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Prevalence of and risk factors for colonization with methicillin-resistant Staphylococcus aureus at the time of hospital admission. Infect. Control Hosp. Epidemiol. 2003, 24, 409–414. [CrossRef] [PubMed] 11 of 11 11 of 11 J. Clin. Med. 2019, 8, 631 34. Lu, P.L.; Chin, L.C.; Peng, C.F.; Chiang, Y.H.; Chen, T.P.; Ma, L.; Siu, L.K. Risk factors and molecular analysis of community methicillin-resistant Staphylococcus aureus carriage. J. Clin. Microbiol. 2005, 43, 132–139. [CrossRef] [PubMed] 35. Sfeir, M.; Obeid, Y.; Eid, C.; Saliby, M.; Farra, A.; Farhat, H.; Mokhbat, J.E. Prevalence of Staphylococcus aureus methicillin-sensitive and methicillin-resistant nasal and pharyngeal colonization in outpatients in Lebanon. Am. J. Infect. Control 2014, 42, 160–163. [CrossRef] [PubMed] 36. Munckhof, W.J.; Nimmo, G.R.; Schooneveldt, J.M.; Schlebusch, S.; Stephens, A.J.; Williams, G.; Huygens, F.; Giffard, P. 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Olsen, K.; Sangvik, M.; Simonsen, G.S.; Sollid, J.U.E.; Sundsfjord, A.; Thune, I.; Furberg, A. Prevalence and population structure of Staphylococcus aureus nasal carriage in healthcare workers in a general population. The Tromsø Staph and Skin Study. Epidemiol. Infect. 2013, 141, 143–152. [CrossRef] 41. Campbell, K.A.; Cunningham, C.; Hasan, S.; Hutzler, L.; Bosco, J.A., 3rd. Risk Factors for Developing Staphylococcus aureus Nasal Colonization in Spine and Arthroplasty Surgery. Bull. Hosp. Jt. Dis. (2013) 2015, 73, 276–281. 42. Durmaz, R.; Tekerekoglu, M.S.; Kalcioglu, T.; Ozturan, O. Nasal carriage of methicillin-resistant Staphylococcus aureus among smokers and cigarette factory workers. New Microbiol. 2001, 24, 143–147. [PubMed] 43. References McEachern, E.K.; Hwang, J.H.; Sladewski, K.M.; Nicatia, S.; Dewitz, C.; Mathew, D.P.; Nizet, V.; Crotty Alexander, L.E. Analysis of the Effects of Cigarette Smoke on Staphylococcal Virulence Phenotypes. Infect. Immun. 2015, 83, 2443–2452. 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This article is an open access article distributed under the terms and conditions of the Creative Commons Attribution (CC BY) license (http://creativecommons.org/licenses/by/4.0/). © 2019 by the authors. Licensee MDPI, Basel, Switzerland. This article is an open access article distributed under the terms and conditions of the Creative Commons Attribution (CC BY) license (http://creativecommons.org/licenses/by/4.0/).
https://openalex.org/W4293661304
https://journal2.unusa.ac.id/index.php/MTPHJ/article/download/3187/1749
Indonesian
null
The Relationship between Snack Consumption Patterns and Nutritional Status in Adolescents in Malang City Indonesia
Medical technology and public health journal
2,022
cc-by-sa
2,785
ABSTRACT Background : The consumption of snacks is often done between meals. About 20% of the daily intake of adolescents comes from snacks. Snacks contribute ‘empty’ energy but provide an excess intake of substances that are not beneficial to the teenager’s body. Objectives; this study aims to determine the pattern of snack consumption and nutritional status of adolescents in Malang City. Material and Method; This study used a cross-sectional approach to determine the relationship between snack consumption patterns and nutritional status in adolescent in Malang City. The population of this study consisted of 128 high school students (aged 15-18 years) who were selected using the multistage random sampling method ( a combination of the cluster method and simple random sampling) from July-September 2015. Results : Respondents who experiences poor nutritional status were 40 pople (8,6%) and overweight as many as 88 people (19%). Respondents who consumed snacks as much as 3-4x/week showed less nutritional status 14people (35%) and overweight 35 people (39,8%). Conclusion; This study concludeds that the frequency of snack consumption affects the nutritional status of underweight and overweight in adolescents aged 15-18 years in Malang City Keywords: consumption pattern, snacks, nutrition status, adolescents Ira Dwijayanti1*, Jane C-J Chao2 Ira Dwijayanti1*, Jane C-J Chao2 1 Program Studi S1 Gizi, Fakultas Kesehatan, Universitas Nahdlatul Ulama Surabaya, Surabaya, Indonesia 2 School of Nutrition and Health Sciences, College of Nutrition, Taipei Medical University, Taipei, Taiwan *iradwijayanti@unusa.ac.id ABSTRAK Pendahuluan : Konsumsi makanan ringan atau camilan sering dilakukan di antara waktu makan utama. Sekitar 20% dari kebutuhan asupan energi harian remaja berasal dari camilan. Camilan menyumbang energi ‘kosong’ tetapi memberikan asupan berlebih zat yang tidak bermanfaat pada tubuh remaja. Penelitian ini bertujuan untuk mengetahui pola konsumsi camilan dan status gizi pada remaja di Kota Malang. Bahan dan Metode : Penelitian ini menggunakan pendekatan cross-sectional untuk mengetahui hubungan pola konsumsi camilan dan status gizi pada remaja di Kota Malang, Indonesia. Populasi penelitian ini terdiri dari 128 siswa SMA (usia 15-18 tahun) yang dipilih menggunakan metode multistage random sampling (gabungan dari metode cluster dan simple random sampling) dari Bulan Juli- September 2015. Hasil : Responden yang mengalami status gizi kurang sebanyak 40 orang (8,6%) dan status gizi lebih sebanyak 88 orang (19%). Reponden yang mengonsumsi camilan sebanyak 3-4x/minggu menunjukkan status gizi kurang 14 orang (35%) dan status gizi lebih 35 orang (39,8%). Ada perbedaan signifikan antara frekuensi konsumsi camilan dan status gizi remaja (p=0.00). Kesimpulan : Penelitian ini menyimpulkan bahwa frekuensi konsumsi camilan berpengaruh terhadap status gizi kurang dan lebih pada remaja usia 15-18 tahun di Kota Malang. Kata Kunci : Pola konsumsi, camilan, status gizi, remaja PENDAHULUAN telah meningkat secara signifikan bersamaan dengan tingkat obesitas pada remaja. Sekitar 20% dari kebutuhan asupan energi harian remaja berasal dari camilan (5). Camilan menyumbang energi ‘kosong’ tetapi memberikan asupan berlebih zat yang tidak bermanfaat pada tubuh remaja (6). Berat badan lebih dan obesitas merupakan permasalahan gizi remaja di Indonesia. Prevalensi remaja usia 16-18 tahun yang mengalami kelebihan berat badan dan obesitas adalah 13,5%. Prevalensi remaja gemuk di Indonesia mengalami peningkatan sebanyak 2,2% dalam waktu 5 tahun. Provinsi Jawa Timur termasuk dalam daerah yang memiliki prevalensi remaja gemuk melebihi prevalensi nasional yaitu 11,3% (1,2) Remaja dengan kelebihan berat badan atau obesitas memiliki risiko yang lebih tinggi untuk mengalami penyakit kronis seprti hipertensi, diabetes tipe 2 dan dislipidemia serta konsekuensi psikososial seperti stres dan depresi. Selain itu, remaja obesitas memiliki risiko tetap mengalami obesitas saat dewasa (3). Sebagian besar remaja di Indonesia menggunakan waktu luang untuk kegiatan tidak aktif, sepertiga remaja makanan camilan buatan pabrik atau makanan olahan, dan sepertiga lainnya sering mengonsumsi kue basah, roti basah, gorengan dan kerupuk (7). Perubahan gaya hidup saat ini membuat remaja semakin terhubung degan akses internet sehingga remaja lebih sering membuat keputusan mandiri dalam memilih makanan. Pilihan makanan yang dibuat seringkali kurang tepat sehingga dapat menyebakan masalah gizi (5)(8). Keadaan status gizi remaja di Indonesia harus diperbaiki. Berdasarkan penelitian sebelumnya, ditemukan adanya perubahan pola makanan dan aktivitas fisik pada remaja (4). Konsumsi makanan ringan atau camilan sering dilakukan di antara waktu makan utama merupakan salah satu pola makan yang banyak ditemui pada remaja. Konsumsi camilan Pendekatan pola makan untuk mengurangi konsumsi camilan sering ditargetkan untuk mencapai pengurangan kalori dalam program pengurangan berat badan remaja. Memahami hubungan antara pola konsumsi camilan dan status gizi pada remaja ini sangat penting. Penelitian sebelumnya hanya fokus terhadap asupan makanan remaja dari laporan atau wawancara orang tua. Potensi bias dalam laporan asupan makan remaja yang tidak dilakukan secara langsung akan membatasi kemampuan peneliti untuk memperkirakan hubungan yang sebenarnya (2). di Kota Malang. Beberapa kriteria inklusi penelitian ini adalah (1) Kriteria umur subjek penelitian adalah 15-18 tahun; (2) Siswa berdomisili di Malang; (3) Siswa wajib mengisi persetujuan orang tua atau wali pada formulir perijinan penelitian; dan (4) Subjek penelitian harus mengisi dan melengkapi kuesioner penelitian secara mandiri. Enam puluh enam siswa tidak mengisi kuesioner secara lengkap sehingga dikeluarkan dari penelitian ini. Penelitian ini telah disetujui oleh Komisi Etika Penelitian Kesehatan Fakultas Kedokteran Universitas Brawijaya (No. 465/EC/KEPK/088/2015). PENDAHULUAN Upaya perbaikan gizi pada remaja yang dilakukan oleh sektor kesehatan tidak akan tercapai maksimal tanpa adanya intervensi sensitif yang sesuai dengan keadaan masyarakat. Perlu dilakukan penelitian untuk mendapatkan gambaran data dasar pola makan dan status gizi remaja. Oleh karena itu, penelitian ini bertujuan untuk mengetahui pola konsumsi camilan dan status gizi pada remaja di Kota Malang. Data tinggi dan berat badan responden diukur untuk menentukan Indeks Massa Tubuh (IMT)/Umur (U). IMT/U dikategorikan berdasarkan nilai persentil sesuai dengan grafik World Health Organization (WHO) yaitu berat badan kurang (IMT <5 th persentile), normal ≥ 5 th - <85 th dan gemuk/obesitas ≥85th persentile (10). Data pola makan dikumpulkan dengan menggunakan kuesioner Semi Quantitative-Food Frequency Questionnaire (SQ-FFQ). Semua bahan makanan diadopsi dari pedoman makan Indonesia yang disajikan dalam tumpeng gizi seimbang. Tumpeng gizi seimbang atau piramida makanan merupakan pedoman pola makan yang digambarkan melalui 4 lapisan piramida dengan tujuan untuk menyeimbangkan gizi METODE Penelitian ini menggunakan pendekatan cross-sectional untuk mengetahui hubungan pola konsumsi camilan dan status gizi pada remaja di Kota Malang, Indonesia. Penelitian cross- sectional memberikan keuntungan peneliti untuk mendapatkan berbagai hasil dengan eksposur secara bersamaan dalam satu waktu (9). Populasi penelitian ini terdiri dari 128 siswa SMA (usia 15-18 tahun) yang dipilih menggunakan metode multistage random sampling (gabungan dari metode cluster dan simple random sampling) dari Bulan Juli-September 2015 ini. Semua analiss statistik dilakukan dengan menggunakan SPSS versi 22 (Armonk, NY:IBM Corp). dan porsi makan harian. Data food recall 24 hours juga dikumpulkan dari 50 orang siswa yang dipilih secara acak untuk menambah daftar makanan lain dan menentukan ukuran porsi rata-rata dalam kuesioner. Frekuensi jenis makanan diukur dengan sebelas kategori yaitu tidak pernah, kurang dari 1 kali sebulan, 1 sampai 3 kali sebulan, 1 kali seminggu, 2 kali seminggu, 3 kali seminggu, 4 kali seminggu, 5 kali seminggu, 6 kali seminggu, 7 kali seminggu dan lebih dari 1 kali sehari. Analisis pola makan menggunakan Principal Component Analysis (PCA). Total 84 jenis makanan yang terdapat di kuesioner dikurangi menjadi 23 jenis makanan berdasarkan faktor loading ≥± 0,30. Empat faktor diidentifikasi berdasarkan nilai eigen lebih besar dari >0,10, bentuk plot scree dan interpretasi pola makan yang bermakna. Interpretasi komponen makanan menggunakan rotasi varimax. Pola makan camilan terdiri dari jenis makanan kue ketan, kue mangkok, cenil, dan kue pukis (11). Analisis data menggunakan HASIL Hubungan antara konsumsi camilan dan status gizi remaja usia 15-18 tahun di Kota Malang (n=128) Variabel Kurus n=40 Gemuk n=88 pa Konsumsi Camilan (n,%) Tidak pernah 0 (0.0) 4 (4.5) 0.00 1-2x/ minggu 6 (15.0) 27 (30.7) 3-4x/ minggu 14 (35.0) 35 (39.8) 5-6x/ minggu 6 (15.0) 10 (11.4) 7x/ minggu 14 (35.0) 12 (13.6) aData analisis menggunakan uji korelatif spearman . Hubungan antara konsumsi camilan dan status gizi remaja usia 15-18 tahun di Kota Malang (n=128) remaja yang cenderung mengonsumsi makanan ringan yang mengandung tinggi lemak dan tinggi kalori dapat mengakibatkan gizi lebih (15). Kebiasaan konsumsi camilan juga dapat menjadikan seseorang melewatkan waktu makan utama sehingga menyumbang asupan energi kosong dan menyebabkan kurangnya asupan zat penting untuk tubuh (16). HASIL Data karakteristik dasar responden terdiri dari 189 laki-lai (40,1%) dan 278 perempuan (59,9%) yang berusia 15 sampai 18 tahun. Pendidikan ayah dan ibu menunjukkan 46,8% dan 47,8% merupakan lulusan sarjana. Ayah bekerja penuh waktu (±≥ 8 jam per hari) sebanyak 67% dan ibu tidak bekerja sebanyak 43,8%. Penghasilan keluarga menunjukkan sebanyak Rp 3.000.000 rupiah sebanyak 55,3%. Tidak ada perbedaan yang signifikan dalam data demografi antara jenis kelamin. Konsumsi camilan dan pengukuran antropometri sesuai dengan IMT menurut usia disajikan dalam tabel 1. Responden melaporkan paling banyak konsumsi camilan 3-4x seminggu (35,8%) dan 7x seminggu (22,2%). Tabel 2 menunjukkan bahwa 8,6% dan 19% responden dikasifikasikan sebagai kurus (IMT menurut usia <5persentile) dan kelebihan berat badan/obesitas (IMT menurut usia >85persentile). Analisis data menggunakan pengukuran non-parametrik karena distribusi data tidak normal dan data merupakan data nominal. Proporsi dan perbandingan antar variabel diukur dengan tes chi square. Korelasi Spearman dan regresi logistik digunakan untuk mengukur korelasi antar variable. Nilai p kurang dari 0,05 dianggap signifikan dalam penelitian Hubungan antara konsumsi camilan dan status gizi remaja usia 15-18 tahun di Kota Malang ditunjukkan pada tabel 2. Responden yang mengalami status gizi kurang (8,6%) dan lebih (19%) sebanyak 128 orang. Reponden yang mengonsumsi signifikan antara frekuensi konsumsi camilan dan status gizi remaja. 3-4x/minggu sebanyak camilan menunjukkan status gizi kurang (35%) dan status gizi lebih (39,8%). Ada perbedaan Tabel 1. Data konsumsi camilan dan status gizi remaja usia 15-18 tahun di Kota Malang (n=128) Variabel Laki-laki Perempuan Total IMT/U (n, %) Kurus (≤5th presentile) 19 (10.2) 21 (7.6) 40 (8.6) Gemuk (≥85th presentile) 45 (24.2) 43 (15.5) 88 (19.0) Tabel 2 Hubungan antara konsumsi camilan dan status gizi remaja usia 15-18 tahun di Kota Tabel 1. Data konsumsi camilan dan status gizi remaja usia 15-18 tahun di Kota Malang (n=128) Variabel Laki-laki Perempuan Total IMT/U (n, %) Kurus (≤5th presentile) 19 (10.2) 21 (7.6) 40 (8.6) Gemuk (≥85th presentile) 45 (24.2) 43 (15.5) 88 (19.0) Tabel 2. Hubungan antara konsumsi camilan dan status gizi remaja usia 15-18 tahun di Kota Malang (n=128) Variabel Kurus n=40 Gemuk n=88 pa Konsumsi Camilan (n,%) Tidak pernah 0 (0.0) 4 (4.5) 0.00 1-2x/ minggu 6 (15.0) 27 (30.7) 3-4x/ minggu 14 (35.0) 35 (39.8) 5-6x/ minggu 6 (15.0) 10 (11.4) 7x/ minggu 14 (35.0) 12 (13.6) aData analisis menggunakan uji korelatif spearman Tabel 2. PEMBAHASAN Konsumsi makanan ringan atau camilan dalam frekuensi sering menyebabkan asupan makanan berlebih dan memiliki potensi untuk meningkatkan berat badan (12). Jumlah camilan yang dikonsumsi cenderung lebih sedikit dibandingkan dengan jumlah menu pada makanan utama, namun frekuensi camilan yang sering itu menjadikan asupan kalori yang setara atau berlebih (13). Kebiasaan mengonsumsi energi melebihi kebutuhan tubuh maka kelebihan energi akan disimpan dalam bentuk lemak sebagai cadangan energi (14). Perubahan gaya hidup pada Pemilihan jenis makanan dan frekuensi konsumsi camilan menjadi sangat berpengaruh terhadap tingkat kecukupan asupan gizi. Responden menyebutkan jenis makanan camilan kue ketan, kue mangkok, cenil, dan kue pukis. Jenis makanan yang mengandung tepung berkontribusi terhadap peningkatan berat badan (17). Seseorang yang mengonsumsi camilan dengan frekuensi yang sering sedangkan tetap mengonsumsi makanan utama maka cenderung akan meningkatkan berat badan. Sebaliknya, jika frekuensi camilan sering namun melewatkan waktu makan utama dan asupan zat gizi tidak terpenuhi makan seseorang cenderung menjadi kurus (18). mineral(21). mineral(21). KESIMPULAN 6. Zuhdy N, Ani LS, Utami WAU. Aktivitas Fisik , Pola Makan dan Status Gizi Pelajar Putri SMA di Denpasar Utara (Physical Activity , Food Consumption and Nutritional Status among Female High School Students in North Denpasar ). Public Heal Prev Med Arch [Internet]. 2015;3(1):78–83. Tersedia pada: https://www.researchgate.net/publicatio n/332348005 Penelitian ini menyimpulkan bahwa frekuensi konsumsi camilan berpengaruh terhadap status gizi kurang dan lebih pada remaja usia 15-18 tahun di Kota Malang. Penelitian ini diharapkan dapat dilanjutkan menggunakan metode prospektif untuk mengetahui efek dalam jangka panjang camilan terhadap status gizi di masa depan. 7. Irdiana W, Nindya TS. Hubungan Kebiasaan Sarapan dan Asupan Zat Gizi dengan Status Gizi Siswi SMAN 3 Surabaya. Amerta Nutr. 2017;1(3):227. mineral(21). Penelitian menunjukkan bahwa konsumsi camilan berfungsi mengganjal rasa lapar di antara waktu makan. Masyarakat Indonesia rata-rata mengonsumsi 3x makanan ringan per hari, melebihi jumlah rata-rata global. Selain itu, tersedianya waktu luang dan rendahnya aktivitas fisik mengarahkan remaja untuk mekan berlebihan terutama camilan setelah mengonsumsi makanan utama atau sekedar pengganti waktu makan. Remaja memiliki risiko yang besar untuk mandapatkan asupan gizi yang tidak seimbang (17). Perbaikan pola makan remaja yang bersifat spesifik dan sensitif gizi perlu diupayakan dan memiliki integrasi yang baik untuk mencapai status gizi remaja yang optimal. Pendidikan gizi untuk meningkatkan pengetahuan, sikap dan efikasi diri remaja terkait makanan sehat harus dilakukan secara kontinyu(22). Penelitian ini menunjukkan bahwa responden yang memiliki status gizi kurang dan lebih didapatkan mengonsumsi camilan dengan frekuensi 3-4x/minggu. Penelitian lain menunjukkan bahwa hasil serupa dimana responden yang terbiasa mengonsumsi camilan berhubungan dengan peningkatan asupan energi dan signifikan terhadap status gizi (19). Penelitian lain menunjukan bahwa ada hubungan antara konsumsi camilan dan status gizi. Responden yang sering mengonsumsi camilan dilaporkan mengalami defisiensi mikronutrien seperti kalsium dan vitamin A sehingga menghambat pertumbuhan. Status gizi diperburuk dengan konsumsi camilan dalam jumlah besar dengan kandungan mikronutrien rendah. Oleh karena itu konsumsi camilan harus dikurangi dan diganti dengan makanan yang mengandung padat energi (20). Penelitian lain juga menunjukkan bahwa remaja lebih menyukai makanan dengan kandungan natrium dan lemak yang tinggi tetapi rendah vitamin dan Keterbatasan dalam penelitian kami adalah penelitian dilakukan di sekolah menengah atas di Kota malang sehingga membatasi untuk generalisasi hasil di kota lain. Jumlah sampel juga mungkin dapat membatasi untuk menemukan hasil yang serupa pada populasi umum. Penyebab langsung antara konsumsi camilan pada remaja usia 15-18 tahun di Kota Malang tidak dapat diukur dari waktu ke watu karena menggunakan desain penelitian cross-sectional. DAFTAR PUSTAKA 1. Setiawati FS, Mahmudiono T, Ramadhani N, Hidayati KF. Intensitas Penggunaan Media Sosial, Kebiasaan Olahraga, dan Obesitas Pada Remaja Di SMA Negeri 6 Surabaya Tahun 2019. Amerta Nutr. 2019;3(3):142. 8. Kumala M, Kumala M, Limanan D, Santoso H. Pemeriksaan Status Gizi Sebagai Upaya Preventif Penyakit Degeneratif Pada Siswa Sekolah BM Jakarta Pusat. J Bakti Masy Indones. 2020;3(1):10–8. 2. Irdianty MS, Sani FN. Perbedaan Aktivitas Fisik Dan Konsumsi Camilan Pada Remaja Obesitas Di Kabupaten Bantul. J Kesehat Kusuma Husada. 2018;91–7. 9. Sedgwick P. Cross sectional studies: Advantages and disadvantages. BMJ [Internet]. 2014;348(February). Tersedia pada: http://dx.doi.org/doi:10.1136/bmj.g227 6 3. Tripicchio GL, Kachurak A, Davey A, Bailey RL, Dabritz LJ, Fisher JO. Associations between snacking and weight status among adolescents 12–19 years in the United States. Vol. 11, Nutrients. 2019. 10. Danun NV, Kaligis SHM, Tiho M. Hubungan Indeks Massa Tubuh (IMT) dengan Kadar Apolipoprotein B (ApoB) pada Remaja Overweight dan Obes. J e-Biomedik. 2016;4(1). 4. Emilia E, Akmal N. ANALISIS KONSUMSI MAKANAN JAJANAN TERHADAP PEMENUHAN GIZI REMAJA. J Nutr Culinary(JNC). 2021;1(1). 11. Dwijayanti I, Chien YW, Poda GG, Chao JCJ. Defining food literacy and dietary patterns among senior high school students in Malang City, East Java. J Gizi Indones (The Indones J Nutr. 2021;10(1):45–53. 5. Kumala AM, Margawati A, Rahadiyanti A. Hubungan Antara Durasi Penggunaan Alat Elektronik (Gadget), Aktivitas Fisik Dan Pola Makan Dengan Status Gizi Pada Remaja Usia 13-15 Tahun. J Nutr Coll. 2019;8(2):73. 12. Margiyanti NJ. Analisis Tingkat Pengetahuan, Body Image dan Pola Makan terhadap Status Gizi Remaja Putri. J Akad Baiturrahim Jambi. 2021;10(1):231. Tata Boga [Internet]. 2021;10(1):129– 37. Tersedia pada: https://ejournal.unesa.ac.id/index.php/j urnal-tata-boga/article/view/38402 13. Hafiza D, Utmi A, Niriyah S. Hubungan Kebiasaan Makan Dengan Status Gizi Pada Remaja Smp Ylpi Pekanbaru. Al-Asalmiya Nurs J Ilmu Keperawatan (Journal Nurs Sci [Internet]. 2021;9(2):86–96. Tersedia pada: https://jurnal.stikes- alinsyirah.ac.id/index.php/keperawatan/ article/view/671 18. Bertéus Forslund H, Lindroos AK, Sjöström L, Lissner L. Meal patterns and obesity in Swedish women - A simple instrument describing usual meal types, frequency and temporal distribution. Eur J Clin Nutr. 2002;56(8):740–7. 14. Mukhlisa WNI, Rahayu LS, Furqan M. Asupan Energi Dan Konsumsi Makanan Ringan Berhubungan Dengan Kejadian Gizi Lebih Pada Remaja. Argipa [Internet]. 2018;3(2):59–66. Tersedia pada: https://journal.uhamka.ac.id/index.php/ argipa/article/download/944/1023 19. Nuryani N, Rahmawati R. Kebiasaan jajan berhubungan dengan status gizi siswa anak sekolah di Kabupaten Gorontalo. J Gizi Indones (The Indones J Nutr. 2018;6(2):114–22. 20. Sekiyama M, Roosita K, Ohtsuka R. DAFTAR PUSTAKA Snack foods consumption contributes to poor nutrition of rural children in West Java, Indonesia. Asia Pac J Clin Nutr. 2012;21(4):558–67. 15. Nasrudin, Rumagit FA, Pascoal ME. Hubungan frekuensi konsumsi makanan jajanan dengan status gizi dan prestasi belajar anak Sekolah Dasar Negeri Malalayang Kota Manado. J Gizido. 2016;8(2):61–70. 21. Maria Goreti Pantaleon. Hubungan Pengetahuan Gizi dan Kebiasaan Makan dengan Status Gizi Remaja Putri di SMA Negeri II Kota Kupang. CHMK Heal J. 2019;3(3):69–76. 16. McCrory MA, Campbell WW. Effects of eating frequency, snacking, and breakfast skipping on energy regulation: Symposium overview1,2. J Nutr. 2011;141(1):144–7. 22. United Nation Children’s Fund. Strategi Komunikasi Perubahan Sosial dan Perilaku: Meningkatkan Gizi Remaja di Indonesia. 2021;1–66. 17. Amaliyah M, Soeyono RD, Nurlaela L, Kritiastuti D. Pola Konsumsi Makan Remaja Di Masa Pandemi Covid-19. J
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Public health application of predictive modeling: an example from farm vehicle crashes
Injury epidemiology.
2,019
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© The Author(s). 2019 Open Access This article is distributed under the terms of the Creative Commons Attribution 4.0 International License (http://creativecommons.org/licenses/by/4.0/), which permits unrestricted use, distribution, and reproduction in any medium, provided you give appropriate credit to the original author(s) and the source, provide a link to the Creative Commons license, and indicate if changes were made. The Creative Commons Public Domain Dedication waiver (http://creativecommons.org/publicdomain/zero/1.0/) applies to the data made available in this article, unless otherwise stated. Abstract Many variables are needed to predict a future event with good accuracy, with an implicit assumption that the predicted event has consistent explanatory factors. For example, to predict tomorrow’s weather in a given city, we will need information on today’s weather in that city, today’s weather in all places surrounding that city, the wind speed, and the season and annual averages based on past weather trends. In the current highly digitized world with Big Data, large sets of variables with sophisticated predictive algorithms may allow forecasting a future event with increasing fidelity. Ranapurwala et al. Injury Epidemiology (2019) 6:31 https://doi.org/10.1186/s40621-019-0208-9 Public health application of predictive modeling: an example from farm vehicle crashes Shabbar I. Ranapurwala1,2* , Joseph E. Cavanaugh3,4, Tracy Young3, Hongqian Wu4, Corinne Peek-Asa3 and Marizen R. Ramirez3,5 Abstract Background: The goal of predictive modelling is to identify the likelihood of future events, such as the predictive modelling used in climate science to forecast weather patterns and significant weather occurrences. In public health, increasingly sophisticated predictive models are used to predict health events in patients and to screen high risk individuals, such as for cardiovascular disease and breast cancer. Although causal modelling is frequently used in epidemiology to identify risk factors, predictive modelling provides highly useful information for individual risk prediction and for informing courses of treatment. Such predictive knowledge is often of great utility to physicians, counsellors, health education specialists, policymakers or other professionals, who may then advice course correction or interventions to prevent adverse health outcomes from occurring. In this manuscript, we use an example dataset that documents farm vehicle crashes and conventional statistical methods to forecast the risk of an injury or death in a farm vehicle crash for a specific individual or a scenario. Results: Using data from 7094 farm crashes that occurred between 2005 and 2010 in nine mid-western states, we demonstrate and discuss predictive model fitting approaches, model validation techniques using external datasets, and the calculation and interpretation of predicted probabilities. We then developed two automated risk prediction tools using readily available software packages. We discuss best practices and common limitations associated with predictive models built from observational datasets. Conclusions: Predictive analysis offers tools that could aid the decision making of policymakers, physicians, and environmental health practitioners to improve public health. Keywords: Forecasting, Predictions, Decision support techniques, Motor vehicles Yahoo, travel websites, or elsewhere. The information needed for such prediction may, for example, come from an individual’s browsing history or grocery shopping habits. Many variables are needed to predict a future event with good accuracy, with an implicit assumption that the predicted event has consistent explanatory factors. For example, to predict tomorrow’s weather in a given city, we will need information on today’s weather in that city, today’s weather in all places surrounding that city, the wind speed, and the season and annual averages based on past weather trends. In the current highly digitized world with Big Data, large sets of variables with sophisticated predictive algorithms may allow forecasting a future event with increasing fidelity. Yahoo, travel websites, or elsewhere. The information needed for such prediction may, for example, come from an individual’s browsing history or grocery shopping habits. * Correspondence: sirana@email.unc.edu 1Injury Prevention Research and Department of Epidemiology, University of North Carolina at Chapel Hill, 137 E Franklin St, Suite 500, CB# 7505, Chapel Hill, NC 27599, USA 2Department of Epidemiology, College of Public Health, University of Iowa, Iowa City, IA, USA Full list of author information is available at the end of the article Introduction Predictive models or algorithms are routinely utilized to predict many everyday events or decisions. The list of such outcomes include daily or inclement weather events (Saha et al. 2014); department store purchases that com- panies use to provide coupons at grocery stores (Wu and Bryniolfsson 2013; Schoen et al. 2013); or targeted adver- tisement and news stories that we receive on Facebook, Page 2 of 11 Page 2 of 11 Ranapurwala et al. Injury Epidemiology (2019) 6:31 Ranapurwala et al. Injury Epidemiology (2019) 6:31 In healthcare settings, medical tests, (e.g., blood pres- sure or cholesterol measurements) are one form of predictive data that allow physicians to assess the pro- bability that the patient may (or may not) have an adverse event in the (near or distant) future. We have utilized many such medical tests to predict good or bad health outcomes, for example, to predict breast cancer (Gail et al. 2007; Quante et al. 2012) and 5-year or 10-year risks of cardiovascular diseases (Keys et al. 1972; Kleinbaum et al. 1971; Wilson et al. 1998). The latter work (Keys et al. 1972; Kleinbaum et al. 1971; Wilson et al. 1998) led to the development of a web-based tool to forecast the 10-year risk of cardiovascular disease, pre- dicted by age, sex, smoking status, blood pressure (in- cluding medications), and cholesterol levels (AHA 2017). Similarly, predictive algorithms are also now being used to estimate the probability that an individual may suffer from an opioid overdose in a defined future period (Glanz et al. 2018). Such predictive algorithms are helpful for a physician, counselor, health education specialist, social worker, police or parole officer, policy maker or other professionals to identify individuals at an increased risk of having certain adverse outcome within a given timeframe. Predictive models can also be used to help educate target populations so that individuals can understand their personal risks given their unique set of demographics, behavioral characteristics or environmen- tal / social conditions. may discover that the risk of a hypothetical adverse event may be twice as high among the exposed than the un- exposed, i.e., a risk ratio of 2. While on a population or sample level this may hold true, the risk of actually ex- periencing the adverse event for each individual in the exposed and unexposed subsets differ. Introduction Indeed, some indi- viduals in the exposed subset will never experience the ad- verse event, and some individuals will experience the adverse event even when they were not exposed. A pre- dictive algorithm allows us to identify or measure subject-specific probabilities of experiencing an adverse events, thereby aiding decision making at an individual or situational level. Predictive models can be developed using the same traditional regression analyses methods used for causal modelling (e.g. linear, log binomial, logistic, Poisson, Cox regression). In this manuscript, we illustrate the de- velopment of a simplistic predictive algorithm for an injury-related outcome that incorporates our prior under- standing of the substantive area and utilizes a large database to predict the probability of an adverse event. Predictive models have widespread applicability in the area of injury and violence prevention, especially as the field continues to develop and implement interventions that target individual behaviors. For example, a physician could use predictive tools based on a patient’s diagnosis, mental and physical health history and previous medi- cation prescriptions to titrate the supply of opioid pain medication for that patient so that the pain is adequately controlled while also reducing the risk of an opioid use disorder. Using similar patient-history driven predictive tools, a mental health counselor could advise reduced access to a firearm for someone at high risk of commit- ting suicide. A particular focus of our research is motor vehicle crashes, a leading cause of death in rural agricul- tural communities. Predictive models can aid prevention efforts that target individual protective behaviors while driving or operating agricultural equipment on road- ways. Examples of protective behaviors include use of seat belts, installation of rollover protective devices in tractors and safe driving behaviors (following traffic regulations). Notably, predictive models serve a different purpose than causal (or associational) models and their resultant measures like relative (ratio) measures of risks, rates, odds or hazards. In multivariable modeling, causal or associational measures (risk ratios, rate ratios, odds ratios or hazard ratios) examine the partial relationship (or association) between a single independent variable with a health outcome (dependent variable) on a ‘popu- lation level.’ Causal measures tell us for example the effectiveness of population level interventions in impro- ving public health or the population level impact of harm- ful exposures and their resulting public health burden. Example data Predictive models were fit using multivariable logistic regression for individual-level data. To accommodate clustering at the crash level, generalized estimating equations (GEEs) with an exchangeable working corre- lation structure were employed to fit the models. Three types of models were formulated. First, we added a set of non-modifiable variables, which cannot be practically intervened on, to determine changes in injury probabil- ity given the non-modifiable factors (model 1). The non-modifiable variables included state, season, weather, time-of-crash, number of involved vehicles, equipment type, and age and sex of the occupant. Next, we added semi-modifiable factors that can be indirectly intervened upon or are consequences of the modifiable factors (model 2). These included ambient light, manner of collision, vehicle action, number of occupants in the vehicle, and the occupant type. Lastly, we added modi- fiable factors, which may be directly intervened on, to the model (model 3). These included driver contributing circumstances and occupant protection. Non-modifiable, semi-modifiable, and modifiable factors were classified a-priori based on previous literature (Hughes and Rodgman 2000; Peek-Asa et al. 2007; Pinzke and Lundqvist 2004; Costello et al. 2003; Gerberich et al. 1996; Jaarsma and De Vries 2014; Marlenga et al. 2006; Gkritza et al. 2010; Maio et al. 1992; Stephan and Newstead 2014; Russo et al. 2014), expert knowledge, and consensus among the research team members. Transportation is the leading cause of agricultural-re- lated death, and crashes with farm equipment on road- ways present a burden for all roadway users (BLS 2013a, b). Predictive algorithms in transportation research could allow us to identify high crash risk scenarios and provide opportunities to intervene. We used secondary crash data from 2005 to 2010 to assess the risk of injury or death in farm vehicle-related crashes (referred hereon after as crash/ farm crash, unless otherwise mentioned), given crash-level, vehicle-level, and individual-level factors. The data were collected from the departments of transportation (DOT) of Illinois (IL), Iowa (IA), Kansas (KS), Minnesota (MN), Missouri (MO), Nebraska (NE), North Dakota (ND), South Dakota (SD), and Wisconsin (WI). The data include all police-reported crashes, including death, injury, or property damage of at least $500 to $1500, depending on the state. Methods to identify and code farm vehicle crashes from these data have been discussed previously (Harland et al. 2014; Ranapurwala et al. 2016). Multilevel crash data include the characteristics of crashes, involved vehicles, and their occupants. Introduction Thus, causal models help in population-level decision making, especially in formulating policy or even treatment decisions that affect large populations (e.g., firearm safety laws, laws mandating use of prescription monitoring pro- grams or standardized treatment regimens for treating cancers or HIV-AIDS). However, causal measures do not quantify the risk of the adverse event for a particular indi- vidual with a particular set of variable values. Predictive models offer a complementary means to aid decision making at the individual or situational level, which causal (or associational) models are unable to address. For example, while comparing a sample of individuals who were exposed to a certain condition to another sample of individuals who were not exposed to that condition, we We used farm vehicle equipment involved motor vehicle crash (henceforth, farm crash) data from nine Midwestern states in the United States from 2005 to 2010 to build models for forecasting the risk of injury or death in a farm crash. For this case example, we employ the logistic regression framework, as this regression technique best addresses the nature of the data. How- ever, the methods described herein can be easily repli- cated in other datasets where outcome distributions favor the use of alternative modeling frameworks. We demonstrate model building approaches for pre- dictive models using prior knowledge of the substantive Ranapurwala et al. Injury Epidemiology (2019) 6:31 Ranapurwala et al. Injury Epidemiology (2019) 6:31 Page 3 of 11 Ranapurwala et al. Injury Epidemiology Page 3 of 11 (Hughes and Rodgman 2000; Peek-Asa et al. 2007; Pinzke and Lundqvist 2004; Costello et al. 2003; Gerberich et al. 1996; Jaarsma and De Vries 2014; Marlenga et al. 2006; Gkritza et al. 2010; Maio et al. 1992; Stephan and Newstead 2014; Russo et al. 2014). Vehicle type was classified as farm vehicle or non-farm vehicle. Number of vehicle occupants was categorized as single or multiple occupant. The crash type was defined as either a single vehicle crash (farm vehicle only), or a multiple vehicle crash (two or more vehicles, one of them being a farm vehicle). area. We further demonstrate model fitting approaches, model validation techniques using external datasets, and the calculation and interpretation of predicted proba- bilities. Additionally, we share two risk prediction tools that can be used by lay and scientific audiences to predict their own or hypothetical risks of being injured should they be involved in a farm crash. Introduction Lastly, we dis- cuss best practices and common limitations associated with predictive models. Results From 2005 to 2010 there were 7094 farm crashes in the nine states, of which 86% (n = 6119) were multiple vehicle crashes and 14% (n = 975) were single vehicle crashes. Of the 7094 crashes, 10 crashes had missing injury or fatality information, and were subsequently excluded from the analyses. A total of 12,936 vehicles were involved in the 7084 crashes, of which 11,961 ve- hicles were involved in multiple vehicle farm crashes. There were 14,834 occupants involved in the 7084 crashes, and of these, 2087 (14.1%) had been injured or killed in the crash. The distribution of the crashes, injuries and deaths by state and year are presented in Table 1. External validation We partitioned 2005–2010 data in three ways to obtain a training dataset and a validation dataset: 1) 2005–2007 data were separated as training data and 2008–2010 as validation data, 2) 2005–2008 data were separated as training data and 2009–2010 as validation data, and 3) 2005–2009 data were separated as training data and 2010 as validation data. We fit models 1–3 using each of the three training datasets and applied the estimated model coefficients to obtain expected outcome counts in the corresponding validation datasets. This allowed us to compare the observed injury/death counts from the validation dataset to the expected injury/death counts from the same validation dataset using predictions based on models 1–3 from the training datasets. Table 2 presents regression coefficients from three fitted models: model 1 included non-modifiable factors, model 2 added the semi-modifiable factors, and model 3 included two additional modifiable factors. Comparing the QICs and c-statistics (estimated AUCs) from the three nested models (models 1, 2 and 3), we see that model 3 was the best fitting and was hence selected as the final model. The comparisons of state-by-state expected injuries/ deaths to the observed injury/ deaths from also implies that model 3 best characterized the observed data as compared to models 1 and 2 (Table 3), thereby suggesting that model 3 best estimated the overall number of injuries and deaths. Example data The crash characteristic variables were year, month, and date of the crash, day of the week, time of the day, the state in which crash occurred, season, num- ber of fatalities, number of injured occupants, number of vehicles involved in the crash, manner of collision, ambient lighting at the time of crash, and weather conditions. The vehicle characteristics included vehicle type, vehicle action at the time of the crash, number of occupants, and driver contributing circumstances. The person-level characteris- tics included age, sex, injury status (no injury or fatal and non-fatal injury), occupant protection information, occu- pant seating, and driver or passenger status. We did not include variables that were highly collinear with other variables that better explained the outcomes. Seating was highly correlated with the driver status (driver/ passenger), and month was highly correlated with season; consequently, these two variables were removed from the predictive model. Day of the week was removed because it did not predict injury. Occupant age in 10-year age categories provided better penalized model fit than continuous or log-transformed age. Multiple imputation (five imputed datasets), using the fully conditional specification methods for logistic re- gression, was performed to impute missing values for vehicular action (5.9%), driver circumstances (14.8%), and occupant protection (21.4%) with no interactions. All Injury severity, coded by the reporting law enforcement officer, was available at the individual-level, defined as no injury, possible injury, non-incapacitating injury, incapaci- tating injury, or fatality. We developed a new binary injury variable for each individual, such that ‘any injury’ cor- responds to non-incapacitating, incapacitating, or fatal injury; and ‘no injury’ corresponds to no or possible injury. Discrete covariate categories were collapsed into broader but meaningful categories based on a-priori knowledge Ranapurwala et al. Injury Epidemiology (2019) 6:31 Ranapurwala et al. Injury Epidemiology Page 4 of 11 Page 4 of 11 models were fit to each of the five imputed datasets. The resulting regression coefficients from the imputed datasets were then pooled to obtain the final coefficients that are reported here. some crash scenarios as examples to demonstrate the calculation and interpretation of predicted probability of injury. In our application, the predicted probability (or risk) of injury or death in a farm crash scenario was calcu- lated for each occupant involved in the crash as: Internal validation P Injury or Death ð Þ ¼ Odds 1 þ Odds ¼ 1 1 þ exp −α þ β1C1 þ β2C2 þ ⋯þ βiCi       Model fit was assessed in three ways. First, by using the quasi-likelihood information criterion (QIC), a variant of AIC designed for models fit using GEEs (Pan 2001). Smaller values of QIC (or AIC or BIC) correspond to models that provide better penalized fit (Akaike 1973; Schwarz 1978). Second, by calculating the concordance statistic (or c-statistic), which estimates the area under the receiver operating characteristic curve (AUC). The c-statistic ranges from 0.50 to 1, with higher values cor- responding to models that provide better discrimination between outcome occurrence and non-occurrence (Gail et al. 2007; Quante et al. 2012). Lastly, model fit was assessed by estimating an expected outcome count using the model coefficients in the same dataset from which the coefficients were obtained (Keys et al. 1972). A chi-squared statistic was calculated to compare the expected and observed outcome count to assess statis- tical difference between the two. Note that all three of these methods are of limited utility because they only evaluate the fitted model based on the same dataset used in the model construction (i.e., the “training data”), and do not examine if the predictive models could accurately predict outcomes in new data (i.e., in “validation data”). Hence validation of the predictive model in external data is also needed. Here, P is the predicted probability or risk of injury or death, C1, C2, … , Ci are the covariates in the logistic regression model, and β1, β2, … , βi are the regression coefficients for those covariates (Keys et al. 1972; Kleinbaum et al. 1971). All analyses were conducted using SAS 9.4 (SAS Institute, Cary, NC). Using the model coefficients, we built two interactive risk prediction tools, one using Microsoft Excel (Microsoft, Inc), and one using R-Shiny (RStudio). The study was considered non-human subjects by the Institutional Review Board at the University of Iowa due to the de-identified nature of the secondary data. Predicted probabilities The observed and expected counts for the validation data were most similar for models 2 and 3 (Table 4). This comparison revealed that our predictive models could accurately predict the number injuries or deaths in future farm crash data from the same nine US states. Predicted probabilities for different regression models can be readily obtained from standard statistical analyses programs such as SAS, STATA, R, etc. For this example, based on the estimated model coefficients from the final fitted logistic regression model (model 3), we constructed Ranapurwala et al. Injury Epidemiology (2019) 6:31 Page 5 of 11 Table 1 Distribution of farm vehicle crashes and resulting injuries and deaths by calendar year and state: 2005–2010 Farm vehicle crashes N (%) Injuries and deaths N (rate per 100 crashes) Deaths N (rate per 100 crashes) Total 7084 2087 (29.5) 163 (2.3) Calendar Year 2005 1166 (16.5) 338 (29.0) 21 (1.8) 2006 1114 (15.7) 318 (28.5) 29 (2.6) 2007 1198 (16.9) 336 (28.0) 26 (2.2) 2008 1160 (16.4) 316 (27.2) 22 (1.9) 2009 1196 (16.9) 407 (34.0) 37 (3.1) 2010 1250 (17.6) 372 (29.8) 28 (2.2) State Iowa 1178 (16.6) 421 (35.7) 35 (3.0) Illinois 1214 (17.1) 421 (34.7) 27 (2.2) Kansas 700 (9.9) 186 (26.6) 19 (2.7) Minnesota 850 (12.0) 199 (23.4) 22 (2.6) Missouri 1084 (15.3) 207 (19.1) 12 (1.1) North Dakota 253 (3.6) 51 (20.2) 12 (4.7) Nebraska 536 (7.6) 189 (35.3) 10 (1.9) South Dakota 232 (3.3) 74 (31.9) 8 (3.4) Wisconsin 1037 (14.6) 339 (32.7) 18 (1.7) Using predicted probabilities based on model 3, we con- structed a hypothetical crash scenario to estimate the probability of injury or death for the involved individuals with specific demographic and driving characteristic profiles. We consider the following non-modifiable characteristics for the scenario: male driver of a farm vehicle in Iowa, aged 25–34 years, was involved in a single vehicle farm crash on a clear morning (6:00–11:59 am) dur- ing the growing season (June–August). Semi-modifiable characteristics: the manner of collision set to non-collision, the vehicle action set to heading straight, and the presence of a male passenger. Modifiable characteristics: Driver contributing circumstances set to none and occupant protection set to all occupants wearing seatbelts (Table 5). In this scenario, the risk of injury or death for the farm vehicle driver would be 16.7% and that for a male passenger would be 17.8%. Predicted probabilities If, however, the driver and passenger were not wearing seatbelts, and the driver disregarded the traffic regulations, their respective risks of injury or death would be 48.5% and 50.4% (Table 5). Suppose this scenario was altered to be a multivehicle crash in which the farm vehicle (with the same male driver and male passenger) was rear-ended by a non-farm vehicle (with a male driver and a male passenger). If all the individuals were wearing seat belts, and both the drivers were following traffic regulations, then the risk of injury or death in the crash would be 7.3% for the farm vehicle driver, 7.8% for the farm vehicle passenger, 28.7% for the non-farm vehicle driver, and 30.3% for the non-farm vehicle passenger. If these individuals were not wearing seatbelts and the drivers did not follow traffic regulations, then their respective risks would be 26.9, 28.4, 65.3, and 67% (Table 5). Examining model fit as shown in Table 3, and validation in an external dataset (external to the training dataset) as shown in Table 4, are important pieces in addressing potential underfitting or overfitting of the predictive models. With an underfitted predictive model, within the training data, the expected model outcome counts will not accurately represent the observed counts due to bias. This may happen due to consideration of a limited num- ber of predictors, or due to collapsing variables with mul- tiple discrete categories into a few large categories that may not represent the finer categories. An adequate model fit will exhibit a close correspondence between the expected and observed counts for the training data (Table 3). On the other hand, one can include too many predictors (and interactions of those predictors) to achieve a nearly exact model fit, so that the expected model outcome counts from the training data nearly replicate the observed outcomes from the training data. When applied to external validation datasets, such overly complex models may not be able to accurately estimate the new observed outcome counts. These types of pre- dictive models are referred to as overfitted or non- generalizable. In this example, the models accurately approximate the observed counts for both the training dataset (Table 3) and the validation dataset (Table 4). The propriety of the model fits is also represented by the AUCs. These risks can be easily estimated by deploying inter- active tools that utilize model coefficients from the final fitted model. Predicted probabilities We developed a Microsoft Excel-based tool using the model 3 coefficients from 2005 to 2010 data that can be downloaded at https://drive.google.com/drive/ folders/0B0B0TgPTSgJ8bjVZYjdGaF96UzQ. We also de- veloped a similar internet-based tool using R-Shiny (Fig. 1); the online tool can be found at https://gpcah-farmcrash- predictive.shinyapps.io/predictiveapp/. These two tools are freely available and can be used by anyone to estimate an individual’s risk of injury in a farm crash. The risk estimates in Table 5 were produced using these tools. Discussion In this manuscript, we demonstrate how the regression coefficients obtained from predictive analyses can be used to forecast the risk of a health outcome for indivi- duals in specific farm crash scenarios. The predictive models allow us to compare the risks in different sce- narios, helping us to appreciate the change in risk with varying circumstances as shown in Table 5. Specifically, Page 6 of 11 Ranapurwala et al. Injury Epidemiology (2019) 6:31 Ranapurwala et al. Injury Epidemiology Table 2 Injury / death status, and estimated regression coefficients for non-modifiable, non + semi-modifiable, and non + semi +modifiable risk factors to predict the risk of injury or death in a farm crash: 2005–2010 Variables Categories Injured or died Model coefficients (std. Discussion Injury Epidemiology (2019) 6:31 Ranapurwala et al. Injury Epidemiology Table 2 Injury / death status, and estimated regression coefficients for non-modifiable, non + semi-modifiable, and non + semi +modifiable risk factors to predict the risk of injury or death in a farm crash: 2005–2010 (Continued) Variables Categories Injured or died Model coefficients (std. error) Yes No Model 1a Model 2b Model 3c Manner of collision Non collision (referent) 294 1301 0 0 Head-on 127 415 0.40 (0.17) 0.40 (0.18) Rear-end 716 2747 0.28 (0.13) 0.28 (0.13) Angle, oncoming left turn 361 2343 0.04 (0.14) 0.10 (0.14) Sideswipe, same direction 192 2698 −0.85 (0.15) −0.73 (0.16) Sideswipe, opposite direction 152 1462 −0.65 (0.16) −0.55 (0.16) Other 245 1781 −0.21 (0.14) −0.21 (0.14) Vehicle action Heading straight (referent) 1510 6934 0 0 Turning 133 2489 −0.55 (0.11) −0.62 (0.11) Overtaking/ passing/ changing lanes 280 1750 −0.25 (0.09) −0.34 (0.10) Slowing/stopping 43 608 −0.89 (0.17) −0.84 (0.18) Other 121 966 −0.30 (0.11) −0.35 (0.11) Multiple passengers No (referent) 1392 9908 0 0 Yes 695 2839 0.28 (0.08) 0.34 (0.08) Driver No (referent) 433 1710 0 0 Yes 1654 11,037 −0.16 (0.09) −0.08 (0.09) Modifiable factors Driver contributing circumstance No contributing action (referent) 871 6508 0 Disregarded traffic regulation 302 1487 0.35 (0.09) Reckless, careless, negligent, aggressive driving 407 2510 0.16 (0.08) Inattentive/distracted driver 256 1093 0.25 (0.09) Other contributing action 251 1149 0.25 (0.10) Occupant Protection None (referent) 870 4534 0 Seat belt 1052 7439 −1.19 (0.09) Child safety restraint 59 384 −1.25 (0.20) Other restraint/ protection 106 390 0.18 (0.16) Quasi-likelihood Information Criterion (QIC) 10,844.4 10,077.4 9676.8 AUC (95% CI) 0.69 (0.68, 0.71) 0.75 (0.74, 0.76) 0.78 (0.76, 0.79) Abbreviations: AUC Area under the receiver operating characteristic (ROC) curve amodel 1 includes non modifiable factors Table 2 Injury / death status, and estimated regression coefficients for non-modifiable, non + semi-modifiable, and non + semi +modifiable risk factors to predict the risk of injury or death in a farm crash: 2005–2010 (Continued) the change in modifiable and semi-modifiable factors may allow us to understand the impact of intervening on these factors on the individual’s risk of injury or death. outcome for every individual differently based on all other individual or scenario-specific coefficients (Table 5). Discussion error) Yes No Model 1a Model 2b Model 3c Intercept Intercept (constant) 2087 12,747 −1.75 (0.18) −1.73 (0.24) −0.97 (0.25) Non-modifiable factors State Iowa 421 1858 0.39 (0.10) 0.46 (0.10) 0.42 (0.10) Illinois 421 2230 0.19 (0.10) 0.20 (0.11) 0.33 (0.11) Kansas 186 1291 −0.19 (0.12) −0.21 (0.12) −0.26 (0.13) Minnesota 199 1688 −0.30 (0.11) −0.24 (0.12) −0.08 (0.12) Missouri 207 1686 −0.09 (0.11) −0.21 (0.12) −0.34 (0.12) North Dakota 51 431 −0.43 (0.21) −0.50 (0.21) −0.56 (0.21) Nebraska 189 970 0.16 (0.12) 0.11 (0.12) 0.08 (0.12) South Dakota 74 379 0.16 (0.17) 0.14 (0.18) −0.06 (0.19) Wisconsin (referent) 339 2214 0 0 0 Season Winter (Jan-Mar) (referent) 192 1510 0 0 0 Planting (Apr-May) 341 2307 0.06 (0.12) 0.20 (0.12) 0.17 (0.12) Growing (Jun-Aug) 635 3492 0.30 (0.11) 0.42 (0.11) 0.38 (0.11) Harvesting (Sep-Dec) 919 5438 0.19 (0.10) 0.16 (0.10) 0.15 (0.10) Weather at the time of crash Clear (referent) 1618 9802 0 0 0 Cloudy 329 2081 −0.01 (0.08) −0.09 (0.08) −0.11 (0.08) Rain 78 376 0.17 (0.15) −0.03 (0.16) 0.03 (0.16) Snow/sleet/hail/freezing rain/drizzle 22 289 −0.74 (0.28) −0.89 (0.28) −0.87 (0.28) Fog/smog/smoke/other 40 199 0.15 (0.22) 0.01 (0.22) −0.08 (0.23) Time of crash 12:00–5:59 am (referent) 131 739 0 0 0 6:00–11:59 am 501 3478 −0.11 (0.13) 0.13 (0.14) 0.17 (0.14) 12:00–5:59 pm 885 6218 −0.14 (0.13) 0.08 (0.13) 0.08 (0.14) 6:00–11:59 pm 570 2312 0.41 (0.14) 0.12 (0.14) 0.11 (0.14) Number of vehicles Single vehicle 238 827 1.37 (0.10) 1.22 (0.13) 1.22 (0.14) Two or more vehicles (referent) 1849 11,920 0 0 0 Equipment type Farm vehicle/equipment 672 6875 −1.14 (0.06) −1.02 (0.06) −1.63 (0.08) Non-farm vehicle (referent) 1415 5872 0 0 0 Age < 16 years age 159 799 0.24 (0.11) 0.04 (0.13) 0.13 (0.14) 16–24 years age 388 2120 0.24 (0.09) 0.21 (0.09) 0.22 (0.10) 25–34 years age (referent) 234 1839 0 0 0 35–44 years age 288 1851 0.15 (0.10) 0.15 (0.10) 0.20 (0.10) 45–54 years age 325 2326 0.11 (0.09) 0.10 (0.10) 0.13 (0.10) 55–64 years age 278 1794 0.22 (0.10) 0.24 (0.10) 0.30 (0.10) 65+ years age 415 2018 0.49 (0.09) 0.51 (0.09) 0.59 (0.10) Sex Female (referent) 626 2663 0 0 0 Male 1461 10,084 −0.19 (0.06) −0.14 (0.06) −0.26 (0.06) Semi-modifiable factors h l h ( f ) Table 2 Injury / death status, and estimated regression coefficients for non-modifiable, non + semi-modifiable, and non + semi +modifiable risk factors to predict the risk of injury or death in a farm crash: 2005–2010 Table 2 Injury / death status, and estimated regression coefficients for non-modifiable, non + semi-modifiable, and non + semi +modifiable risk factors to predict the risk of injury or death in a farm crash: 2005–2010 Page 7 of 11 Page 7 of 11 Ranapurwala et al. Discussion Average predicted probability, AUC Area under the receiver operating curve, QIC Quasi-likelihood information criteria this example, other characteristics were considered con- stant for all the vehicle occupants, but they may be changed easily (and will result in different risk esti- mates) to determine risks for other individuals in different scenarios. 2010; Maio et al. 1992; Stephan and Newstead 2014; Russo et al. 2014). However, these studies focused on reporting effect estimates (odds ratios or rate ratios) of association between individual risk factors and injury or death in a farm crash. Such models also assume that an individual’s outcomes do not depend on another individ- ual’s outcomes (no interference assumption, also known as the stable unit treatment value assumption) (Schwartz et al. 2012). This is another differentiating feature, that while causal (or associational) modeling assumes (rather requires) no interference, predictive modeling embraces interference. The non-modifiable, semi-modifiable, and modifiable factors used to predict the injury or death in our example dataset have been previously identified as risk factors for a farm crash (Ranapurwala et al. 2016; Hughes and Rodgman 2000; Peek-Asa et al. 2007; Pinzke and Lundqvist 2004; Costello et al. 2003; Gerberich et al. 1996; Jaarsma and De Vries 2014), and injury (Hughes and Rodgman 2000; Marlenga et al. 2006; Gkritza et al. p g Using different combinations of predictors could allow one to estimate the predictive capability (or sensitivity) Table 4 Validation of the predictive models Validation data years (Training data years) Total validation data occupants (training data occupant) Observed injuries or deaths in validation data (N) Non-modifiable (Model 1) Non + semi-modifiable (Model 2) Non + semi + modifiable (Model 3) Avg. pred. Prob. Expected injuries/ deaths (N) Avg. pred. Prob. Expected injuries/ deaths (N) Avg. pred. Prob. Expected injuries/ deaths (N) 2008-‘10 (‘05-‘07) 7624 (7210) 1095 0.1407b 1073 0.1445e 1102 0.1437h 1095 2009-‘10 (‘05-‘08) 5216 (9618) 779 0.1383c 721a 0.1405f 733 0.1397i 729 2010 (‘05-‘09) 2615 (12,219) 372 0.1424d 372 0.1410g 369 0.1401j 366 Abbreviations: Avg. pred. Prob. Average predicted probability ap-value = 0.0327 (Chi Sq = 4.56, df = 1), suggesting that expected injuries and deaths (n = 721) were significantly different than the observed (n = 779). Discussion Predictive modeling is used to ultimately answer the questions like “what is the probability (or risk) of an injury for an individual given a crash and the combi- nation of all circumstantial factors?” It is noteworthy that the risks estimates presented in Table 5 do not entirely depend on driver behavior. They also depend on the total contribution of risk factors such as driver or passenger (semi-modifiable factor), farm or non-farm vehicle (non-modifiable factor), and single vehicle crash or multivehicle crash (non-modifiable factor). In addition, in In predictive analyses, the estimated model coefficients may be used to suggest that a certain variable is a strong or weak predictor of the outcome; however, the coeffi- cient estimate is not used to characterize the effect of a variable on the outcome (which is the aim of causal modeling). This fundamentally differentiates predictive modeling from causal modeling, with the latter being ubiquitous in the public health literature. In predictive modeling, a coefficient affects the probability of the Ranapurwala et al. Injury Epidemiology (2019) 6:31 Page 8 of 11 Ranapurwala et al. Injury Epidemiology Table 3 Comparison of the expected (model-based) to observed number of injuries in the nine states from 2005 to 2010 State Total occupants (N) Observed injuries or deaths (N) Non-modifiable (Model 1) QIC = 10,844.4 AUC = 0.69 (0.68, 0.71) Non + semi-modifiable (Model 2) QIC = 10,077.4 AUC = 0.75 (0.74, 0.76) Non + semi + modifiable (Model 3) QIC = 9676.8 AUC = 0.78 (0.76, 0.79) Avg. pred. Prob. Expected injuries/ deaths (N) Avg. pred. Prob. Expected injuries/ deaths (N) Avg. pred. Prob. Expected injuries/ deaths (N) IA 2279 421 0.1804 411 0.1820 415 0.1818 414 IL 2651 421 0.1600 424 0.1602 425 0.1594 422 KS 1477 186 0.1251 185 0.1246 184 0.1242 183 MN 1887 199 0.1008 190 0.1044 197 0.1017 192 MO 1893 207 0.1135 215 0.1169 221 0.1197 227 ND 482 51 0.1052 51 0.1066 51 0.1064 51 NE 1159 189 0.1634 189 0.1641 190 0.1619 188 SD 453 74 0.1549 70 0.1586 72 0.1592 72 WI 2553 339 0.1383 353 0.1367 349 0.1365 348 Total 14,834 2087 0.1408 2089 0.1419 2104 0.1415 2099 Abbreviations: Avg. pred. Prob. Discussion All other expected to observed differences were non-significant bAUC = 0.69 based on training data 2005–2007 cAUC = 0.70 based on training data 2005–2008 dAUC = 0.69 based on training data 2005–2009 eAUC = 0.75 based on training data 2005–2007 fAUC = 0.75 based on training data 2005–2008 gAUC = 0.74 based on training data 2005–2009 hAUC = 0.77 based on training data 2005–2007 iAUC = 0.77 based on training data 2005–2008 jAUC = 0.77 based on training data 2005–2009 Table 4 Validation of the predictive models Table 4 Validation of the predictive models Page 9 of 11 Page 9 of 11 Ranapurwala et al. Injury Epidemiology (2019) 6:31 Ranapurwala et al. Injury Epidemiology Table 5 Predicted probabilities of injury or death for drivers and passengers in varying farm crash scenarios using the model 3 estimated coefficients from Table 1 The risk or injury or death in a farm crash in Iowa for a 25–34 year old, male, in growing season, clear weather, between 6:00–11:59 am, daylight, heading straight, passengers on board (for single vehicle crash, manner of collision = non collision; for multiple vehicle crash, manner of collision = rear end) Vehicle/ occupant type Seat belt Driver contributing circumstances Risk of injury or death (%) Single vehicle crash Multiple vehicle crash Farm vehicle driver Yes None 16.7% 7.3% Farm vehicle passenger 17.8% 7.8% Non-farm vehicle driver 28.7% Non-farm vehicle passenger 30.3% Farm vehicle driver Yes Disregarded traffic regulations 22.2% 10.1% Farm vehicle passenger 23.6% 10.8% Non-farm vehicle driver 36.4% Non-farm vehicle passenger 38.2% Farm vehicle driver No None 39.8% 20.6% Farm vehicle passenger 41.6% 21.8% Non-farm vehicle driver 57.0% Non-farm vehicle passenger 58.8% Farm vehicle driver No Disregarded traffic regulations 48.5% 26.9% Farm vehicle passenger 50.4% 28.4% Non-farm vehicle driver 65.3% Non-farm vehicle passenger 67.0% Table 5 Predicted probabilities of injury or death for drivers and passengers in varying farm crash scenarios using the model 3 estimated coefficients from Table 1 simulate potential effects of evidence-based inter- ventions in well-defined populations and conduct cost- benefit analysis for widespread policy initiatives. For example, if an intervention is known to improve seat-belt compliance by 60% among a specific group of low compliant drivers, it could be simulated using our predictive model, which will allow us to estimate the number of overall injuries and deaths prevented. Discussion We can then estimate the cost of the prevented injuries and deaths and compare that against the cost of implemen- ting the intervention. Similar predictive models could be developed in other research areas to help physicians make more informed decisions about prescribing medications to their patients. For example, a risk prediction tool, like the one we devel- oped, could inform physicians of their patient’s risk of suffering from an opioid use disorder in the future due to exposure to prescription opioid medication. Such in- formation will help the physician to better titrate the patient’s pain medications. Eventually, such tools may not only aid physicians but also improve health out- comes for patients. Now, with the advancement of analytical approaches and superior computing power, and their integration in medicine, such approaches can be feasibly deployed and used. y p y Some best practices to develop and improve predictive models may involve the following, although this is not an exhaustive list. First, the overall data sample should be carefully selected so that it represents the relevant source population. Second, the training dataset should be large enough so as to observe all possible combi- nations of predictors. Third, the validation dataset should represent the same data generating mechanism as the training dataset, which can be accomplished through random selection of the observations for the validation dataset from the overall sample. Fourth, for quantitative variables, the most appropriate functional or categorical representation must be utilized. For example, age broken into 5-year categories may sometimes provide better prediction than 10-year age categories or age treated as a continuous variable. Fifth, when predictive outcomes exhibit secular time trends, appropriately accounting for such trends in the modeling structure may help improve the prediction. Sixth, when combining finer categorical variables into larger categories, it is prudent to combine finer categories that produce similar model coefficients and avoid combining categories that vary considerably in their model coefficients. The use of a model selection criterion, such as QIC, AIC, or BIC, can often facilitate the fourth, fifth and sixth objectives. Lastly, for a predictive model to be utilized in practice, there must be a temporal ordering between the predictors and the outcome; specifically, all predictors must be observed before the occurrence of the outcome. of the predictive models. Discussion For example, we identified real- istic scenarios where the risk of injury given a farm crash was as low as 3% for some occupants, and as high as 93% for others, a difference of up to 90% of injury and death risk. This suggests good predictive capability of the model, since the predictive model covers most of the probability range between 0 and 1 (or 0 and 100%). Such a sensitive tool may be useful for policy makers to Ranapurwala et al. Injury Epidemiology (2019) 6:31 Ranapurwala et al. Injury Epidemiology Page 10 of 11 (2019) 6:31 Fig. 1 Screenshot of the online tool to calculate risk of injury or death in a farm crash Fig. 1 Screenshot of the online tool to calculate risk of injury or death in a farm crash These individual-level predictive models are extended further into more complex modeling frameworks like agent-based models that predict systems level changes. Historical and existing data sources can be used to develop predictive models, which can incorporate more data over time to improve the model’s predictive capabi- lities. Such iterative updating can be viewed as a type of “machine learning” (Wicks et al. 2016). Advances in computing have already taken predictive modeling to the next stage, via machine learning and precision medicine. Methods such as random forests, neural net- works, and Q-learning utilize observational or experi- mental data, superimpose predictive modeling theory, and not only predict potential future outcomes, but also allow us to make evidence-based decisions to optimize the best possible outcomes in the future. However, some machine learning methods have been characterized as “black box” techniques that obfuscate rather than illuminate the dynamics of the underlying phenomenon. A clear advantage of the predictive modeling approach presented here is that it provides an interpretable and transparent characterization of these dynamics (Keil and Edwards 2018). US states. Second, the crashes included in these data are those that were reported to the police. Some crashes may never be reported, misreported as non-farm crashes, or be excluded due to missing information. Similarly, mistakes may materialize in the reporting of injury severity by the responding police officers. Such exclusions and measure- ment errors may lead to misclassification of the predicted probabilities. Conclusion Predictive analysis offers elegant tools that may help us understand the multidimensionality of the occurrence of different health outcomes and allow individual-level risk assessment. Algorithms and tools derived from such analyses could support policymakers, physicians, and environmental health practitioners in developing and implementing tailored prevention strategies to improve health outcomes for their patients or clients. Discussion Third, we developed the predictive models using commonly available variables form the nine states; however, there may be other unmeasured variables that may predict the outcome and hence may have an impor- tant role in determining the risk of injury for an indivi- dual. Generalizability, misclassification, and unmeasured predictors are three main limitations that may exist with most predictive models. Limitations We encountered a number of limitations in our example dataset that commonly arise in predictive modeling regardless of the data source or substantive area of research. First, these data were collected from nine US states; hence, the results are not generalizable to other g The authors thank the Great Plains Center for Agricultural Health and Dr. Karisa Harland for their continued support and guidance in continuing this work. We also thank Dr. Elizabeth T. Rogawski and Dr. Stephen W. Marshall for their review of earlier versions of this manuscript. Competing interests Pinzke S, Lundqvist P. Slow-moving vehicles in Swedish traffic. J Agric Saf Health. 2004;10(2):121–6. The authors declare that they have no competing interests. assessment across the risk continuum: genetic and nongenetic risk factors contributing to differential model performance. Breast Cancer Res. 2012;14(6):R144. Ranapurwala SI, Mello ER, Ramirez MR. A GIS-based matched case-control study of road characteristics in farm vehicle crashes from nine Midwestern US states. Epidemiology. 2016;27(6):827–34. Russo F, Biancardo SA, Dell’acqua G. Road safety from the perspective of driver gender and age as related to the injury crash frequency and road scenario. Traffic Inj Prev. 2014;15(1):25–33. S h S M thi S W X t l Th NCEP li t f t t i 2 J Author details 1 Russo F, Biancardo SA, Dell’acqua G. Road safety from the perspective of driver gender and age as related to the injury crash frequency and road scenario. Traffic Inj Prev. 2014;15(1):25–33. Russo F, Biancardo SA, Dell’acqua G. Road safety from the perspective of driver gender and age as related to the injury crash frequency and road scenario. Traffic Inj Prev. 2014;15(1):25–33. 1Injury Prevention Research and Department of Epidemiology, University of North Carolina at Chapel Hill, 137 E Franklin St, Suite 500, CB# 7505, Chapel Hill, NC 27599, USA. 2Department of Epidemiology, College of Public Health, University of Iowa, Iowa City, IA, USA. 3Injury Prevention Research Center, Department of Occupational and Environmental Health, College of Public Health, University of Iowa, Iowa City, IA, USA. 4Department of Biostatistics, University of Iowa, Iowa City, IA, USA. 5Division of Environmental Health Sciences, University of Minnesota, Minneapolis, MN, USA. Schoen H, Gayo-Avello D, Metaxas PT, et al. The power of prediction with social media. Internet Res. 2013;23(5):528–43. Received: 20 December 2018 Accepted: 29 April 2019 Authors’ contributions Harland KK, Greenan M, Ramirez M. Not just a rural occurrence: differences in agricultural equipment crash characteristics by rural-urban crash site and proximity to town. Accid Anal Prev. 2014;70:8–13. SIR conceptualized and analyzed the data, created the Excel-based tool, interpreted the results, and wrote the first draft of the manuscript. MRR and CPA provided the substantive grounding in farm vehicle crash research. TY acquired the data and conducted analyses. HW conducted analyses, especially the multiple imputations, and created the R-Shiny tool. JEC provided guidance toward the deep understanding of the analytic methods. JEC, CPA, MRR acquired the funding, data, and interpreted the results. All authors critically reviewed all drafts of the manuscript and approved the final draft. Hughes R, Rodgman E. Crashes involving farm tractors and other farm vehicles/ equipment in North Carolina 1995–1999. Chapel Hill: Highway Safety Research Center; 2000. Available at: https://trid.trb.org/view. aspx?id=673852. Accessed 7 Jan 2015. Hughes R, Rodgman E. Crashes involving farm tractors and other farm vehicles/ equipment in North Carolina 1995–1999. Chapel Hill: Highway Safety Research Center; 2000. Available at: https://trid.trb.org/view. aspx?id=673852. Accessed 7 Jan 2015. Hughes R, Rodgman E. Crashes involving farm tractors and other farm vehicles/ equipment in North Carolina 1995–1999. Chapel Hill: Highway Safety Research Center; 2000. Available at: https://trid.trb.org/view. aspx?id=673852. Accessed 7 Jan 2015. Jaarsma CF, De Vries JR. Agricultural vehicles and rural road safety: tackling a persistent problem. Traffic Inj Prev. 2014;15(1):94–101. Jaarsma CF, De Vries JR. Agricultural vehicles and rural road safety: tackling a persistent problem. Traffic Inj Prev. 2014;15(1):94–101. Keil AP, Edwards JK. You are smarter than you think: (super) machine learning in context. Eur J Epidemiol 2018 https://doiorg/101007/s10654-018-0405-9 [Epub ahead of print] Keil AP, Edwards JK. You are smarter than you think: (super) machine learning in context. Eur J Epidemiol. 2018. https://doi.org/10.1007/s10654-018-0405-9 [Epub ahead of print]. Ethics approval and consent to participate Keys A, Aravanis C, Blackburn H, et al. Probability of middle-aged men developing coronary heart disease in five years. Circulation. 1972;45(4):815–28. The study was considered non-human subjects by the Institutional Review Board at the University of Iowa due to the de-identified nature of the secondary data. Kleinbaum DG, Kupper LL, Cassel JC, Tyroler HA. Multivariate analysis of risk of coronary heart disease in Evans County, Georgia. Arch Intern Med. 1971;128:943–8. heart disease in Evans County, Georgia. Arch Intern Med. 1971;128:943–8. Maio RF, Green PE, Becker MP, Burney RE, Compton C. Rural motor vehicle crash mortality: the role of crash severity and medical resources. Accid Anal Prev. 1992;24(6):631–42. Publisher’s Note Springer Nature remains neutral with regard to jurisdictional claims in published maps and institutional affiliations. Funding DC: Bureau of Labor Statistics (BLS); 2013a. http://data.bls.gov/gqt/ InitialPage. Accessed 29 Jan 2015 This work was funded by the Great Plains Center for Agricultural Health which is funded through the National Institute for Occupational Health and Safety grant # U50 OH007548–11. The funding for publication was provided by the University of North Carolina at Chapel Hill Injury Prevention Research Center, which is funded by the Centers for Disease Control and Prevention grant #R49 CE002479. Bureau of Labor Statistics, U.S. Department of Labor. Census of nonfatal occupational injuries and illnesses: agricultural workers (code 452000). Washington, DC: Bureau of Labor Statistics (BLS); 2013b. http://data.bls.gov/ gqt/InitialPage. Accessed 29 Jan 2015 Costello TM, Schulman MD, Luginbuhl RC. Understanding public health impacts of farm vehicle public road crashes in North Carolina. J Agric Saf Health. 2003;9(1):19–32. Gail MH, Costantino JP, Pee D, et al. Projecting individualized absolute invasive breast cancer risk in African American women. J Natl Cancer Inst. 2007;99(23):1782–92. Gerberich SG, Robertson LS, Gibson RW, Renier C. An epidemiological study of roadway fatalities related to farm vehicles: United States, 1988 to1993. J Occup Env Med. 1996;38(11):1135–40. Gkritza K Kinzenbaw CR Hallmak S Hawkins N An empirical analysis of farm Availability of data and materials All data generated or analyzed during this study are included in this published article in aggregate form (Tables 1 and 2) which can be easily used to reproduce the study results. Line level data are available from the Departments of Transportation of the nine states listed in this study, but restrictions apply to the availability of these data, which were used under license for the current study, and so are not publicly available. Data are however available from the authors upon reasonable request and with permission of the respective Departments of Transportation. Gkritza K, Kinzenbaw CR, Hallmak S, Hawkins N. An empirical analysis of farm vehicle crash injury severities on Iowa’s public road system. Accid Anal Prev. 2010;42(4):1392-7. Glanz JM, Narwaney KJ, Mueller SR, et al. Prediction model for two-year risk of opioid overdose among patients prescribed chronic opioid therapy. J Gen Intern Med. 2018; [Epub ahead of print]. Acknowledgements The authors thank the Great Plains Center for Agricultural Health and Dr. Karisa Harland for their continued support and guidance in continuing this work. We also thank Dr. Elizabeth T. Rogawski and Dr. Stephen W. Marshall for their review of earlier versions of this manuscript. Page 11 of 11 Page 11 of 11 Ranapurwala et al. Injury Epidemiology (2019) 6:31 Ranapurwala et al. Injury Epidemiology Ranapurwala et al. Injury Epidemiology (2019) 6:31 (2019) 6:31 Consent for publication The data was collected on human beings, however, it was collected as an administrative data by law enforcement at the scene of a crash. The data was pre-existing at the time of this research and the study team did not receive and identifiable information on any individuals and there was no direct contact with people whose data are included in the study. Hence, we could not have possibly taken a consent for publication. Marlenga B, Doty BC, Berg RL, Linneman JG. Evaluation of a policy to reduce youth tractor crashes on public roads. Inj Prev. 2006;12:46–51. Marlenga B, Doty BC, Berg RL, Linneman JG. Evaluation of a policy to reduce youth tractor crashes on public roads. Inj Prev. 2006;12:46–51. Pan W. Akaike’s information criterion in generalized estimating equations. Biometrics. 2001;57(1):120–5. Peek-Asa C, Sprince NL, Whitten PS, et al. Characteristics of crashes with farm equipment that increase the potential for injury. J Rural Health. 2007;23(4):339–47 Pinzke S, Lundqvist P. Slow-moving vehicles in Swedish traffic. J Agric Saf Health. 2004;10(2):121–6. Pan W. Akaike’s information criterion in generalized estimating equations. Biometrics. 2001;57(1):120–5. Peek-Asa C, Sprince NL, Whitten PS, et al. Characteristics of crashes with farm equipment that increase the potential for injury. J Rural Health. 2007;23(4):339–47. References Akaike H. Information theory and an extension of the maximum likelihood principle. In: Proc. 2nd inter. symposium on information theory, 267–281, Budapest; 1973. American Heart Association (AHA). Atherosclerotic cardiovascular disease (ASCVD) risk calculator. Available at: http://static.heart.org/riskcalc/app/index.html#!/ baseline-risk. Accessed 5 July 2017. Wu L, Bryniolfsson E. The future of prediction: how Google searches foreshadow housing prices and sales: SSRN; 2013. https://doi.org/10.2139/ssrn.2022293. Bureau of Labor Statistics, U.S. Department of Labor. Census of fatal occupational injuries: agricultural workers (code 452XXX). Washington,
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Middle Jurassic black shales (Skrzypny Shale Formation) – palaeoenvironmental significance of one of the oldest deposits of the Pieniny Klippen Belt.
Geotourism/Geoturystyka
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Środkowojurajskie czarne łupki (formacja łupków ze Skrzypnego) – znaczenie paleośrodowiskowe jednych z najstarszych osadów pienińskiego pasa skałkowego Katarzyna Górniak1, Krzysztof Bahranowski1, Adam Gaweł1, Leszek Marynowski2 & Tadeusz Szydłak1 1 AGH University of Science and Technology, Department of Mineralogy, Petrography and Geochemistry, 30-059 Kraków, al. Mickiewicza 30, Poland; e-mail: gorniak@agh.edu.pl 2 University of Silesia, Faculty of Earth Sciences, 41-200 Sosnowiec, ul. Będzińska 60, Poland e-mail: marynows@wnoz.us.edu.pl Abstract: This paper deals with a fragment of geological history of Middle Jurassic black spherosideritic shales outcropping in upper part of the Homole Gorge (Skrzypny Shale Formation, Niedzica succession). These important deposits belong to the Flackenmergel facies, widespread in oceanic Tethyan deposits. This formation represents one of the oldest rocks of the Pieniny Klippen Belt. Dark colour of these rocks (total organic carbon content in them amounts to about 1 wt.%) and the occurrence of siderite concretions indicate reducing conditions of their origin but oxidizing environment of waters above them in sedimentary basin. Oxic conditions of organic matter sedimentation are confirmed by the elevated values of pristine to phytane ratio. Moreover the results based on n-alkanes indicate mixed terrestrial/marine origin of organic matter with prevalence of the last one These information are slightly obliterated because of advanced thermal maturity of organic matter caused by post- sedimentation processes. Microstructural features of deposits of the Skrzypny Shale Formation and the composition of clay minerals occurring in them indicate late diagenetic conditions. In SEM images we observe one-directional orientation of clay minerals and rotation of mica flakes, often chloritized. Clay minerals are represented by high-illitic illite/smectite, micas, post-biotitic chlorite and subordinately kaolinite. As follows from authors studies, these macroscopically nearly “mute” clay rocks, little interesting in outcrops, particularly when they occur in such attractive geological objects as the Homole Gorge, can be the source of important information concerning the history of sedimentation basins. Key words: Pieniny Klippen Belt, middle Jurassic, black shales, Warszawa Kraków Warszawa Kraków Warszawa Kraków Warszawa Kraków Zakopane Chabówka Czarny Dunajec Niedzica Szczawnica Nowy Targ Warszawa Kraków Warszawa Kraków Warszawa Kraków Warszawa Kraków Zakopane Chabówka Czarny Dunajec Niedzica Szczawnica Nowy Targ ukazujących się w niewielkim odsłonięciu w górnej części wąwozu Homole (formacja łupków ze Skrzypnego, sukcesja niedzicka). Te ważne utwory należą do facji Fleckenmergiel szeroko rozpowszechnionej w oceanie Tetydy. Formacja łupków ze Skrzypnego reprezentuje jedne z najstarszych skał pienińskiego pasa skałkowego. Środkowojurajskie czarne łupki (formacja łupków ze Skrzypnego) – znaczenie paleośrodowiskowe jednych z najstarszych osadów pienińskiego pasa skałkowego Ciemna barwa badanych skał (zawartość całkowitego węgla organicznego około 1% wag.) oraz występowanie w nich konkrecji syderytowych, wskazują na warunki redukcyjne panujące w osadzie, natomiast na obecność natlenionych wód nad osadem w środowisku ich sedymentacji. Sedymentację substancji organicznej w warunkach raczej utleniających potwierdzają badania związków organicznych, tj. wysokie wartości stosunku pristan/fitan. Wyniki tych badań wskazują ponadto na mieszane, lądowo/morskie pochodzenie substancji organicznej z przewagą ostatniej z wymienionych. Informacje te są nieco zatarte z powodu dużej dojrzałości termicznej substancji organicznej spowodowanej procesami postsedymentacyjnymi. Cechy mikrostrukturalne utworów formacji łupków ze Skrzyponego oraz skład występujących w nich minerałów ilastych wskazują na warunki odpowiadające późnej diagenezie. W obrazach elektronomikroskopowych (SEM) widoczna jest bowiem jednokierunkowa orientacja minerałów ilastych i rotacja blaszek mik, często schlorytyzowanych. W składzie minerałów ilastych występuje zaś wysokoillitowy illit/ smektyt, miki, pobiotytowy chloryt i podrzędnie kaolinit. Z przeprowadzonych badań wynika, że makroskopowo niemal „nieme” skały ilaste, niepozorne wyglądające w odsłonięciu, szczególnie jeśli występują obok atrakcyjnych obiektów geologicznych jak to ma miejsce w wąwozie Homole, są źródłem ważnych informacji dotyczących historii basenów sedymentacyjnych. Warto więc zwrócić na nie uwagę. Słowa kluczowe: Pieniński Pas Skałkowy jura środkowa czarne Warszawa Warszawa Warszawa Warszawa Abstract: This paper deals with a fragment of geological history of Middle Jurassic black spherosideritic shales outcropping in upper part of the Homole Gorge (Skrzypny Shale Formation, Niedzica succession). These important deposits belong to the Flackenmergel facies, widespread in oceanic Tethyan deposits. This formation represents one of the oldest rocks of the Pieniny Klippen Belt. Dark colour of these rocks (total organic carbon content in them amounts to about 1 wt.%) and the occurrence of siderite concretions indicate reducing conditions of their origin but oxidizing environment of waters above them in sedimentary basin. Oxic conditions of organic matter sedimentation are confirmed by the elevated values of pristine to phytane ratio. Moreover the results based on n-alkanes indicate mixed terrestrial/marine origin of organic matter with prevalence of the last one These information are slightly obliterated because of advanced thermal maturity of organic matter caused by post- sedimentation processes. Microstructural features of deposits of the Skrzypny Shale Formation and the composition of clay minerals occurring in them indicate late diagenetic conditions. In SEM images we observe one-directional orientation of clay minerals and rotation of mica flakes, often chloritized. Clay minerals are represented by high-illitic illite/smectite, micas, post-biotitic chlorite and subordinately kaolinite. Środkowojurajskie czarne łupki (formacja łupków ze Skrzypnego) – znaczenie paleośrodowiskowe jednych z najstarszych osadów pienińskiego pasa skałkowego As follows from authors studies, these macroscopically nearly “mute” clay rocks, little interesting in outcrops, particularly when they occur in such attractive geological objects as the Homole Gorge, can be the source of important information concerning the history of sedimentation basins. Abstract: This paper deals with a fragment of geological history of Middle Jurassic black spherosideritic shales outcropping in upper part of the Homole Gorge (Skrzypny Shale Formation, Niedzica succession). These important deposits belong to the Flackenmergel facies, widespread in oceanic Tethyan deposits. This formation represents one of the oldest rocks of the Pieniny Klippen Belt. Dark colour of these rocks (total organic carbon content in them amounts to about 1 wt.%) and the occurrence of siderite concretions indicate reducing conditions of their origin but oxidizing environment of waters above them in sedimentary basin. Oxic conditions of organic matter sedimentation are confirmed by the elevated values of pristine to phytane ratio. Moreover the results based on n-alkanes indicate mixed terrestrial/marine origin of organic matter with prevalence of the last one These information are slightly obliterated because of advanced thermal maturity of organic matter caused by post- sedimentation processes. Microstructural features of deposits of the Skrzypny Shale Formation and the composition of clay minerals occurring in them indicate late diagenetic conditions. In SEM images we observe one-directional orientation of clay minerals and rotation of mica flakes, often chloritized. Clay minerals are represented by high-illitic illite/smectite, micas, post-biotitic chlorite and subordinately kaolinite. As follows from authors studies, these macroscopically nearly “mute” clay rocks, little interesting in outcrops, particularly when they occur in such attractive geological objects as the Homole Gorge, can be the source of important information concerning the history of sedimentation basins. Słowa kluczowe: Pieniński Pas Skałkowy, jura środkowa, czarne łupki, mikrostruktura, minerały ilaste Geoturystyka 2 (13) 2008: 19-24 Geoturystyka 2 (13) 2008: 19-24 Introduction Early Jurassic dark marly facies (of Fleckenmergel type) are particularly interesting for researchers since in them there are recorded the initial stages of evolution of the Pieniny basin (Birkenmajer, 1986, Krobicki & Golonka 2008a and the refer- ences in these papers). In Polish part of the Pieniny Klippen Belt one of the oldest rocks belonging to this facies are dark sphaerosideritic shale with Bositra bivalve (Skrzypny Shale Formation) of Late Aalenian-Early Bajocian age (Krobicki Key words: Pieniny Klippen Belt, middle Jurassic, black shales, mikrostructure, clay minerals Treść: Praca przedstawia fragment historii geologicznej środkowojurajskich czarnych łupków sferosyderytowych 19 Middle Jurassic black shales (Skrzypny Shale Formation) Middle Jurassic black shales (Skrzypny Shale Formation) transporting oxygen-deficient waters are entering the conti- nental slope. This model corresponds to the conditions dominating in Middle Jurassic Pieniny basin. Dark marly facies sediments of this age appear in rocks of the Niedzica succession deposited on continental slope and in those of the Czorsztyn succession accumulated on the slope of Czorsztyn ridge. As already informed, the former are one of the oldest deposits forming the fold of the Czajakowa Skała Klippe and are observed in uppermost part of the Homole Gorge. The dark shales of the Czorsztyn succession are exposed in the Krupianka stream (Krobicki & Golonka, 2008a, Fig. 14A, and references in this paper). Bathymetric conditions of for- mation of sediments of the Fleckenmergel facies, estimated by Tyszka (2001) on the ground of microfossil studies, cor- respond to the slope zone. & Golonka, 2008a, Fig. 7). This formation is described as a series of clay rocks of black and black-greenish colour, con- taining sphaerosideritic concretions and sometimes ammo- nites. The thickness of this formation amounts to approxi- mately 5 m. These interesting deposits are outcropping in upper part of the Homole Gorge, at the pass below Czajakowa Skała Klippe (Krobicki & Golonka, 2008a, Fig. 12D). & Golonka, 2008a, Fig. 7). This formation is described as a series of clay rocks of black and black-greenish colour, con- taining sphaerosideritic concretions and sometimes ammo- nites. The thickness of this formation amounts to approxi- mately 5 m. These interesting deposits are outcropping in upper part of the Homole Gorge, at the pass below Czajakowa Skała Klippe (Krobicki & Golonka, 2008a, Fig. 12D). Outcrop at the pass below Czajakowa Skała Klippe The Skrzypny Shale Formation outcropping here belongs to the Niedzica succession. The deposits of this succession occur in the form of a slice contacting tectonically with un- derlying rocks. This slice represents an overturned fold, strongly tectonically folded and forming the Czajakowa Skała Klippe. The Skrzypny Shale Formation occurs in the bottom of the Niedzica succession rock complex. A fragment of Czajakowa Skała fold was displaced on a bed of these shales, representing sliding plane, and actually they occur in a land- slide in upper part of the Homole Gorge (Krobicki & Golon- ka, 2008a, Fig. 12D). In this small outcrop the shales in question represent fine slaty, dark gray-olive shales contain- ing mica flakes up to 0.5 mm in size and brown sideritic concretions. These shales are soft, poorly calcareous, contain- ing no sand fraction. The size of sphaerosiderites does not exceed a dozen cm. They are hard, containing fractures filled with calcite. These carbonate concretions from other occur- rence of these shales of the Niedzica succession in Kapuśnica near Niedzica castle were examined in detail by W. Narębski (Birkenmajer & Narębski, 1957). They were found to contain, apart from sideroplesitic (Mg-sideritic) component, significant admixture of calcite, indicating their deposition in rather deep-water and more offshore marine environment than these shales of the Czorsztyn and Branisko successions. The geo- chemical character of concretions and shales containing them clearly suggests their early diagenetic origin under conditions of moderately reducing sideritic geochemical facies (Birken- majer & Narębski, 1957). This conclusion is confirmed by their loaf-shaped forms (Seilacher, 2001). The series of dark clayey rocks accumulating in oxygen- deficient environments are usually but apparently monotonous. Complex sedimentologic, palaeontologic and geochemical studies of the Lower Jurassic sediments in Yorkshire, United Kingdom, allowed to distinguish three facies occurring in the basins dominated by generally oxygen-deficient waters. They have resulted in distinguishing normal, restricted and bitumi- nous shales (Morris, 1979). This facial variability is caused by cyclic change of position of the boundary between oxygen- ated and oxygen-deficient zones in the water column above the sediment. Lithological features of sediments of the Skrzypny Shale Formation of the Niedzica succession outcrop- ping at the pass below Czajakowa Skała Klippe represent the normal shales facies. This conclusion results from the com- parison of their properties with those of sediments reported by Morris (1979) for individual facies distinguished. Outcrop at the pass below Czajakowa Skała Klippe One-directional orientation of flakes of clay minerals bent on a quartz grain (Q) due to compaction (SEM/EDX) • Jednokierunkowa orientacja blaszek minerałów ilastych ugiętych na ziarnie kwarcu wyniku kompakcji widoczna w badaniach elektronomikroskopo- wych świeżego przełamu próbki. (SEM/EDX) Q Fig. 4. One-directional orientation of flakes of clay minerals bent on a quartz grain (Q) due to compaction (SEM/EDX) • Jednokierunkowa orientacja blaszek minerałów ilastych ugiętych na ziarnie kwarcu wyniku kompakcji widoczna w badaniach elektronomikroskopo- wych świeżego przełamu próbki. (SEM/EDX) Fig. 4. One-directional orientation of flakes of clay minerals bent on a quartz grain (Q) due to compaction (SEM/EDX) • Jednokierunkowa orientacja blaszek minerałów ilastych ugiętych na ziarnie kwarcu wyniku kompakcji widoczna w badaniach elektronomikroskopo- wych świeżego przełamu próbki. (SEM/EDX) Because of complicated geological history of the Pieniny Klippen Belt, not only organic matter but also primary features of the Skrzypne Shale Formation could be signifi- cantly changed by diagenetic processes and tectonic defor- mations. The rocks of the Skrzypny Shale Formation of the Niedzica succession, outcropping at the pass below the Cza- jakowa Skała Klippe are composed mainly of clay minerals and micas (about 77 wt.%). Clay minerals are represented by high-illitic illite/smectite, illite, chlorite and kaolinite. Mo- reover, these shales contain quartz (about 20 wt.%), trace amounts of feldspars and calcite (about 3 wt.%) (Fig. 2). strongly chlorotized, mica flakes and kaolinite plates up to a dozen or even several ten micrometers in size (Fig. 5), exhibit rotation by 30 to 90º relative to one-directional ori- entation of fine flakes of clay minerals (Fig. 6). According to Weaver (1989) criteria, the above described microstructure, clay mineral composition and the state of preservation of organic matter indicate that the rocks studied were subjected to processes characteristic of late stage of diagenesis. The Skrzypny shales in this outcrop were till now not yet studied in detail. Their geologic history can be reconstructed on the ground of X-ray, geochemical and electron microscope stud- ies using SEM/EDX technique. Compaction process resulted in one-directional orientation of flakes of clay minerals (illite/smectite, illite) surrounding fine quartz grains and rare feldspars and bioclasts (Fig. 3). Quartz and feldspar grains (Fig. 4) are very fine, up to a dozen micrometers in size. Bioclasts are strongly destroyed, showing numerous dissolution caverns (Fig. 3). Outcrop at the pass below Czajakowa Skała Klippe Semi-quantitative mineral composition of rocks of the Skrzypny Shale Formation from the outcrop at the pass below Czajakowa Skała Klippe (Niedzica succession) based on standard X-ray data • Półilościowy skład mineralny utworów formacji łupków ze Skrzypnego w z odsłonięcia na przełęczy pod Czajakową Skałą (sukcesja niedzicka) określony w oparciu o standardowe badania rentgenogarficzne B Fig. 3. Bioclasts (B) showing dissolution caverns surrounded by flakes of clay minerals (SEM/EDX) • Bioklast z kawernami z rozpuszczania otoczony przez blaszki minerałów ilastych. (SEM/EDX) Fig. 2. Semi-quantitative mineral composition of rocks of the Skrzypny Shale Formation from the outcrop at the pass below Czajakowa Skała Klippe (Niedzica succession) based on standard X-ray data • Półilościowy skład mineralny utworów formacji łupków ze Skrzypnego w z odsłonięcia na przełęczy pod Czajakową Skałą (sukcesja niedzicka) określony w oparciu o standardowe badania rentgenogarficzne Fig. 3. Bioclasts (B) showing dissolution caverns surrounded by flakes of clay minerals (SEM/EDX) • Bioklast z kawernami z rozpuszczania otoczony przez blaszki minerałów ilastych. (SEM/EDX) pH of pore waters in this sediments (Birkenmajer & Narębski 1957). Biomarkers are the indices of redox conditions in sedi- mentation environment of rocks enriched in organic matter. They represent “molecular fossils” i.e. fragments of organic matter which preserved the internal structure of initial biomol- ecules. Two isoprenoids: pristine (Pr) and phytane (Ph) were used to determination of water column conditions during Skrzypny shales sedimentation. Elevated Pr/Ph ratio amounts to 1.8 suggest rather oxic conditions, but this data should be treated as preliminary. Taking into account relatively high TOC content and rather oxic conditions of the organic matter sedi- mentation, all this suggest that suboxic to anoxic conditions took place during early stage of diagenesis (Fig. 1). Unfortu- nately, due to relatively high thermal maturity of the samples and their partial weathering such biomarkers as terpenoids and steroids were not found. The prevalence of short-chain n- alkenes, with maximum at n-C16 indicates rather marine character of organic matter. However, in the range of long-chain n-alkanes we observe slight prevalence of odd carbon chain n-alkanes (n‑C27, n-C29, n-C31) what, in turn, indicates a ter- restrial component of organic matter. Consequently, these data indicate a mixed terrestrial/marine character of organic matter with distinct prevalence of the marine one. Fig. 4. Outcrop at the pass below Czajakowa Skała Klippe They were formed under conditions when the boundary of oxidizing and reducing conditions was situated within the sediment and oxidized water above the latter. Such environment is favour the development of epifauna and infauna and its accumulation in the sediment (Fig.1). On the other side, in such geochemical facies siderite-bearing concretions can be formed (Birkenma- jer & Narębski, 1957; Potter et al., 2005). The sediments of the Skrzypny shale formation contain about 1 wt.% of total organic carbon (TOC) and trace amounts of sulphur. Taking into account weathering of the investigated material, not altered TOC content seems to be higher, which indicate oxygene depletion during deposition or early diagen- esis. Increased content of calcite in them indicates rather high Dark colour of sediments initiating sedimentation in the Pieniny basin suggests their deposition under oxygen-deficient conditions enabling preservation of organic matter. Such conditions are characteristic of a source rocks for oil and gas deposits. In Demaison and Moore (1980) opinion, oxygen- deficient environment is characterized by the content of less than 0.5 ml oxygen in one litre of water. Such conditions are formed when the request for oxygen in water is higher than its supply. This request depends on the productivity of surface waters and the supply of oxygen to sediments on the circula- tion of currents. It is considered that oxygen-deficient condi- tions were particularly common in Upper Jurassic and Lower Cretaceous period because of global worming of cli- mate and an increase of water level in the oceans. The source rocks of the largest known oil and gas deposits are of this age. Moreover, anoxic conditions can be formed but locally. By analogy to actual oceanic environment (continental slope zone of the Indian Ocean) it is considered that oxygen-deficient sediments are accumulating in the areas, where currents Fig. 1. Presentation of sedimentary conditions of deposits of the Skrzypny Shale Formation based on lithologic and biomar- ker data • Ilustracja warunków sedymentacji utworów formacji łupków ze Skrzypnego w oparciu o badania cech litologicznych i biomarkerów Fig. 1. Presentation of sedimentary conditions of deposits of the Skrzypny Shale Formation based on lithologic and biomar- ker data • Ilustracja warunków sedymentacji utworów formacji łupków ze Skrzypnego w oparciu o badania cech litologicznych i biomarkerów 20 Middle Jurassic black shales (Skrzypny Shale Formation) Fig. 2. Outcrop at the pass below Czajakowa Skała Klippe Large, often As follows from the obtained results, the deposition of sediments of the Skrzypny Shale Formation of the Niedzica succession took place under conditions of oxidized waters 21 Middle Jurassic black shales (Skrzypny Shale Formation) Bt Ko Ch Bt e biotite (Bt) and chloritized biotite (Ch) flakes and o) plates among fine flakes of high-illitic illite/smectite EM/EDX) • Duże blaszki biotytu zubożonego w Fe, wanego biotytu i płytki kaolinitu wśród drobnych bla- oillitowego illitu/smektytu i illitu. (SEM/EDX) Fig. 6. Rotation of large mica (M) and post-mica chlorite plates by 30-90º relative to one-directional orienta- tion of fine flakes of clay minerals (CM). F – feldspar (SEM/EDX) • Rotacja dużych blaszek mik i chlorytu pomikowego o kąt 30-90º w stosunku do jednokierun- kowego ułożenia drobnych blaszek minerałów ilastych. (SEM/EDX) F CM M ediments but pore waters impoverished in oxygen. atter contained in them is of continental/marine predominance of the latter. During late diagen- mented by the composition of clay minerals and tural features of the shales in question, organic gained considerable thermal maturity. The occur- igh-illitic illite/smectite, Fe-impoverished and biotite and the microstructures, characterizing by onal orientation of clay minerals and rotation of itized mica flakes the result of post-sedimentary under conditions of late diagenesis.  ledgements: This work was partially financial by AGH University of Science and Technology .140.158. Bt Ko Ch Bt d e e, - Fig. 6. Rotation of large mica (M) and post-mica chlorite plates by 30-90º relative to one-directional orienta- tion of fine flakes of clay minerals (CM). F – feldspar (SEM/EDX) • Rotacja dużych blaszek mik i chlorytu pomikowego o kąt 30-90º w stosunku do jednokierun- kowego ułożenia drobnych blaszek minerałów ilastych. (SEM/EDX) F CM M n. e - d c - d y f y l y Fig. 5. Large biotite (Bt) and chloritized biotite (Ch) flakes and kaolinite (Ko) plates among fine flakes of high-illitic illite/smectite and illite (SEM/EDX) • Duże blaszki biotytu zubożonego w Fe, schlorytyzowanego biotytu i płytki kaolinitu wśród drobnych bla- szek wysokoillitowego illitu/smektytu i illitu. (SEM/EDX) Fig. 5. Large biotite (Bt) and chloritized biotite (Ch) flakes and kaolinite (Ko) plates among fine flakes of high-illitic illite/smectite and illite (SEM/EDX) • Duże blaszki biotytu zubożonego w Fe, schlorytyzowanego biotytu i płytki kaolinitu wśród drobnych bla- szek wysokoillitowego illitu/smektytu i illitu. (SEM/EDX) Bt Ko Ch Bt above the sediments but pore waters impoverished in oxygen. Outcrop at the pass below Czajakowa Skała Klippe Organic matter contained in them is of continental/marine origin with predominance of the latter. During late diagen- esis, documented by the composition of clay minerals and microstructural features of the shales in question, organic matter has gained considerable thermal maturity. The occur- rence of high-illitic illite/smectite, Fe-impoverished and chloritized biotite and the microstructures, characterizing by one-directional orientation of clay minerals and rotation of often chloritized mica flakes the result of post-sedimentary processes under conditions of late diagenesis.  Ko Bt Acknowledgements: This work was partially financial supported by AGH University of Science and Technology grant: 11.11.140.158. Fig. 6. Rotation of large mica (M) and post-mica chlorite plates by 30-90º relative to one-directional orienta- tion of fine flakes of clay minerals (CM). F – feldspar (SEM/EDX) • Rotacja dużych blaszek mik i chlorytu pomikowego o kąt 30-90º w stosunku do jednokierun- kowego ułożenia drobnych blaszek minerałów ilastych. (SEM/EDX) F CM M Fig. 6. Rotation of large mica (M) and post-mica chlorite plates by 30-90º relative to one-directional orienta- tion of fine flakes of clay minerals (CM). F – feldspar (SEM/EDX) • Rotacja dużych blaszek mik i chlorytu pomikowego o kąt 30-90º w stosunku do jednokierun- kowego ułożenia drobnych blaszek minerałów ilastych F CM M Fig. 6. Rotation of large mica (M) and post-mica chlorite plates by 30-90º relative to one-directional orienta- tion of fine flakes of clay minerals (CM). F – feldspar (SEM/EDX) • Rotacja dużych blaszek mik i chlorytu pomikowego o kąt 30-90º w stosunku do jednokierun- kowego ułożenia drobnych blaszek minerałów ilastych. (SEM/EDX) F CM M M Fig. 6. Rotation of large mica (M) and post-mica chlorite plates by 30-90º relative to one-directional orienta- tion of fine flakes of clay minerals (CM). F – feldspar (SEM/EDX) • Rotacja dużych blaszek mik i chlorytu pomikowego o kąt 30-90º w stosunku do jednokierun- kowego ułożenia drobnych blaszek minerałów ilastych. (SEM/EDX) Streszczenie Środkowojurajskie czarne łupki (formacja łupków ze Skrzypnego) – znaczenie paleośrodowiskowe jednych z najstarszych osadów pienińskiego pasa skałkowego Katarzyna Górniak, Krzysztof Bahranowski, Adam Gaweł, Leszek Marynowski & Tadeusz Szydłak w utworach tych zapisane są początkowe fazy rozwoju ba- senu pienińskiego (Birkenmajer, 1986, Krobicki & Golonka, 2008 a i literatura tam cytowana). W polskiej części Pieniń- skiego Pasa Skałkowego, jednymi z najstarszych skał nale- żących do tych facji są czarne łupki sferosyderytowe z mał- żami Bositra (formacja łupków ze Skrzypnego) wieku późny aalen-wczesny bajos (Krobicki & Golonka, 2008a, Fig. 7). Formacja łupków ze Skrzypnego jest opisywana jako seria skał ilastych o barwie czarnej i czarnozielonej z konkrecjami syderytowymi w których spotyka się amonity. Miąższość formacji określa się na około 5 m. Te interesujące utwory od- słaniają się w górnej części wąwozu Homole, na przełęczy pod Czajakową Skałą (Krobicki & Golonka, 2008a, Fig. 12D). Ciemne wczesnojurajskie facje margliste (typu Flecken- mergiel) budzą szczególne zainteresowanie badaczy, ponieważ 22 Middle Jurassic black shales (Skrzypny Shale Formation) ka, 2008a, Fot. 14A i literatura tam cytowana). Warunki ba- tymetryczne dla facji Fleckemergiel podane przez Tyszkę (2001) w oparciu o badania mikroskamieniałości odpowia- dają strefie zewnętrznego szelfu i skłonu. Przełęcz pod Czajakową Skałą – odsłonięcie utworów formacji łupków ze Skrzypnego j p yp g Formacja łupków ze Skrzypnego odsłaniająca się na prze- łęczy pod Czajakową Skałą należy do sukcesji niedzickiej. Utwory sukcesji niedzickiej w wąwozie Homole występują w formie płatu kontaktującego z podłożem tektonicznie. Płat ten tworzy fałd obalony, silnie wtórnie sfałdowany nazywa- ny Czajakową Skałą. Formacja łupków ze Skrzypnego wy- stępuje w spągu sukcesji niedzickiej. Fragment struktury fałdu Czajakowej Skały został przemieszczony po warstwie tych łupków stanowiących powierzchnię poślizgu i obecnie widoczne są one w osuwisku znajdującym się w górnej czę- ści wąwozu Homole (Krobicki & Golonka, 2008a, Fot. 12D). Utwory formacji łupków ze Skrzypnego w tym niewielkim odsłonięciu wykształcone są jako drobnołupiące się, ciem- noszarooliwkowe łupki z blaszkami miki o wielkości do 0.5 mm i z brązowymi konkrecjami syderytowymi. Łupki są miękkie, słabo wapniste, bezpiaszczyste. Wielkość sferosy- derytów nie przekracza kilkunastu centymetrów. Są one twarde, pryskliwe, ze szczelinami zabliźnionymi kalcytem. Jak wynika ze szczegółowych badań W. Narębskiego (Bir- kenmajer & Narębski, 1957) konkrecje te są zbudowane z syderoplezytu (Mg-syderyt) ze znacząca domieszką kalcytu. Skład chemiczny konkrecji wskazuje, że powstały one w czasie wczesnej diagenezy w słabo redukcyjnych warunkach facji syderytowej (Birkenmajer & Narębski, 1957). Bochen- kowaty kształt również wskazuje na ich wczesnodiagenetycz- ną genezę (Seilacher, 2001). Streszczenie Serie ciemnych skał ilastych gromadzące się w środowi- skach zubożonych w tlen zwykle pozornie są monotonne. Powiązanie badań sedymentologicznych, paleontologicznych i geochemicznych przeprowadzonych dla utworów dolnoju- rajskich (Yorkshire, Wielka Brytania) pozwoliło zdefiniować trzy facje występujące w basenach zdominowanych przez wody generalnie niedotlenione. Są to facje: łupków normal- nych (normal shale), łupków słabo bitumicznych (restricted shale) i łupków bitumicznych (bituminous shale) (Morris 1979). Przyczyną tego zróżnicowania facjalnego jest cyklicz- nie zmieniające się położenia granicy między strefą natlenio- ną i zubożoną w tlen w słupie wody na osadem. Cechy litologiczne utworów formacji łupków ze Skrzypne- go sukcesji niedzickiej odsłaniające się na przełęczy pod Czajakowa Skałą reprezentują fację łupków normalnych. Wy- nika to z porównania ich cech z cechami osadów podanymi przez Morrisa (1979) dla poszczególnych facji. Środowisko w jakim powstaje facja łupków normalnych to: położenie grani- cy między warunkami utleniającymi i redukcyjnymi w obrębie osadu i natlenionych wód nad osadem. W takim środowisku możliwy jest rozwój epifauny i infauny oraz nagromadzenie jej w osadzie (Fig. 1). Jednocześnie są to warunki geochemicz- ne w których mogą tworzyć się konkrecje syderytowe (Birken- majer & Narębski, 1957; Potter et al., 2005). Badane utwory formacji łupków ze Skrzypnego zawiera- ją około 1% całkowitego węgla organicznego (TOC) oraz śladowe ilości siarki. Biorąc pod uwagę przypowierzchniowe zwietrzenie badanego materiału, pierwotna ilość TOC była zapewne wyższa, co wskazuje na sedymentację w warunkach zubożonych w tlen. Wskaźnikiem warunków redoks panują- cych w środowisku sedymentacji skał zasobnych w substan- cje organiczną są biomarkery. Biomarkery pozwalają określić również pochodzenie substancji organicznej nagromadzonej w osadach. Biomarkery to „molekularne skamieniałości” (molecular fossils) czyli fragmenty substancji organicznej, które zachowują budowę wewnętrzna wyjściowych biomo- lekuł. Stopień natlenienia kolumny wody podczas sedymen- tacji łupków ze Skrzypnego określa stosunek pristanu do fi- tanu czyli izoprenoidów obecnych w badanych próbkach. Wartość tego stosunku wynosi 1,8, co sugeruje raczej tleno- we warunki sedymentacji substancji organicznej, przy czym nie są to dane jednoznaczne i należy je traktować jako wyj- ściowe do dalszych badań. Zatem, stosunkowo wysoka za- wartość węgla organicznego w badanych skałach oraz raczej tlenowe warunki sedymentacji, sugerują występowanie wa- runków suboksycznych do anoksycznych już w osadzie na wczesnym etapie diagenezy (Fig. 1). Z powodu stosunkowo wysokiej dojrzałości termicznej materii organicznej oraz jej częściowego utlenienia w próbkach nie stwierdzono terpeno- idów i steroidów. Przewaga n-alkanów krótkołańcuchowych, z maksimum przy n-C16 wskazuje na raczej morski charakter materii organicznej. Streszczenie Jednak w zakresie n-alkanów długołań- cuchowych uwidacznia się lekka przewaga n-alkanów nie- parzysto węglowych (n-C27, n-C29, n-C31), co z kolei wskazu- je na składnik lądowej materii organicznej. Z danych tych ą g ę ( , ) Ciemna barwa osadów rozpoczynających sedymentację w basenie pienińskim sugeruje, że są to utwory deponowane w warunkach niedoboru tlenu, umożliwiających zachowanie w osadzie substancji organicznej. Z takimi niedotlenowymi środowiskami wiąże się powstanie skał macierzystych dla złóż ropy naftowej i gazu. Demaison i Moore (1980) uważa- ją środowisko za niedotlenione jeśli w jednym litrze wody rozpuszczone jest mniej niż 0.5 mililitra tlenu. Takie warun- ki tworzą się wówczas kiedy zapotrzebowanie na tlen wodzie jest większe niż jego dostawa. Zapotrzebowanie na tlen za- leży od produktywności wód powierzchniowych, a dostawa tlenu do osadu od cyrkulacji prądów. Uważa się, że warunki zubożone w tlen szczególnie powszechnie panowały w póź- nej jurze i wczesnej kredzie z powodu globalnego ocieplenia klimatu i podniesienia poziomu wód w oceanach. Skały macierzyste największych znanych złóż ropy i gazu są tego wieku. Warunki anoksyczne mogą tworzyć się też lokalnie. Przez analogię do współczesnego środowiska oceanicznego (strefa skłonu kontynentalnego Oceanu Indyjskiego) uważa się, że osady niedotlenione gromadzą się w obszarach, w których prądy niosące wody zubożone w tlen (nie więcej niż 0.5 ml tlenu/litr wody) wkraczają na skłon kontynentalny. Model ten odpowiada warunkom panującym w basenie pie- nińskim w środkowej jurze. Ciemne facje margliste tego wieku pojawiają się wśród utworów sukcesji niedzickiej de- ponowanej na skraju szelfu kontynentalnego i sukcesji czor- sztyńskiej gromadzonej na skłonie grzbietu czorsztyńskiego. Pierwsze z wymienionych, jak wspomniano powyżej, są jednymi z najstarszych utworów budujących fałd Czajakowej Skały i widoczne są w najwyższej części wąwozu Homole, drugie odsłaniają się w potoku Krupianka (Krobicki & Golon- 23 Middle Jurassic black shales (Skrzypny Shale Formation) wynika, że materia organiczna badanych skał jest mieszane- go, lądowo/morskiego pochodzenia z wyraźną przewagą morskiej substancji organicznej. wynika, że materia organiczna badanych skał jest mieszane- go, lądowo/morskiego pochodzenia z wyraźną przewagą morskiej substancji organicznej. Mikrostruktura, skład minerałów ilastych oraz stan za- chowania substancji organicznej opisane powyżej wskazują, zgodnie z kryteriami Weavera (1989), że badane utwory formacji łupków ze Skrzypnego ulegały procesom charakte- rystycznym dla późnego etapu diagenezy. Złożona historia geologiczna Pienińskiego Pasa Skałko- wego powoduje, że nie tylko substancja organiczna ale i pierwotne cechy utworów formacji łupków ze Skrzypnego są w znacznym stopniu zmienione przez procesy diagenezy i deformacje tektoniczne. Streszczenie Utwory formacji łupków ze Skrzypnego sukcesji niedzickiej odsłaniające się na przełę- czy pod Czajakową Skałą zbudowane są głównie z minerałów ilastych i mik (około 77% wag.). Minerały ilaste są repre- zentowane przez wysokoillitowy illit/smektyt, illit, chloryt i kaolinit. Ponadto w badanych skałach występuje kwarc (około 20% wag.), śladowo skalenie oraz kalcyt (około 3% wag.) (Fig. 2). Utwory formacji łupków ze Skrzypnego z omawianego odsłonięcia nie były jak dotąd przedmiotem tak szczegółowych badań. Swoją historię geologiczną ujawniają po przeprowa- dzeniu badań rentgenograficznych, geochemicznych i przy użyciu mikroskopu elektronowego (SEM/EDX). Z badań tych wynika, że sedymentacja utworów formacji łupków ze Skrzyp- nego sukcesji niedzickiej przebiegała w warunkach natlenio- nych wód nad osadem i zubożonych w tlen wód porowych. Substancja organiczna w nich zawarta jest pochodzenia lądo- wo/morskiego z wyraźną przewagą ostatniej z wymienionych. W warunkach później diagenezy zamanifestowanej w składzie minerałów ilastych i w cechach mikrostrukturalnych badanych utworów substancja organiczna osiągnęła znaczną dojrzałość termiczną. Występowanie wysokoillitowego illitu/smektytu, zubożonego w Fe i schlorytyzowanego biotytu oraz mikro- struktur charakteryzujących się jednokierunkowym ułożeniem blaszek minerałów ilastych i rotacją blaszek mik, często schlo- rytzowanych, jest skutkiem procesów posedymentacyjnych odpowiadających warunkom późnej diagenezy. i Skutkiem procesu kompakcji jest jednokierunkowa orien- tacja blaszek minerałów ilastych (illit/smektyt, illit) obleka- jących drobne ziarna kwarcu oraz nieliczne skalenie i biokla- sty (Fig. 3). Ziarna kwarcu (Fig. 4) i skaleni są bardzo drobne. Mają wielkość nie przekraczającą kilkunastu mikrometrów. Bioklasty są silnie zniszczone, z licznymi kawernami z roz- puszczania (Fig. 3). Duże blaszki miki, często schlorytyzo- wanej oraz płytki kaolinitu o wielkości osiągającej od kilku- nastu do kilkudziesięciu mikrometrów (Fig. 5) wykazują rotację o kąt od 30 do 90° w stosunku do jednokierunkowego położenia drobnych blaszek minerałów ilastych (Fig. 6). Podziękowania: Praca była częściowo finansowana przez Akademię Górniczo-Hutniczą, umowa nr 11.11.140.158. References (Literatura) Birkenmajer, K., 1986. Stages of structural evolution of the Pieniny Klippen Belt, Carpathians. Studia Geologica Polonica, 88: 7-32. Potter, P. E., Maynard, J.B. & Depetris, P.J., 2005. Mud and Mudstones. Springer 297pp. Moore, D.M. & Reynolds, R.C., Jr. 1997. X-ray diffraction and the identi- fication and analysis of Clay Minerals.Oxford University Press. New York, 378. l Birkenmajer, K. & Narębski, W., 1958. Konkrecje węglanowe łupków aale- nu pienińskiego pasa skałkowego Polski. Rocznik Polskiego Towarzystwa Geologicznego, XXVII, 51-74. Seilacher, A., 2001. Concretion morphologies reflecting diagenetic and epigenetic pathways. Sedimentary Geology, 143, 41-57. Demaison, G. J. & Moore, G.T. 1980. Anoxic Environments and oil source bed genesis. Streszczenie The American Association of Petroleum Geologists Bul- letin, 64, 8: 1179-1209. Tyszka, J., 2001. Microfossil assambladges as bathymetic indicators of the Toarcian/Aalenian “Fleckenmergiel” – facies in the Carpathian Pieniny Klippen Belt. Geologica Carpathica, 52: 147-158. Krobicki, M. & Golonka, J., 2008. Geological history of the Pieniny Klip- pen Belt and Middle Jurassic black shales as one of the oldest deposits of this region – stratigraphical position and palaeoenvironmental sig- nificance. Geoturystyka (ten tom). Weaver, C.E., 1989. Clays, muds and shales. Development in Sedimento- logy 44. Elsevier, Amsterdam, 819pp. logy 44. Elsevier, Amsterdam, 819pp. Morris, K. A., 1979. A classifcation of Jurassic marine shale sequence: an example from the Toarcin (Lower Jurassic) of Great Britain. Paleoge- ography, Paleoclimatology, paleoecology, v.26:117-120.
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Traducción comentada de un acta notarial de celebración de matrimonio civil del español al árabe
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ARTÍCULOS Anaquel de Estudios Árabes ISSN: 1130-3964 https://dx.doi.org/10.5209/anqe.66701 Traducción comentada de un acta notarial de celebración de matrimonio civil del español al árabe Beatriz Soto Aranda1; Rabab Kabbour2 Recibido: 2 de diciembre de 2019 / Aceptado: 29 de febrero de 2020 Resumen. Las actas notariales españolas de celebración de matrimonio civil tienen su origen en la Ley 15/2015, de 2 de julio, de la Jurisdicción Voluntaria, que permite a los notarios, en tanto que juristas y titulares de la fe pública, intervenir con plena efectividad y sin merma de garantías, en algunos de los actos de jurisdicción voluntaria que hasta la fecha se encomendaban a los jueces, como es el matrimonio. Esta circunstancia ha supuesto una diversificación del tipo de documentos que se traducen al árabe para constatar el estado civil de un ciudadano residente en España, como son la certificación de matrimonio expedida por el Registro Civil y el certificado de estar inscrito en el Registro de Uniones de Hecho, cuya regulación es autonómica, no estatal. El presente estudio presenta una propuesta de traducción comentada al árabe de esta tipología documental de carácter jurídico-administrativo, con especial atención a los conocimientos lingüísticos, jurídicos y culturales necesarios para abordar este tipo de traducción especializada, y las macroestructuras de los textos original y meta. Por último, y en lo referente a la terminología y los aspectos formales exigidos por la legislación del ordenamiento jurídico meta, habida cuenta las diferencias existentes entre los ordenamientos jurídicos árabes, y al no tratarse de un estudio comparativo, se ha seleccionado Marruecos como contexto de recepción de un hipotético encargo de traducción, por ser este el país árabe con mayor número de inmigrantes en España. Palabras clave: actas notariales, traducción jurídica, español, árabe. [en] Annotated translation of a notarial act of civil marriage from Spanish into Arabic Abstract. The Spanish civil marriage notarial acts stem from Law 15/2015 of 2 July 2015 of the Voluntary Jurisdiction. Up to then, voluntary jurisdiction acts could only be performed by judges, however, since the entry into force of Law 15/2015, notary publics, in their capacity as jurists, can intervene in some of these acts, such as a marriage act, with full effectiveness. This has led to a diversification of the type of documents that are translated into Arabic to prove the marital status of a citizen residing in Spain - documents such as marriage certificates, which are issued by the Civil Registry, and de facto union certificates, which are a matter of Autonomous Communities competence, and do not depend on state regulation. This paper presents a commented translation into Arabic of this type of legal-administrative documents, paying special attention to the linguistics, legal and cultural knowledge that this type of specialized translation requires. And it focuses as well in the macro-structures of both the original and the target texts. Finally, regarding the terminology and formal aspects stipulated by law in the target legal system, 1 2 Institución: Universidad Rey Juan Carlos E-mail: beatriz.soto@urjc.es Institución: Universidad Autónoma de Madrid E-mail: kabbour.rabab@gmail.com Anaquel estud. árabes 31 2020: 179-205 179 Soto Aranda, B.; Kabbour, R. Anaquel estud. árabes 31 2020: 179-205 180 given the differences between the Arab legal systems, and since this is not a comparative study, Morocco has been selected as the reception context of a hypothetical translation order, as this is the Arab country with the largest number of immigrants in Spain. Keywords: notarial acts; legal translation; Spanish; Arabic. Sumario: 1. Introducción. 2. Aproximaciones a la traducción jurídica árabe-español. 3. Conocimientos para afrontar la traducción de un acta notarial de matrimonio del español al árabe. 4. Propuesta de traducción comentada. 4.1. Menciones preliminares. 4.2. Comparecencia. 4.3. Exposición. 4.4. Estipulaciones y otorgamiento. 4.5. Autorización. 5. Conclusiones. Cómo citar: Soto Aranda, B., Kabbour, R. (2020): Traducción comentada de un acta notarial de celebración de matrimonio civil del español al árabe, en Anaquel de Estudios Árabes 31, 179-205. 1. Introducción Tanto el estudio de los textos relativos al derecho contemporáneo de los países árabes3 como aquellos dedicados al derecho comparado4 han tenido un importante desarrollo en las últimas décadas en España; ello a la vez que aumentaba el número de investigaciones sobre la traducción jurídica del árabe al español, así como aquellos con una orientación didáctica más específica5, junto con manuales de traducción jurídica y administrativa6 y diccionarios especializados7. 3 4 5 6 7 Pueden consultarse las obras que Caridad Ruíz-Almodóvar ha dedicado a diferentes aspectos del derecho de los países árabes, en especial, RUÍZ ALMODÓVAR, Caridad, Códigos de estatuto personal: el derecho privado en los países árabes, Granada, 2005. RUÍZ ALMODÓVAR, Caridad, “El testamento en los códigos de estatuto personal de los países árabes”, en Anaquel de estudios árabes, 26, 2015, (DOI: https://doi.org/10.5209/rev_ ANQE.2015.v26.47696). Con anterioridad a la firma del acuerdo con las comunidades islámicas en materia de matrimonio, ver FIERRO, Maribel (Coord.), Repertorio bibliográfico de derecho islámico, Murcia 1999. Entre otros, puede consultarse BLÁZQUEZ RODRÍGUEZ, Irene, (2004) “Pluralidad de formas de celebración y matrimonio musulmán. Una perspectiva desde el derecho internacional privado español”, en Boletín Mexicano de Derecho Comparado [En línea], 110, disponible en https://helvia.uco.es/xmlui/bitstream/handle/10396/2198/art1.pdf?sequence=1&isAllowed=y [Último acceso el 30 de octubre de 2019]; y ROCA, Encarna, ‘‘Las consecuencias del acuerdo con las comunidades islámicas en materia de matrimonio’’, en El islam jurídico y Europa. A. Borrás y S. Meinissi (eds.) (1998) 245-464. LAPIEDRA GUTIÉRREZ, Eva, “La traducción del derecho árabe: planteamientos y propuestas”, en Traducir del árabe. M. de Epalza y Mª. J. Rubiera (coords.) (2004) 215-260. AGUESSIM EL GHAZOUANI, Abdellatif, “La enseñanza de la traducción jurídica, económica y comercial en la combinación lingüística: árabe “c” /español”, en El Mundo profesional y las lenguas: enseñar según el MCER. M. Montoro (coord.) (2009) 149-166; GOGAZEH, Ziyad, (2014) “La problemática de la traducción de la terminología jurídica en el libro Manual de traducción jurídica árabe-español”, en Philologia Hispalensis 28/1-2, 2014 (Doi: http//dx.doi.org/10.12795. PH.2014.v28.i01.02); ABDEL-AZIZ, Khaled, “Clasificación del texto jurídico árabe (desde el punto de vista de la traducción jurídica)”, en Quaderns: Revista de traducción, 13 (2006) 169-179 y BOUTEFFAH AGHBALOU, Bouchra, La didáctica de las traducciones jurídica y jurada en la combinación lingüística árabe-español: propuesta de un manual didáctico. Málaga, 2017. MOHAMED SAAD, Saad. Curso de traducción judicial, Vol. I, árabe/español, Madrid 2017. MOHAMED SAAD, Saad. Curso práctico de traducción legal: Volumen I. Árabe/Español, Madrid 2015. MOHAMED SAAD, Saad. Curso de traducción administrativa: Volumen I, Árabe-Español. Madrid 2010. AGUESSIM EL GHAZOUANI, Abdellatif, Diccionario jurídico español/árabe. Granada 2006; FERIA GARCÍA, Manuel, Diccionario de términos jurídicos árabe-español. Barcelona 2006. KHAY, Sarah, Traducción judicial en España y Marruecos. Propuesta de elaboración de un diccionario jurídico en materia penal árabefrancés-español, Alcalá 2016. EL KRIRH, Khatima. Sobre la traducción jurídica y judicial. Estudio de la variación aplicado a la terminología del Derecho penal y procesal penal en español, francés y árabe, Málaga 2016. Soto Aranda, B.; Kabbour, R. Anaquel estud. árabes 31 2020: 179-205 181 Dentro de este marco general de tratamiento de la traducción jurídica en la dirección árabe-español, destacan los estudios dedicados al derecho de familia y a los estatutos árabes de código personal8 y, en particular, aquellos centrados en la traducción de documentación relativa a la filiación expedida por los registros civiles9, así como las actas de matrimonio10, y, más recientemente, las sentencias judiciales de divorcio11. Ello se debe a que es en este ámbito del derecho donde parece generarse mayor número de anisomorfismos culturales y asimetrías terminológicas entre el ordenamiento jurídico español y los de los países árabes12. Ello supone un reto para el traductor que debe procurarse amplios conocimientos de las culturas y ordena8 9 10 11 12 Véase, entre otros, EL-MADKOURI MAATAOUI MAATAOUI MAATAOUI, Mohamed, “Los aspectos lingüísticos y culturales de la traducción jurídica árabe (El caso del estatuto personal marroquí)”, en Revista del Instituto Egipcio de Estudios Islámicos, 39 (2011) 75-96. ALLOUCHI, Outman, “Nociones de derecho marroquí de familia imprescindibles para abordar el estudio de la traducción de sentencias judiciales marroquíes en materia de disolución del matrimonio”, en Tonos digital: Revista de estudios filológicos [En línea] Nª 30, disponible en http://hdl.handle.net/10201/47882 [Último acceso el 30 de octubre de 2019]. BENYAICH, Sokaina (2014), Estudio terminológico de la Mudawana (Código de Familia Marroquí). Máster Universitario en Comunicación Intercultural, Traducción e Interpretación en los Servicios Públicos, Universidad de Alcalá. FERIA GARCÍA, Manuel, “Reflexiones en torno a las últimas reformas del derecho de familia en Marruecos”, en Hesperia culturas del Mediterráneo 2 (2005) 21-36. AGUESSIM EL GHAZOUANI, (2013) Abdellatif, “Aproximación a la traducción jurídico-administrativa de documentos del registro civil marroquí: certificaciones de nacimiento” en Tonos digital: Revista de estudios filológicos [En línea] Nª 25, disponible en https://digitum.um.es/digitum/handle/10201/38087 [Último acceso el 30 de octubre de 2019] Sobre la traducción de actas de matrimonio egipcias, puede consultarse ABDEL-AZIZ, Khaled, (2005) “El lenguaje jurídico del derecho egipcio de familia (un enfoque descriptivo)” en Sendebar: Revista de la Facultad de Traducción e Interpretación [En línea]. Nº 16, disponible en https://revistaseug.ugr.es/index.php/sendebar/ article/view/1049/1247 [Último acceso el 30 de octubre de 2019] y ABDEL-AZIZ, Khaled, Un estudio descriptivo de la Ley egipcia de Estatuto Personal número 100 de 1985: un enfoque traductológico. Barcelona 2003. Sobre la traducción jurada de actas de matrimonio marroquíes, véase FERIA GARCÍA, M.C. “La traducción jurada de actas matrimoniales marroquíes”, en Traducir para la justicia. M. Feria García (ed.) (1999) 221-258; FERIA GARCÍA, Manuel, La traducción fehaciente del árabe: fundamentos históricos, jurídicos y metodológicos. Málaga 2002; y FERIA GARCÍA, Manuel, “Convivencia e interacción de tradiciones jurídicas en Marruecos: la fe pública: sofrim, notarios latinos y adules”, en Awraq: Estudios sobre el mundo árabe e islámico contemporáneo, 24 (2007), 241-286. Con relación a la traducción de actas de matrimonio tunecinas, véase BEN OTHMAN, Fathen, “Análisis lingüístico-pragmático y cultural de problemas de traducción en un documento jurídico (árabe-español). Acta de matrimonio”. Actas del XXIV Congreso Internacional de AESLA [Recurso electrónico]: aprendizaje de lenguas, uso del lenguaje y modelación cognitiva : perspectivas aplicadas entre disciplinas, 2007. ALLOUCHI, Outman, La sentencia de divorcio (árabe/español): Estudio jurídico y textual preliminar. Granada 2010. ALLOUCHI, Outman, (2016) “Nociones de derecho marroquí de familia imprescindibles para abordar el estudio de la traducción de sentencias judiciales marroquíes en materia de disolución del matrimonio” en Tonos digital: Revista de estudios filológicos [En línea], Nº 30, disponible en en http://www.tonosdigital.es/ojs/index. php/tonos/article/view/1413 [Último acceso el 30 de octubre de 2019]. EL-MADKOURI MAATAOUI, Mohamed, “La traducción del árabe al español y el exequátur en materia de divorcio”, Anaquel De Estudios Árabes, 30, 2019 (Doi: https://doi.org/10.5209/ANQE.62685). A cerca de los parámetros culturales que condicionan los documentos jurídicos y administrativos poscoloniales, expedidos por países con población musulmana, véase MAYORAL, Roberto, Translating Official Documents (Translation practicies explained), Londres 2003. Con relación a la hibridación terminológica de los documentos africanos, véase El-MADKOURI MAATAOUI, Mohamed y SOTO ARANDA, Beatriz (2012) “Aspectos lingüísticos y extralingüísticos de la traducción jurídico-administrativa de documentos africanos al francés”, en Synergies Espagne [en línea], Nº 5, 111-128, disponible en http://gerflint.fr/Base/Espagne5/el_madkouri.pdf [Último acceso el 30 de octubre de 2019]. KARIM, Hasna, La traducción de los anisomorfismos culturales en el ámbito jurídico: aplicaciones al derecho marroquí en comparación con el derecho francés y español. Málaga 2010. EL JIRARI, Abderrahim, “La influencia de las asimetrías institucionales en la traducción jurídica y administrativa: estudio de caso: Marruecos y España”, en FITISPos international journal: public service interpreting and translation, 5 (2018) 159-169. 182 Soto Aranda, B.; Kabbour, R. Anaquel estud. árabes 31 2020: 179-205 mientos jurídico-administrativos asociados a las lenguas que el proceso traductor pone en contacto, además de desarrollar una capacidad de documentación adecuada. Como señala El-Madkouri Maataoui, incluso la competencia de un traductor cuya L1 es el árabe: depende de si traduce documentos de países cuya legislación, instituciones y terminología domina y que suelen coincidir con sus países de origen, tránsito o estudios. […] Las dificultades se dan en este caso no por desconocimiento de la lengua árabe, sino por no poder aprehender el mundo conceptual y referencial de las palabras13. En este sentido, cabe recordar que los ordenamientos jurídicos son propios de cada país, y distintos del resto de ordenamientos de otros países, con independencia de que la lengua vehicular y sus bases sean comunes. Junto a ello, los procesos migratorios, en general, y la residencia y el arraigo de la población migrante en los países de acogida, en particular, llevan consigo necesariamente la interacción entre las administraciones del país de origen y de acogida, que resultarían imposibles sin la mediación de la traducción14. Así, la labor traductora constituye una herramienta imprescindible en trámites judiciales, administrativos y notariales para los inmigrantes15. Lapiedra señala sobre esta cuestión que: Hay que tener también en cuenta que el mayor contacto entre el derecho de los países árabes y el derecho tanto español como europeo en general se da justamente en el ámbito del Derecho de Familia. […] Y es lógico que así sea, ya que el derecho tiene un componente territorial sobradamente conocido, al que escapa sin embargo el Estatuto Personal. Así lo dispone el art. 9 del Código civil español: “la ley personal correspondiente a las personas físicas es la determinada por su nacionalidad. Dicha ley regirá la capacidad y el estado civil, los derechos y deberás de la familia y la sucesión por causa de muerte…”16. En este contexto social, y lejos de visiones románticas de la traducción que cuestionan la traducción inversa realizada por no nativos17, el dominio de la traducción 13 14 15 16 17 EL-MADKOURI MAATAOUI MAATAOUI, Mohamed, “Aspectos lingüísticos y extralingüísticos de la traducción jurídico-administrativa del árabe al español”, en Estudios de lingüística y traductología árabe. S. M. Saad (coord.) (2010), p. 236. Véase p. e. el convenio hispano-marroquí de cooperación judicial en materia civil, mercantil y judicial firmado el 30 de mayo de 1997 (BOE 151, de 25 de junio de 1997), disponible en https://www.boe.es/boe/dias/1997/06/25/ pdfs/A19583-19587.pdf, [Último acceso el 30 de octubre de 2019], que incluye un artículo sobre la traducción de documentación judicial. EL-MADKOURI MAATAOUI MAATAOUI, Mohamed, “El discurso del lenguaje jurídico-administrativo español: análisis y perspectivas.”, en Estudio multilingüe e implicaciones culturales. Mª M. Errutia (coord.) (2016)127-164. NEBOT, ROSER, Nicolás. “El lenguaje jurídico en español y en árabe. Una misma situación con enfoques divergentes. Una misma necesidad de traducción con soluciones diferentes”, en Recursos documentales y tecnológicos para la traducción del texto jurídico. G. Corpas Pastor (ed.) (2003) 297-309. LAPIEDRA GUTIÉRREZ, Eva, op. cit. p. 217. KOCIJANČIČ POKORN, Nike, Challenging the Traditional Axioms: Translation into a Non-Mother Tongue. Amsterdam 2005. Kelly, Dorothy, “The Wrong Way Round? Consideraciones sobre la cuestión de la direccionalidad en la traducción profesional y la formación de traductores”, en Experiencias de traducción. Reflexiones desde la práctica traductora. C. García de Toro e I. García Izquierdo (eds.) (2005), 129-146. CAMPBELL, Stewart: Translation into the Second Language, Londres 1998. Soto Aranda, B.; Kabbour, R. Anaquel estud. árabes 31 2020: 179-205 183 hacia el árabe es una necesidad formativa para los estudiantes de traducción en un mercado laboral cada vez más especializado, y en constante cambio a nivel social, económico y político18. 2. Aproximaciones a la traducción jurídica español-árabe La traducción inversa en la combinación lingüística español-árabe ha recibido cierta atención académica en España, con especial referencia a la conocida como Ley de Extranjería19 y a un tipo de acta notarial de dominio para reanudación del tracto sucesivo, esto es, un requisito para la transmisión de derechos o bienes20. En ambos casos, se trata de documentación con un importante calado social; la traducción de una ley es relevante porque todo ciudadano con independencia de su estatus debe conocer sus derechos y obligaciones, y ese es un paso clave en el proceso de integración social; la traducción de un acta notarial lo es porque los instrumentos notariales relacionados con compra-venta de inmuebles y herencias da cuenta de los procesos de integración social de la población inmigrada21, sin olvidar que la asistencia de los traductores-intérpretes en las notarías es obligatoria por ley cuando el cliente no tiene la competencia lingüística suficiente en la lengua vehicular. En los dos estudios, queda patente que la traducción es un acto de comunicación intercultural22, y que la labor del traductor debe regirse por la deontología profesional, máxime cuando la agencia de este tipo de traducciones corresponde principalmente a la figura del traductor jurado. Las investigaciones realizadas sobre la calidad tanto de la traducción jurídica como de la traducción inversa coinciden en señalar que el éxito de una traducción no depende tanto de la competencia lingüística en ambas lenguas, que se le presupone a todo estudiante que se enfrenta a una traducción especializada, como del desarrollo de las competencias extralingüística, de transferencia y documental. Siguiendo la taxonomía propuesta por Borja23, la competencia extralingüística incluye conocimientos enciclopédicos del mundo, conocimientos jurídicos y del lenguaje jurídico, del derecho de las lenguas A y B, de derecho comparado y de derecho internacional. Esta descripción es más amplia que la definición propuesta por el Grupo PACTE, esto es, “conocimientos acerca de la organización del mundo en general y de ámbitos particulares”24, y está más en consonancia con el concepto de competencia intercul18 19 20 21 22 23 24 HUSNI, Ronak y Newman, Daniel, Arabic-English-Arabic Translation: Issues and Strategies, Londres, 2015, XIV. AGUESSIM EL GHAZOUANI, Abdellatif, Traducción jurídico-administrativa e inmigración. Propuesta de traducción al árabe de la ley de extranjería en vigor. Granada, 2004. https://hera.ugr.es/tesisugr/1540576x.pdf [Último acceso el 30 de octubre de 2019]; y AGUESSIM EL GHAZOUANI, Abdellatif, “Traducción jurídicoadministrativa e inmigración: propuesta de traducción al árabe de la Ley de extranjería en vigor”, Traducción como mediación entre lenguas y culturas = Translation as mediation or how to bridge linguistic and cultural gaps [Recurso electrónico] / Carmen Valero Garcés (ed. lit.) (2005), 272-279. NEBOT, ROSER, Nicolás. op. cit. DEL POZO TRIVIÑO, Maribel (2008) “La interpretación en las notarías españolas”, en Actas del III Congreso Internacional de la Asociación Ibérica de Estudios de Traducción e Interpretación. La traducción del futuro: mediación lingüística y cultural en el siglo XXI. L. Pegenaute, (ed.) (2008) vol. 2, 491-504. disponible en: http:// www.aieti.eu/wp-content/uploads/AIETI_3_MPT_Interpretacion.pdf [Último acceso el 30 de octubre de 2019]. HURTADO ALBIR, A. Traducción y traductología. Madrid 2001, p. 607. BORJA ALBI, Anabel, “Organización del conocimiento para la traducción jurídica a través de sistemas expertos basados en el concepto de género textual”, en El género textual y la traducción. Reflexiones teóricas y aplicaciones pedagógicas. I. García Izquierdo (ed.) (2005), 37-67. Grupo PACTE, “La competencia traductora y su adquisición” en Quaderns. Revista de traducció 6, (2001), 41. 184 Soto Aranda, B.; Kabbour, R. Anaquel estud. árabes 31 2020: 179-205 tural, en el sentido de que ha de incluir conocimientos de ambas culturas, además de los enciclopédicos y los temáticos, puesto que no se trasladan palabras, sino discursos, que cumplen una función concreta en el contexto sociocultural de partida, al contexto de llegada con una función equivalente25. Sin olvidar que la traducción jurídica es un caso particular de traducción de textos especializados, pues como señala El-Farahty, el vocabulario legal es específico de cada cultura y está vinculado al propio sistema cultural. Así, “el trabajo del traductor jurídico no consiste solo en transcodificar el significado legal, sino en transferir también su efecto legal”26. A su vez, la competencia de transferencia puede definirse como la capacidad de comprensión y reexpresión de los textos, teniendo en cuenta la finalidad de la traducción y el destinatario de esta. Esta incluye la competencia textual formada por el conocimiento de las taxonomías textuales de partida y de llegada, los aspectos formales exigidos por la legislación en cada ordenamiento jurídico, función y eficacia jurídica de los géneros en cada ordenamiento jurídico, así como la fraseología y terminología propias de cada texto27. En cuanto a la competencia documental, Sales la define como la capacidad de búsqueda, recuperación y tratamiento de información necesaria para llevar a cabo una traducción28. Resulta llamativo que los estudios para la combinación español-árabe antes citados señalen como retos para la traducción jurídica, cuestiones ligadas directamente con estas tres competencias: 1. Ausencia de diccionarios especializados. 2. Falta de unificación de términos jurídicos en los países árabes. 3. Importancia de la relación forma-contenido en el lenguaje jurídico árabe. 4. Conocimiento de las diferencias entre los ordenamientos jurídicos árabes y la terminología empleada en cada uno de ellos. 5. Conocimiento tanto de las bases religiosas del derecho de los países árabes como de los ordenamientos occidentales que han influido en ellos. 6. Dificultad del propio discurso jurídico en cualquier idioma pues se trata de un discurso culturalmente marcado. 7. Estrategias para la traducción de los anisomorfismos. 8. Conocimiento de textos de partida y de llegada, Aspectos formales exigidos por la legislación en cada ordenamiento jurídico. Así, el árabe es lengua oficial de veintidós países con bagajes históricos y culturales muy diversos, a los que cabe sumar algunos países como Chad que tienen el árabe como lengua cooficial29. En muchos casos, se trata de ordenamientos jurídicos híbridos30, característicos de sociedades poscoloniales que han heredado el derecho positivo de las ex metrópolis o de países con un destacado peso histórico, como es el 25 26 27 28 29 30 El-MADKOURI MAATAOUI, Mohamed y SOTO ARANDA, Beatriz, op. cit., p. 127. EL-FARAHATY, Hanem. International Journal of the Semiotics of Law 29 (2016), https://doi.org/10.1007/ s11196-016-9460-2, p. 475. Aunque existen otras definiciones, la propuesta por Anabel Borja Albí (2005), en su artículo “Organización del conocimiento para la traducción jurídica a través de sistemas expertos basados en el concepto de género textual”, ya citado, es muy útil y completa desde el punto de vista de la didáctica de la traducción jurídica. SALES SALVADOR, Dora, Documentación aplicada a la traducción: presente y futuro de una disciplina, Gijón 2006. SHIYAB, Said “The dilema of legal terminology in the Arab World”. Handbook of Terminology. Vol. 2 Terminology in the Arab World. Abied Alsulaiman y Ahmed Allaithty (eds), (2019), p. 140. EL-MADKOURI MAATAOUI, Mohamed y SOTO ARANDA, Beatriz, op. cit. p. 121. Soto Aranda, B.; Kabbour, R. Anaquel estud. árabes 31 2020: 179-205 185 caso de Francia en Egipto; derecho, de influencia anglosajona o continental, que conjugan con algunos principios islámicos. En palabras de Nebot, “los ordenamientos legales vigentes en estos países son transpolaciones, a veces simples traducciones, de los códigos occidentales, adaptadas a las condiciones dispuestas por algunos de los principios islámicos, pero no todos”31. Abdel-Aziz coincide con esta afirmación cuando señala que, en el caso egipcio, Actualmente, las leyes del derecho islámico tienen poca influencia en el sistema jurídico egipcio (el derecho egipcio actual ha conservado algunos aspectos de alšarî‘a al-islamiyya, que se manifiestan básicamente en la aplicación del derecho de familia (Código de Estatuto Personal) y lo que llamamos el espíritu de las leyes islámicas, a través de islamizar algunas de las leyes civiles, no en su contenido sino en su forma externa; por ejemplo, al- šarî‘a al-islamiyya prohíbe la usura; de hecho, las leyes civiles evitaron mencionar el término usura a la hora de egipcializar el derecho francés32. En este sentido, se puede afirmar que el derecho francés ha influido notoriamente en los ordenamientos jurídicos árabes por dos vías: directamente, como es el caso de la Ley libanesa de 9 de marzo de 1932 relativa al Código de obligaciones y contratos33, aún en vigor, que no solo se basa en el código civil francés sino también en la doctrina y jurisprudencia francesas, siendo que el texto original se redactó en francés y el texto en árabe fue una traducción posterior34. Otra, indirecta, merced al hecho de que el código civil egipcio de 1948 elaborado por al-Sunhûrî, de inspiración francesa, sirvió de modelo para el código sirio (1949), el de Iraq (1953), el de Libia (1954), el de Argelia (1975) y el de Jordania (1977). Asimismo, las disposiciones jurídicas que regulan las obligaciones en el código civil egipcio fueron incorporadas al Código de Comercio de Kuwait en 194835. Otros autores como Fakhri36 van más allá, y afirman que el lenguaje jurídico marroquí ha sufrido un cambio sustancial por influencia del francés, pues mientras los textos jurídicos tradicionales seguían una estructura narrativa, la organización actual es cronológica, con oraciones y cláusulas que dan cuenta de una secuencia de hechos y circunstancias ordenados temporalmente, junto con personajes y sus funciones. En un trabajo posterior, Fakhri sostiene que las sentencias del Tribunal de Casación marroquí reflejan prácticas retóricas híbridas, que han tomado prestado modelos occidentales37. Así, ...‫ من قانون المسطرة المدنية‬543 ‫( بناء على أن الفصل‬Visto el artículo 345 del Ley de Enjui31 32 33 34 35 36 37 NEBOT ROSER, Nicolás. op. cit., p. 300 ABDEL-AZIZ, Khaled, op. cit, (2003), p. 176. https://www.ilo.org/dyn/natlex/docs/ELECTRONIC/57070/134590/F-755173354/LBN57070%20ARA_FR.pdf [Último acceso el 21 de noviembre de 2011]. EL CHAZLI, Karim. “Le role des langues dans la connassaince et le devéloppement des droits des pays árabes”, en General Reports of the XIXth Congress of the International Academy of Comparative Law. Martin Schaouer y Bea Verschraegen (eds.) Dordrecht (2017), p. 598. ALUFFI, Roberta, “Unity and varieties of Arabic as a legal language. Practices of interpretation and translation”, en Handbook of communication in the Legal sphere, Jacqueline Visconti (ed.), Der Gruyter La Haya (2018) p. 431. FAKHRI, Ahmed, “Borrowing Discourse patterns: French rhetoric in Arabic legal texts”. Dilworth B. Parkinson, Elabbas Benmamoun (eds.) (2002), p. 161. FAKHRI, Ahmed, “Citations in Arabic Legal Opinion. `Iftaa´versus qadaa´”. Perspectives on Arabic Linguistics Dilworth. B. Parkinson (ed.) (2008), p. 126 Soto Aranda, B.; Kabbour, R. Anaquel estud. árabes 31 2020: 179-205 186 ciamiento Civil …) corresponde a expresiones del francés jurídico como Vu l´article 1382 du code civil, mientras que ‫ من قانون االلتزامات والعقود‬929 ‫( عمالً بمقتضيات الفصل‬En aplicación de lo estipulado en el artículo 929 del Código de Obligaciones y Contratos) corresponde a expresiones como En application des articles R. 142-1 et R. 142-18 du code de la securité. Desde una perspectiva complementaria, Lapiedra subraya el hecho de que la šarî‘a es un conjunto de normas más propiamente religiosas o morales, y que, debido a su carácter no jurídico, las normas de contenido jurídico que se fundamentan en elementos de la šarî‘a, tendrán en cada país la fuerza que su ordenamiento jurídico haya establecido: desde una remisión directa, a la plasmación gradual en leyes, decretos, etc.38. Estas diferencias se observan, por ejemplo, en las constituciones saudí y marroquí. En la primera, aprobada en 1992 y modificada en 2005, denominada ‫النظام األساسي للحكم بالمملكة السعودية‬, sus artículos 1 y 8 dicen: ‫ ودستورها كتاب هللا‬،‫ دينها اإلسالم‬،‫ ذات سيادة تامة‬،‫ المملكة العربية السعودية دولة إسالمية‬.‫المادة األولى‬ )...( .‫تعالى وسنة الرسول صلى هللا عليه وسلم‬ Art. 1. El Reino de Arabia Saudí es un Estado islámico, totalmente soberano, su religión es el islam, y su constitución es el Libro de Allah, Todopoderoso, y la Sunnah del Profeta, la paz sea con él. (...) ‫ يقوم الحكم في المملكة العربية السعودية على أساس العدل والشورى والمساواة وفق الشـريعة‬.٨ ‫المادة‬ .‫اإلسالمية‬ Art. 8 – El Gobierno del Reino de Arabia Saudí se basa en la justicia, la consulta y la igualdad de acuerdo con la šarî‘a islámica. Por su parte, en la constitución marroquí de 2011, denominada ‫دستور المملكة المغربية‬, los artículos 3, 6 y 16 fueron redactados de la siguiente forma: .‫ والدولة تضمن لكل واحد حرية ممارسة شؤونه الدينية‬،‫ اإلسالم دين الدولة‬.3 ‫الفصل‬ Art. 3. El islam es la religión del Estado y es el Estado el que garantiza a cada uno la libertad para practicar sus cultos religiosos. ‫ بما فيهم‬،‫ أشخاصا ذاتيين أو اعتباريين‬،‫ والجميع‬.‫ القانون هو أسمى تعبير عن إرادة األمة‬.6 ‫الفصل‬ .‫ وملزمون باالمتثال له‬،‫ متساوون أمامه‬،‫السلطات العمومية‬ Art. 6. La Ley es la expresión suprema de la voluntad de la nación. Toda persona, física o jurídica, incluidas las autoridades públicas [los poderes públicos], son iguales ante ella y están obligados a cumplirla. ‫ تعمل المملكة المغربية على حماية الحقوق والمصالح المشروعة للمواطنات والمواطنين‬.61 ‫الفصل‬ ‫ في إطار احترام القانون الدولي والقوانين الجاري بها العمل في بلدان‬،‫المغاربة المقيمين في الخارج‬ .)...( ‫االستقبال‬ Art. 16. El Reino de Marruecos protegerá los derechos e intereses legítimos de los ciudadanos marroquíes que residen en el extranjero, en el marco del respeto al derecho internacional y las leyes vigentes en los países de acogida (…). 38 LAPIEDRA, Eva, op. cit. p. 216. Soto Aranda, B.; Kabbour, R. Anaquel estud. árabes 31 2020: 179-205 187 Así, mientras Arabia Saudí fundamenta sus leyes sobre una determinada lectura del Corán y de la tradición profética (Sunnah)39, otros países como Marruecos, aun reconociendo el islam como religión del Estado, supeditan sus normas al derecho internacional y al de los países donde viven ciudadanos marroquíes. El lenguaje jurídico árabe se caracteriza pues por la pluralidad de fuentes del derecho (continental, Common Law, islámico, consuetudinario), la diversidad de sistemas de terminología legal, todo ello acentuado por las influencias externas en cuanto a préstamos, y el hecho de que otros organismos internacionales como la ONU y la OTAN también producen su propia terminología en árabe, basada en criterios locales y la preferencia de los traductores40. Por ejemplo, el término `artículo´ se traducirá por ‫ مادة‬si la traducción ha de surtir efecto en Jordania, Siria, Irak, Egipto y países del Gofo, mientras que también se puede utilizar el término ‫ فصل‬para Marruecos, como se puede apreciar en los ejemplos extraídos de las constituciones. En el caso de ´garante´, es ‫ كفيل‬en Jordania, Siria o Irak, y ‫ ضا ِمن‬en Egipto y el Magreb41. No obstante, sí hay un acuerdo en que el lenguaje jurídico en lengua árabe presenta características comunes como la existencia de términos técnicos como ‫قضية‬ (causa), ‫( دعوى‬demanda) y ‫( محكمة‬tribunal), semitécnicos como ‫( تعويض‬compensación) y ‫( دليل‬prueba), junto con palabras comunes como ‫( طرف‬parte), ‫( ملف‬expediente) y ‫( تقرير‬informe), que adoptan una acepción especializada en este tipo de textos. Asimismo, son frecuentes las metáforas y otros recursos estilísticos. En cuanto a arcaísmos, El-Farahaty señala términos como ‫( الحالة المعتبرة شرعا‬cumpliendo todos los requisitos legales) o ‫( المذكور أعاله‬arriba mencionado), aunque otros han caído en desuso como ‫( المزبور‬el arriba mencionado) y ‫( المسفور‬declarado). Igualmente se caracteriza por el uso de la nominalización y de dobletes. Según Fakhri, la nominalización cumple dos funciones en las sentencias judiciales: una defensa sólida y una referencia anafórica concisa, como se observa en la siguiente expresión formularia característica de los fallos del Tribunal Supremo marroquí, para indicar que considera nulo y sin efecto el fallo de un Tribunal inferior. ‫يعيب الطالب‬ .‫ سوء ونقصان التعليل الموازي النعدامه‬... ‫( على القرار المطعون فيه‬Se rechaza la solicitud impugnada… por error de interpretación y falta de fundamento equivalente a su ausencia)42. En cuanto a los culturemas, tenemos términos como ‫( عدة‬período de espera antes de contraer nuevas nupcias) o ‫( خلع‬divorcio a petición de la esposa). De lo dicho cabe extraer tres principios importantes para la traducción al árabe. Por un lado, a pesar de la variabilidad, existen un conjunto de rasgos comunes que caracterizan los textos legales y que deben tenerse en cuenta, en la medida de lo posible. Por otro, a lo largo de la Historia, el lenguaje jurídico ha consolidado la aplicación de una serie de procedimientos léxicos para dar cuenta de los cambios producidos en las sociedades árabes; y por ende, la incorporación de términos extranjeros al lenguaje jurídico árabe tiene lugar preferentemente dentro de la tradición gramatical de la lengua árabe43: 39 40 41 42 43 EL-FARAHATY, Hanem. op. cit. (2016), 473 DARWISH, Ali Terminology and Translation. A Phonological-Semantic Approach to Arabic Terminology, Melbourne 2009, p. 24. SHIYAB, Said, op. cit. p. 142. FAKHRI, Ahmed, “Nominalization in Arabic Discourse: A Genre Analysis perspective”. Arabic Language and Linguistics. R. Bassiouney y ‎E. G. Katz (eds.) (2012), 145-157 Procedimientos de acuñación e incorporación de terminología jurídica al árabe según Roser NEBOT, op. cit. 188 Soto Aranda, B.; Kabbour, R. Anaquel estud. árabes 31 2020: 179-205 – Derivación léxica: a. Recuperación de términos jurídicos en desuso, e investirlos de una nueva acepción. b. Enriquecimiento polisémico de un vocablo jurídico homologado con un nuevo significado según el contexto. c. Creación de vocablos derivados de términos que comparten un campo semántico. – Traducción explicativa: a. Doblete de nombre y adjetivo, recoge el significado de la expresión original y no sus componentes léxicos inmediatos. Ejemplos de incorporación de términos mediante alguna de estas técnicas son las voces ‫( دستور‬constitución), ‫( جنسية‬nacionalidad) y ‫( مواطنة‬ciudadanía). El primer término fue adoptado por primera vez en Túnez (1861), y se tomó prestado del persa, referido a persona que ejerce la autoridad, y a una regla o regulación que conlleva una pena44. El segundo término se tomó prestado del griego yenos, en la que indicaba `raza´. El tercer término procede de ‫( مواطن‬ciudadano), derivado de ‫وطن‬, que en origen significaba `lugar de nacimiento o residencia´. Por otro, la preferencia por estos procedimientos, frente a la paráfrasis o a la mera transcripción, cuando ello es posible, es correlativa a la importancia que se le otorga a la corrección formal de los enunciados jurídicos45, si bien en el caso de culturemas, se tiende a recurrir a estas dos técnicas, además de la nota a pie de página. En este sentido, el traductor debe prestar atención no solo a la correcta transmisión del contenido y a la terminología empleada en la traducción, sino también la redacción y adecuación estilística del texto meta, al objeto de que la administración destinataria la acepte. Así, el objetivo de una traducción especializada como esta es la de producir un texto meta que desempeñe la misma función legal y produzca el mismo efecto legal46, por lo que además de informar, tienen una función performativa47. En lo concerniente a textos relacionados con los códigos de estatuto personal, y si bien es cierto que son los que mayor número de anisomorfismos presentan debido a la influencia que los principios de la šarî‘a ejercen sobre ellos, hay que observar esta cuestión con cierta precaución. Sin ir más lejos, Abdel-Aziz apunta que el propio término ‘estatuto personal’ “no es un término legal propio del derecho islámico ni de la literatura religiosa árabe-islámica tradicional, sino un término jurídico traducido literalmente del inglés Personal Status y del francés Statut Personnel, según han demostrado ‘Abd-Allah y Qamhawl (1986)”48. Esta traducción fue acuñada por Muhammad Qadrî Bâšâ en 1875 y asumida por el resto de los países árabes49. En el marco de un discurso culturalmente marcado por su inspiración religiosa, los términos se adaptan, se reinterpretan o se crean, en función de los intereses y objetivos del legislador. Para ello se recurre al iytihad, la potestad interpretativa que tienen los especialistas en el fiqh, la jurisprudencia islámica. Ello ocurre, bien por 44 45 46 47 48 49 ALUFFI, Roberta, op. cit. p. 426 NEBOT ROSER, Nicolás, op. cit. p. 301 EL-FARAHATY, Hanem. op. cit. p. 473. MEDIOUNI, Mohamed, “Towards a functional approach to Arabic-English legal translation: The role of comparable /parallel texts”. New insights into Arabic translation and interpreting. M. Taibi (ed,) (2016), 115-160. ABDEL-AZIZ, Khaled, (2003) op. cit., p. 176. ALUFFI, Roberta, op. cit. p. 426 Soto Aranda, B.; Kabbour, R. Anaquel estud. árabes 31 2020: 179-205 189 impulsos sociales internos -tal es el caso de los movimientos feministas-, bien por cuestiones derivadas de la aplicación del derecho internacional; aunque también existe otro factor a tener en cuenta, como es el peso y repercusiones de la emigración a países europeos50. Un claro ejemplo de ello es la redacción del Código de Familia marroquí (en adelante, CF) aprobado en 200451. En lo concerniente a las cuestiones de fondo, el término ‫( ولي‬tutor matrimonial), uno de los requisitos para la validez del matrimonio islámico, deja de ser una figura obligatoria. Así, el nuevo CF ha hecho opcional una condición hasta ahora obligatoria para la validez del matrimonio islámico según la antigua Mudawwana, como es la tutela matrimonial para la mujer ejercida por el wali (tutor), y le ha dado a la mujer mayor de edad la capacidad total de decidir casarse. Ella conserva la posibilidad de ser asistida por un tutor, pero ahora es libre de usar esta facultad o no. Esto ha supuesto que la referencia al wali desaparezca de la mayoría de las actas matrimoniales que se expiden en los últimos años. En lo concerniente a las cuestiones de forma, según el nuevo texto, el matrimonio de los ciudadanos marroquíes en el extranjero puede llevarse a cabo siguiendo las formas del derecho local. En este caso, el CF no requiere que se cumplan las condiciones de validez de un matrimonio dentro de Marruecos, salvo la presencia de dos testigos (en las actas marroquíes esa función es desempeñada por los dos notarios que deben levantar acta). Las actas españolas también incluyen testigos, pero, en el caso de que no sean varones musulmanes, para que sean aceptadas por el consulado correspondiente, será requerida un acta complementaria de testificación que se levanta en el propio consulado. 3. Conocimientos previos para afrontar la traducción de un acta notarial de matrimonio civil al árabe Para traducir un acta notarial de matrimonio, debe tenerse en cuenta la tipología textual, las características de las actas notariales en el contexto meta y el lenguaje que las caracterizan. Los instrumentos notariales se estudian dentro de los lenguajes de especialidad, en concreto, en el marco del lenguaje jurídico. En cuanto al género, se trata de documentos públicos, esto es, autorizados, expedidos o intervenidos por un funcionario público competente, y entran dentro de la categoría de textos de aplicación del Derecho, junto con contratos, testamentos, poderes de representación, etc.52. Borja incluye dentro de los textos jurídicos tanto aquellos que se utilizan en las relaciones entre el poder público y los ciudadanos como aquellos textos codificados, empleados para regular las relaciones entre particulares con transcendencia jurídica (que dan lugar a 50 51 52 La realidad de la inmigración también ha influido en otros cambios legislativos como el art. 65, cap. II de la Ley de nacionalidad consolidada a 26.11.2011., por el que la nacionalidad se obtiene por vía de padre y madre, indistintamente. Antes de la reforma, la nacionalidad por vía materna para los hijos de ciudadanos residentes en el extranjero solo se obtenía en casos de padre desconocido o apátrida. Siguiendo la terminología empleada por Estaban de la Rosa et al. en su traducción comentada del nuevo Código de Familia marroquí, y teniendo en cuenta los sustanciales cambios de fondo que éste presenta respecto del Código del Estatuto Personal y de las Sucesiones, más conocido como antigua Mudawwana (1957-58), optamos por referirnos al primero por sus siglas CF. Véase JUNTA DE ANDALUCÍA. La nueva Mudawwana marroquí: entre tradición y modernidad (Traducción comentada del Código de Familia de 2004), Sevilla 2009. BORJA, Anabel, op.cit, 2005. Soto Aranda, B.; Kabbour, R. Anaquel estud. árabes 31 2020: 179-205 190 contratos, testamentos o poderes)53. El documento propuesto pertenece al segundo grupo. Desde el punto de vista árabe54, el acta objeto de este estudio se puede clasificar del siguiente modo: Acta notarial de celebración de matrimonio civil Género Documento notarial Fuente emisora Notario Receptor Administración Función Instructiva y expositiva, con la finalidad de establecer y legalizar las relaciones particulares según lo establecido y guardar los derechos a largo plazo. Modalidad Escrito para ser leído en silencio Tenor Estándar fosilizado Elaborado a partir de la propuesta de Abdel-Aziz sobre categorización de tipologías textuales jurídicas egipcias (2006) En lo referente a la función de la traducción, hay que tener presente que este tipo de textos han de surtir efecto en una comunidad lingüística y cultural y un ordenamiento jurídico propio, distintos de aquella en la que se originaron, y funcionar de facto como un original55, demostrando el vínculo matrimonial entre dos personas. Como se observará más adelante, para que esto ocurra y el destinatario -en este caso la administración marroquí-, el traductor deberá prestar especial atención al lenguaje empleado y la corrección terminológica. Frente a otros países árabes donde los documentos matrimoniales suelen ser certificados expedidos por un juez o un funcionario autorizado, en el caso de Marruecos se emiten en forma de actas notariales. La razón de esta singularidad puede deberse a que la institución que se encarga de la fe pública en Marruecos, el notariado, destaca por un desarrollo histórico propio, diferenciado del de los países europeos, y de otros países árabes. Marruecos no formó parte del Imperio otomano, mientras que el resto de los países árabes conjugaron las estructuras administrativas heredadas de la época colonial con el sistema jurídico otomano, cuya codificación del derecho de familia comienza con la Ley Otomana de Derecho de Familia promulgada en 191756, que estableció la posibilidad de que las cuestiones relacionadas con la familia se desarrollaran bajo la jurisdicción de tribunales de derecho civil o de derecho islámico. Marruecos siguió un sistema notarial propio influido por el desarrollo que este había tenido en al-Ándalus, y por la jurisprudencia desarrollada por la escuela malikí. Desde un punto de vista histórico, el notariado adular tiene sus raíces en la re53 54 55 56 BORJA, Anabel, El texto jurídico inglés y su traducción al español. Barcelona, Ariel, 2000 ABDEL-AZIZ, Khaled, “Clasificación del texto jurídico árabe (desde el punto de vista de la traducción jurídica”, Quaderns: Revista de traducción, (2006), vol. 13, 169-179. MAYORAL ASENSIO, Alberto, “Las fidelidades del traductor jurado: una batalla indecisa”, en Traducir para la justicia. M.C. Feria García (ed.) (1999). Disponible en: http://www.ugr.es/~greti/puentes/puentes2/07-articulo.pdf [Último acceso el 21 de noviembre de 2019]. TORRES GARCÍA, Ana. op. cit. p. 380. Soto Aranda, B.; Kabbour, R. Anaquel estud. árabes 31 2020: 179-205 191 cepción de las primeras revelaciones del Corán, por las que ordena a los musulmanes formalizar sus actos y negocios jurídicos en dos azoras (2: 282 y 24: 33) 57. Así, su creación tuvo por objeto proteger y defender los derechos y bienes de los individuos, regular y otorgar seguridad jurídica a las relaciones familiares mediante instrumentos públicos y dejar constancia de ellas de forma escrita. Para Nebot58, el origen coránico del lenguaje de especialidad de la jurisprudencia en árabe determina que los campos semánticos, así como los términos jurídicos especializados, tengan que preservar una relación íntima e indestructible de significado con el lenguaje coránico, así como sus aplicaciones y derivaciones prácticas. Si los instrumentos notariales hasta el siglo X se caracterizaron por ser cortos, concisos y resumidos, a partir de esa fecha, empiezan a presentar grandes cambios en al-Ándalus y Marruecos, siendo la característica más importante de los instrumentos de aquella época las anotaciones marginales y las explicaciones jurisprudenciales, aunque estas desaparecen en el siglo XIII d. C., apareciendo publicaciones que describen el objeto de cada instrumento. Los Protectorados francés y español59 reconfiguraron el sistema judicial vigente en Marruecos hasta principios del siglo XX con el fin de afianzar su control sobre el territorio, hecho que también afectó al sistema notarial, al añadir a los notariados islámico y rabínico ya existentes, el notariado latino, con el objeto de cubrir las necesidades de los colonos. Los gobiernos coloniales justificaron su creación alegando la necesidad de modernizar la administración indígena, lo que, en la práctica, favoreció a los súbditos europeos que vivían en Marruecos y se sometían a la competencia de los tribunales franceses, españoles y mixtos —en el caso de Tánger que era una zona internacional—. La creación de esta institución paralela a la del notariado tradicional islámico cabe inscribirse dentro de la política colonial de sustitución y suplantación de las lenguas vernáculas, particularmente por lo que hace referencia a los países del Magreb, que en el momento de su colonización ya disponían de una lengua de uso jurídico y administrativo, el árabe. En el caso francés, este carácter intervencionista alcanzaría su máxima expresión con la firma el 16 de marzo de 1930 del denominado dahir bereber por el que se pretendía dividir el territorio marroquí bajo el mando de Francia en dos zonas jurídicamente diferenciadas independientes: la parte urbana y arabizada se regiría por la šarî‘a —la ley islámica—, estaría bajo el control del sultán, y los crímenes serían juzgados por el Tribunal Cheránico, mientras que, en las zonas rurales, de mayoría bereber, pasaría a aplicarse el derecho consuetudinario, con el objetivo de ir introduciendo las leyes francesas. Tras las protestas, en 1936 se firma un dahir derogando el artículo 6 del primero, que instauraba el sistema legal francés en las zonas bereberes. Con el dahir de 7 julio de 1914, a la vez que se regulaba la profesión del notariado adular o islámico, se crea de facto el notariado latino para tratar los asuntos de los colonos, y de este modo y al igual que ocurrió en otras colonias, los asuntos relacionados con la familia, la herencia, etc., quedaron relegados a los tribunales de jurisdicción religiosa. Durante la época colonial, la organización del notariado adular se fue regulando a través de diversos dahires o decretos reales (1938, 1943) hasta el dahir de 7 de 57 58 59 .2014 )‫ (مجلة إلكترونية‬،‫ المسطرة المدنية‬- ‫ القانون المدني‬،‫ عدنان‬،‫المرابط‬ ROSER NEBOT, Nicolás . op.cit. Para un análisis del Protectorado español y de los traductores, véase, entre otros, FERIA, Manuel, “Conflictos de legitimidad jurídica en Marruecos. El impacto del Protectorado”, El protectorado español en Marruecos: gestión colonial e identidades, F. Rodríguez y H. de Felipe (eds.) (2002). 192 Soto Aranda, B.; Kabbour, R. Anaquel estud. árabes 31 2020: 179-205 febrero de 1944, por el que se hizo obligatoria la incorporación de una nota marginal con las referencias necesarias (tomo, folio, asiento y fecha) al instrumento original. La normativa colonial no se modificó hasta 1982, veinte años después de haber alcanzado la independencia, cuando se incluyó como novedades la forma de otorgar y redactar los testimonios, mientras que el dahir de 1983 regulaba el nombramiento de los adules, la supervisión de su trabajo, la custodia y redacción de los instrumentos, así como los honorarios de los adules, entre otras. El Dahir 16.03, en vigor desde el día 03 de marzo de 2006, es la que regula el acceso y el ejercicio de la profesión, la competencia territorial de los adules, sus responsabilidades y la creación de un colegio nacional de adules y colegios regionales. Por último, desde enero de 2018, las mujeres ya pueden ejercer como notarias en el marco del notariado islámico. El actual notariado marroquí es por lo tanto heredero del sistema colonial y está formado por una estructura bipartita: el notariado confesional, que se encarga de asuntos relativos al estatuto personal y sucesorio, y el notariado de derecho civil, encargado de contratos relativos al derecho común. Dentro del notariado confesional se distinguen a su vez tres categorías60: – El notariado adular, competente para los asuntos religiosos de la población musulmana. Para tener validez, las actas adulares necesitan de la firma de dos notarios y la homologación de un juez. – El notariado que se encarga de la fe pública rabínica, que sigue las disposiciones de la del Talmud y el código Caro61. Los instrumentos notariales deben estar firmados por dos soffrims y homologados por el juez rabínico62. – El notariado latino, que es competente para los actos de personas que no pertenecen a los grupos anteriores, también denominado notariado europeo. Se encargan de la redacción de contratos notariales y no necesitan de homologación alguna. En el proceso de construcción nacional, y en aras de la unión nacional, las cuestiones relativas a la familia y a la religión fueron asumidas como parte de las bases de la reafirmación identitaria y un factor legitimador del Estado, por lo que se consideraron prioritarias y trataron de blindarse institucionalmente tanto por parte de la élite nacionalista del partido Istiqlal como por la élite religiosa63; de hecho, el Código de Estatuto Personal y de Sucesiones (1957-1958), la antigua Mudawwana, fue aprobado con anterioridad a la constitución marroquí, que data de 196264, y su contenido y orientación fue el resultado de las deliberaciones de un comité de diez expertos entre los que figuraban entre autoridades religiosas y funcionarios del ministerio de Justicia65. 60 61 62 63 64 65 Para un análisis exhaustivo del Notariado marroquí, véase FERIA GARCÍA, Manuel, “Convivencia e interacción de tradiciones jurídicas en Marruecos: la fe pública: sofrims, notarios latinos y adules”, en Awraq: Estudios sobre el mundo árabe e islámico contemporáneo, 24 (2007) 241-286. Para un estudio más detallado del notariado rabínico, ZAFRANI, Haïm, Deux mille ans de vie juive au Maroc: histoire et culture, religion et magie, Casablanca 2000. En el caso de Casablanca, es la cámara hebrea del tribunal de primera instancia la competente para su homologación. TORRES GARCÍA, Ana, op. cit. p. 380. EL HAMOUD, Omar, “La constitución marroquí desde el punto de vista de los jóvenes”, en LEX SOCIAL-Revista de los Derechos Sociales 1 (2011) 228. MERNISSI, Fatima, Beyond the Veil. Male-Female Dynamics in Modern Muslim Society. Revised edition, Bloomington 1987. Soto Aranda, B.; Kabbour, R. Anaquel estud. árabes 31 2020: 179-205 193 En él se impuso una visión conservadora de la sociedad y una lectura restrictiva de la jurisprudencia malikí respecto de los principios que sostienen la familia y el papel de la mujer, que era diametralmente opuesto al papel que habían desempeñado las mujeres en la lucha por la independencia, organizadas en torno a la sección femenina del partido Istiqlal y de la asociación Akhawat Saffa. Las actas matrimoniales se regulan por este código, por lo que las modificaciones sufridas por la Mudawwana en 1993 y la nueva redacción del CF de 2004 han afectado en distinto grado a las actas notariales de matrimonio y a su expedición. Así, la reforma de 1993 introdujo la obligatoriedad de registrar el matrimonio (Art. 43) para su reconocimiento oficial y reguló el procedimiento administrativo a seguir. Asimismo, introdujo la obligatoriedad de registrar el divorcio (Arts. 60, 80 y 81) para su reconocimiento oficial66. Ya la Mudawwana de 1958, en su Art. 81 decía: 1º. El acta de repudio debe mencionar, para cada uno de los esposos, su nombre, filiación, domicilió y su identificación mediante el documento individual o un certificado administrativo de identidad. 2º Debe hacer referencia al acta de matrimonio, con indicación del número, folio y fecha, precisando que esta acta se encuentra debajo o detrás del acta de repudio. Esto supuso de facto que muchos matrimonios anteriores a la promulgación de la Mudawwana tuvieran que reconocerse mediante actas alternativas, tales como el acta testifical de continuidad en matrimonio. En 1958, la antigua Mudawwana estableció que las actas notariales de matrimonio debían incluir la siguiente información: 1. Los nombres, apellidos, filiación, domicilio e identidad completa de los esposos, con mención que están en posesión de sus facultades, así como el nombre del Wali. 2. La fecha del acta de matrimonio, el lugar de realización, con indicación de que los esposos y del tutor actúan con conocimiento de causa. 3. Todas las referencias útiles relativas a la edad de la esposa, si es virgen o no, si tiene o no padre, provista o no de un tutor testamentario, si es divorciada o viuda, y si ha observado la idda. 4. La mención del certificado administrativo con su número de orden. 5. La cantidad de la dote67, precisando lo que debe entregarse, si se entrega delante de los adules o si solo hay reconocimiento ante estos de una entrega anterior. 6. La firma de los adules y la homologación del juez con su sello. El Art. 43, por su parte, señalaba que el acta de matrimonio se consignaría en el registro que existe para tal efecto en el tribunal. Un ejemplar de esta acta debía enviarse al Registro Civil: el original del acta se debía enviar a la esposa o a su repre66 67 TORRES CALZADA, Katjia, “Poder y religión en los 50 años del Reino de Marruecos”, 50 años del Reino de Marruecos: análisis sobre el Marruecos Actual. K. Torres Calzada (ed.), (2006), p. 22. La dote es uno de los requisitos para que el matrimonio islámico sea válido. Se trata de un regalo con el que debe obsequiar el esposo a la esposa para formalizar la unión matrimonial. Puede aplazarse la entrega de parte ; este hecho deberá mencionarse expresamente en el acta de matrimonio. El término ‫ صداق‬se traduce por dote en español, aunque en puridad se trata de un anisomorfismo, pues en la cultura popular española, la dote corresponde al ajuar que lleva la esposa al matrimonio). 194 Soto Aranda, B.; Kabbour, R. Anaquel estud. árabes 31 2020: 179-205 sentante en un período máximo de 15 días a contar desde su fecha. El esposo tenía derecho a tener una copia del acta. En el CF de 2004, en el capítulo denominado Título VI. De los procedimientos administrativos y formalidades para la conclusión (establecimiento) del acta matrimonial, indica que el expediente para el matrimonio debe ser autorizado por el juez de familia correspondiente, quien autorizará a los notarios del partido judicial en el que se va a registrar el acta, a levantarla. En ella, los notarios deberán consignar68: 1. La referencia a la autorización del juez, su número y fecha, así como el número de orden del expediente que contiene los documentos proporcionados para el matrimonio y el Tribunal en el que se encuentran en depósito. 2. El apellido y el nombre de los dos esposos, el domicilio o el lugar de residencia de cada uno ellos, el lugar y la fecha de nacimiento, los números de su carné de identidad nacional o lo que proceda y su nacionalidad. 3. El nombre del tutor cuando proceda. 4. El consentimiento mutuo de los dos contrayentes que gozan de capacidad, de discernimiento y la libertad de elegir. 5. En caso de poder otorgado para concluir un matrimonio, el nombre del mandatario, el número de su carné de identidad nacional y la fecha y el lugar en el que se otorgó el poder. 6. La referencia a la situación jurídica del que o de la que ya haya contraído un matrimonio. 7. El importe de la dote cuando se fija, precisando la parte pagada de antemano y aquella al vencimiento de un plazo, y si su percepción tuvo lugar ante los adules o por reconocimiento. Las cláusulas estipuladas por las dos partes. 8. Las firmas de los esposos y del tutor, cuando proceda. 9. El apellido y nombre de los adules y la firma de cada uno ellos y la fecha a la en la cual concluyeron el acta. 10. La homologación por el juez, con su sello, del acta de matrimonio. La lista de los documentos constitutivos del expediente del acta de matrimonio, así como su contenido, pueden ser modificados y completados por decreto del Ministro de Justicia. A ello hay que añadir referencias que suelen aparecer en las actas notariales a los certificados de estado civil y médico que han de presentarse para completar el expediente judicial. Por último, el Art. 68 señala que el contenido del acta de matrimonio ha de transcribirse en el registro existente a tal efecto, en la Sección de Familia. Un testimonio de este se remitirá al Encargado del Registro Civil del lugar de nacimiento de los esposos, acompañado de un acuse de recibo, en el plazo de quince días a partir de la fecha de homologación del matrimonio por el juez. No obstante, si uno de los dos esposos o los dos no hubieran nacido en Marruecos, dicho testimonio se remitirá al Procurador del Rey del Tribunal de Primera Instancia de Rabat. El Encargado del Registro Civil debe incluir todas las menciones del testimonio como anotaciones marginales de la inscripción de nacimiento de cada uno de los cónyuges. La forma, el contenido del registro previsto en el primer párrafo (arriba), así como las menciones antes citadas, son fijados por resolución del Ministro de Justicia. 68 Se ha utilizado la traducción de la Junta de Andalucía (2009). Soto Aranda, B.; Kabbour, R. Anaquel estud. árabes 31 2020: 179-205 195 El lenguaje notarial árabe marroquí comparte con el lenguaje notarial español69 una serie de rasgos, entre los que cabe mencionar los siguientes: Uso construcciones nominales del tipo ‫( المنتصبين لإلشهاد‬asignados para [recibir] las declaraciones), ‫( الحامل للبطاقة الوطنية‬titular del documento nacional de identidad), ‫( تامة العقل‬con sus facultades mentales) y locuciones prepositivas, como )‫من والده(ها‬ (hijo/a de), ‫( على صداق مبارك‬con una dote…). En lo referente al léxico, destaca el uso de tecnicismos como ‫نسخة رسم ثابت مستقل‬ (copia de una certificación auténtica e independiente), ‫( صداق‬dote) y ‫( ولي‬tutor), como de palabras de uso común que en este tipo de documentos adquieren un significado especializado, tal es el caso de ‫( قبل البناء‬antes de la consumación del matrimonio) y ‫ة‬/‫( الساكن‬domiciliado/a). El término ‫ الكتاب‬se refiere al Corán. Es habitual el recurso a la sinonimia (total o parcial), es el caso de expresiones como ‫( الحل للنكاح‬apta para el matrimonio), así como al uso de dobletes y tripletes para reforzar el significado de una determinada expresión. Es el caso de ‫بإذنها ورضاها‬ ‫( وتوكيلها‬con la autorización de la esposa), ‫( بعد باإليجاب والقبول‬acuerdo y aprobación). Fórmulas de respeto. Es el caso de ‫( سيد‬señor/D.) para el esposo, ‫( آنسة‬señorita) y ‫( سيدة‬señora) para la esposa; expresiones formularias del tipo ‫( عبد ربه تعالى‬lit. siervo de su Señor, Todopoderoso), para referirse a los notarios con respeto. Por el contrario, en los instrumentos notariales se usa el tiempo perfectivo, que generalmente tiene valor de pasado (ej.: ‫ حضر‬،‫)تلقى‬, si bien suelen traducirse en función de los requerimientos de la lengua meta. También es habitual que las actas no hagan uso de signos de puntuación como la coma, el punto y coma y el punto, como puede observarse en el siguiente ejemplo extraído de un acta fechada en 2010: ‫الحمد هلل وحده بناء على اإلذن بتوثيق عقد الزواج من قاضي األسرة المكلف بالزواج رقم *** بتاريخ‬ ‫ ملف بمستندات الزواج رقم *** المودع بقسم قضاء األسرة بالمحكمة االبتدائية بالحسيمة‬27/01/2010 ‫نحن *** و *** العدالن المنتصبان لإلشهاد بدائرة المحكمة المذكورة تلقينا بمذكرة الحفظ ألول شهيديه رقم‬ ‫ على الساعة الرابعة والنصف عشيا من يوم األربعاء حادي عشر صفر‬152 ‫ صفحة‬167 ‫ تحت عدد‬32 )...( ‫الخير‬ aunque las actas expedidas por el Consulado de Marruecos sí hacen uso de ello, como puede observarse en el siguiente ejemplo extraído de un acta de 2001. ‫) نهار يوم الجمعة رابع محرم عام اثنين و عشرين و أربعمائة وألف‬10( ‫ ففي الساعة العاشرة‬،‫الحمد هلل وحده‬ ‫ تزوج على بركة هللا تعالى السيد *** المولود بابني‬،‫للهجرة الموافق لثالثين مارس سنة ألفين وواحد للميالد‬ ‫ حسب بطاقة شخصية للحالة المدنية لموجز عقد‬،*** ‫ والدته ميلودة‬،1975 ‫ أكتوبر‬7 ‫حديفة الحسيمة بتاريخ‬ )...( ،.*** ‫ المؤرخة في‬،*** ‫ بطاقة الوطنية رقم‬،‫ جنسيته مغربي‬،‫الوالدة رقم ***؛ مهنته أجير باسبانيا‬ En ocasiones, los notarios de tipo tradicional o adular —ya que trabajan en parejas— recurren a descripciones físicas de los contrayentes de tono arcaizante como las siguientes: ‫( وصفها مائلة للبياض أسيلة متوسطة االمتالء والطول محجوجبة‬su descripción: blanca, de nariz erguida, de talla mediana y con cejas pronunciadas). Por los motivos antes expuestos, era habitual que el tenor de las actas notariales soliera tener numerosas referencias religiosas, como citas del Corán, aunque dichos elementos intertextuales varían, no tanto en función del año de emisión del acta, 69 ETXEBARRIA AROSTEGUI, Maitena (1997), “El lenguaje jurídico y administrativo. Propuestas para su modernización y normalización”, en Revista Española de Lingüística [En línea]. Nº 27, 2, disponible en: http:// www.sel.edu.es/pdf/jul-dic-97/27-2-Etxebarria.pdf [Último acceso el 21 de noviembre de 2019]. 196 Soto Aranda, B.; Kabbour, R. Anaquel estud. árabes 31 2020: 179-205 como del estilo particular adoptado por los notarios en cada caso. Así, hay actas recientes que sí las incluyen y otras que no. Las actas notariales de matrimonio venían caracterizándose por la inclusión de la basmala70 (en el nombre de Dios, el Clemente, el Misericordioso), como elemento de apertura, rasgo que las diferencia de las sentencias judiciales, cuyo encabezamiento es ‫( باسم جاللة الملك وطبقا للقانون‬en nombre su majestad el Rey y de conformidad con la Ley). No obstante, este elemento singularizador no aparece de forma sistemática en todas ellas, siendo que, en algunos casos como las actas expedidas por el Consulado de Marruecos en Madrid, aparecen en forma de logo debajo del escudo del país. Del mismo modo, se recurre a fraseologismos de marcado carácter religioso, entre otros, ‫وهللا سبحانه يؤلف بينهما‬, (que Dios Todopoderoso les congenie), en referencia a los cónyuges, y por el uso de expresiones formularias del tipo ‫( الحمد هلل وحده‬loor a Dios Único). En el caso de esta última, por ejemplo, Mayoral se refiere a ella como una fórmula de salutación, una referencia intertextual significativa en un texto árabe, pero cuya referencia se pierde al traducirla en un contexto cultural no islámico, por lo que muchos traductores omiten su traducción71. Este tipo de referencias religiosas en forma de basmala, citas del Corán, etc., caracterizadoras de las actas marroquíes, no aparecen en las certificaciones de matrimonio de otros países como es el caso de Egipto, aunque el Código de Estatuto Personal egipcio también tenga una fundamentación religiosa, luego se trata de un elemento discursivo, meramente intertextual en este tipo de documentos. 4. Propuesta de traducción comentada Las escrituras públicas, según el artículo 144-2 del Reglamento Notarial, tienen como contenido las declaraciones de voluntad negociables de los interesados, los actos jurídicos que impliquen prestación de consentimiento, los contratos y negocios jurídicos de todas las clases. Su finalidad principal es la dación de consentimiento. En el caso propuesto, los notarios tienen potestad para casar y divorciar en virtud de la Ley 15/2005, de 12 de julio, de la Jurisdicción Voluntaria que: opta por atribuir el conocimiento de un número significativo de asuntos que tradicionalmente se incluían bajo la rúbrica de la jurisdicción voluntaria a operadores jurídicos no investidos de potestad jurisdiccional, tales como los secretarios judiciales, Notarios y registradores de la propiedad y Mercantiles, compartiendo con carácter general la competencia para su conocimiento. Estos profesionales que aúnan la condición de juristas y de titulares de la fe pública reúnen sobrada capacidad para actuar, con plena efectividad y sin merma de garantías, en alguno de los actos de Jurisdicción Voluntaria que hasta ahora encomendaban a los Jueces […] El acta de matrimonio civil tiene una estructura fija formada por los siguientes apartados: 70 71 Se trata de una fórmula de invocación que constituye el primer versículo de todas las azoras del Corán, excepto en el caso de la núm. 9. MAYORAL ALONSO, Roberto, Translating Official Documents (Translation Practices Explained), Londres, 2003. Soto Aranda, B.; Kabbour, R. Anaquel estud. árabes 31 2020: 179-205 197 – Menciones preliminares. En este apartado se hace constar el tipo de instrumento público, el número de protocolo, el lugar y la fecha de su autorización y la identificación del notario que otorga la fe pública. – Comparecencia. En él, se hacen constar los datos personales de los otorgantes y, en su caso, de los demás intervinientes (testigos, abogados, etc.). Incluye también la identificación de éstos por los medios establecidos en la legislación notarial, así como la reseña y juicio de suficiencia de la representación alegada y el juicio de capacidad notarial para el acto documentado. – Exposición. Este apartado hace referencia a los antecedentes necesarios para la documentación del acto. Por ejemplo, en caso de que contenga bienes inmuebles, estos deben ser descritos conforme lo dispuesto en la legislación notarial, hipotecaria y especial. – Estipulaciones y otorgamiento. Es el apartado más importante de las escrituras, ya que contiene las declaraciones de voluntad, así como los pactos, cláusulas y condiciones que las partes desean elevar a público. – Autorización. Contiene la referencia a las advertencias y reservas legales y fiscales, la lectura del documento (por el notario o por la parte otorgante), el consentimiento de los otorgantes y la cláusula de autorización notarial. Al final, se transcriben la firma de los otorgantes y la firma, signo, rúbrica y sello del notario que da fe de la escritura pública. A continuación, presentamos una propuesta de traducción comentada, vertebrada en torno al desarrollo de: a) la competencia extralingüística (con especial atención a los conocimientos jurídicos y culturales) y b) la competencia de transferencia (macroestructura del TO y del TM; terminología y aspectos formales exigidos por la legislación del ordenamiento jurídico meta), a partir de la propuesta de Borja (2005) sobre competencias para el ejercicio la traducción jurídica. 4.1. Menciones preliminares Texto original: ESCRITURA DE CELEBRACIÓN DE MATRIMONIO CIVIL -------------------NÚMERO (Número de Protocolo)72 -----------------------------------------------------En Madrid, a (Fecha de autorización). ---------------------------------------------------Ante mí, IÑIGO CASLA URIARTE, Notario de Madrid y de su Ilustre Colegio con residencia en esta Capital, -------------------------- Propuesta de traducción: ---------------------------------------------------------------------------- ‫عقد زواج مدني رسمي‬ -------------------------------------------------- .‫ من سجل الحفظ‬00000000 ‫ض ِّمن تحت عدد‬ ُ ------------------------------------------------------------------------ .)‫ في (تاريخ تلقي العقد‬،‫مدريد‬ ----- ،‫ والمقيم بنفس هذه المدينة‬،‫ الموثق بهيئة الموثقين في مدريد‬،‫ نحن األستاذ إينييغو كاسال أوريارتي‬،‫أمامنا‬ 72 Los elementos marcados en cursiva en el texto original tienen carácter orientativo para el notario o su(s) ayudante(s) a la hora de elaborar un instrumento. En la versión árabe, estos elementos irán de igual modo en cursiva, pero en algunos casos, irán entre paréntesis para evitar confusiones. Soto Aranda, B.; Kabbour, R. Anaquel estud. árabes 31 2020: 179-205 198 Se ha optado por una traducción funcional del título, en la que se refleja en árabe que se trata de un “matrimonio civil” (‫ )زواج مدني‬celebrado de forma oficial mediante “escritura pública” (‫)عقد رسمي‬. Como ya se ha indicado, En Marruecos, de las certificaciones de matrimonio se encargan los notarios, también denominados adules, requiriendo la autorización previa y la homologación posterior de un juez, luego se consideran de por sí escrituras notariales; a diferencia del resto de países árabes, que han optado por modelos semejantes a la certificación de matrimonio expedida por el Registro Civil español. Como ya se ha mencionado, el CF marroquí (2004)73, permite a los ciudadanos marroquíes residentes en el extranjero contraer matrimonio según la forma prevista por las leyes del país de residencia, incluyendo el matrimonio civil74. 4.2. Comparecencia: Texto original: ------------------------------------- COMPARECEN ------------------------------------Los contrayentes: ---------------------------------------------------------------------------*, mayor de edad, nacido en *, el día *, hijo de don * y doña *, inscrito en el Registro Civil Central *, al tomo *, página *, de nacionalidad *, estado civil *, vecino de *, con domicilio en calle *, número *. -----------------------------------------------Me exhibe NIF/CIF número *. -----------------------------------------------------------*, mayor de edad, nacido en *, el día *, hijo de don * y doña *, inscrito en el Registro Civil Central *, al tomo *, página *, de nacionalidad *, estado civil *, vecino de *, con domicilio en calle *, número *. -----------------------------------------------Me exhibe NIF/CIF número *. ------------------------------------------------------------ Propuesta de traducción: ---------------------------------------------- ‫ حضر‬------------------------------------------------------------------------------------------------------------------------------------ :‫الزوجان‬ ،)‫ من والده (اسم األب‬،)‫الوالدة‬/‫ بتاريخ (تاريخ االزدياد‬،)‫ المولود في (المدينة‬/ ‫ المزداد‬،‫ الراشد‬،* ‫السيد‬ ،*‫ الحامل للجنسية‬،* ‫ صفحة عدد‬،* ‫ كناش عدد‬،* ‫ المقيّد في السجل المدني المركزي‬،)‫ووالدته (اسم األم‬ --------------------- .* ‫ رقم‬،* ‫ سكناه في شارع‬،* ‫ المقيم في‬،)‫ أرمل‬/ ‫ مطلق‬/ ‫حالته العائلية * (عازب‬ -------------------------------------- .* ‫ رمز التعريف الضريبي رقم‬/ ‫وأدلى برقم التعريف الضريبي‬ ‫ من والدها (اسم‬،)‫الوالدة‬/‫ بتاريخ (تاريخ االزدياد‬،)‫ المولودة في (المدينة‬/ ‫ المزدادة‬،‫ الراشدة‬،* ‫والسيدة‬ ‫ الحاملة‬،* ‫ صفحة عدد‬،* ‫ كناش عدد‬،* ‫ المقيّدة في السجل المدني المركزي‬،)‫ ووالدتها (اسم األم‬،)‫األب‬ ------- ، *‫ رقم‬،* ‫ سكناها في شارع‬،* ‫ المقيمة في‬،)‫ أرملة‬/ ‫ مطلقة‬/ ‫ حالتها العائلية * (عازبة‬،*‫للجنسية‬ -------------------------------------- .* ‫ رمز التعريف الضريبي رقم‬/ ‫وأدلت برقم التعريف الضريبي‬ Para proceder a la traducción de este apartado hay que tener en cuenta, en primer lugar, los valores asimétricos de los tiempos verbales en español y en árabe. Las actas notariales en español recurren al presente simple para indicar que, en el 73 74 Puede consultarse en Junta de Andalucía: La nueva Mudawwana marroquí: entre tradición y modernidad. Traducción comentada del Código de Familia marroquí). https://www.juntadeandalucia.es/export/drupaljda/1_2030_ nueva_mudawwana_marroqui.pdf FOBLETS, Mª Claire y LOUKILI, Mohamed, “Mariage et divorce dans le nouveau Code marocain de la famille: quelles implications pour les marocains en Europe?”, en Revue Critique de droit international privé 3 (2006) p. 530. Soto Aranda, B.; Kabbour, R. Anaquel estud. árabes 31 2020: 179-205 199 momento de levantar el acta, las personas concernidas o sus representantes legales se encontraban presentes físicamente ante el notario. Sin embargo, la norma de redacción de las actas marroquíes es el uso del pasado ‫ الماضي‬con un valor perfectivo, que indica que las personas concernidas han hecho acto de presencia ante el notario. En segundo lugar, hay que tener en cuenta las peculiaridades de la terminología marroquí, sobre todo en cuanto a términos como ‫ كناش‬y ‫ المزداد‬cuyo uso es más habitual que ‫ دفتر‬y ‫المولود‬, respectivamente. También se opta por la expresión )‫من والده(ها‬ para traducir `hijo/a de´, por ser de uso habitual en Marruecos. En cuanto a abreviaturas como “NIF” y “CIF”, se ha optado por una traducción explícita para diferenciar entre Número y Código, aunque en España desaparece el denominado “Código de Identificación Fiscal”, mediante la Orden EHA/451/2008, de 20 de febrero, por la que se regula la composición del número de identificación fiscal de las personas jurídicas y entidades sin personalidad jurídica. Texto original: Y como testigos instrumentales idóneos, designados por los contrayentes: --------------------------------------------------------------------------------------------------------** ---------------------------------------------------------------------------------------------** ---------------------------------------------------------------------------------------------Identifico a los comparecientes por el medio supletorio del apartado c) del artículo 23 de la Ley Orgánica del Notariado, resultando sus circunstancias personales de sus propias manifestaciones. Propuesta de traducción: ----------------------------------------------------- :‫ اللذان عينهما الزوجان‬،‫وشاهدا العقد المناسبان‬ ---------------------------------------------------------------------------------------------- ** ---------------------------------------------------------------------------------------------- ** ‫ على التعريف باألطراف الحاضرة أمامنا بالطريقة االستثنائية المنصوص عليها في‬،‫ نحن الموثق‬،‫ونشهد‬ ‫ وأن ظروفهم الشخصية هي المستمدة من‬،‫ من القانون التنظيمي المتعلق بالتوثيق‬32 ‫النقطة (ج) من المادة‬ .‫تصريحاتهم‬ Los términos jurídicos más destacados en este apartado son testigos instrumentales y medio supletorio. Respecto del primer término, hay que tener en cuenta que toda reglamentación que organiza una sociedad o las relaciones entre sus miembros —el derecho privado— dimana del marco cultural que le sirve de base, que en el caso del texto meta es el islam. Así, a pesar de tener una equivalencia funcional en árabe, por la que optamos en la traducción, hay que saber que el término testigo difiere conceptualmente en el derecho marroquí (inspirados en el Derecho islámico), pues los testigos instrumentales (‫ )شهود العقد‬han de ser dos hombres o un hombre y dos mujeres, mientras que en el Derecho español no se establece esta distinción por razón de sexo. Por ello, en el momento de presentar el acta ante la administración marroquí, será requerida la presencia y declaración de dos testigos varones, que se adjuntará al expediente administrativo correspondiente, pues su presencia “es indispensable ya que se trata de una exigencia ad solemnitatem y no de una simple cuestión formal”75 para que el acto 75 BLÁZQUEZ RODRÍGUEZ, Irene, op. cit. p. 171. Soto Aranda, B.; Kabbour, R. Anaquel estud. árabes 31 2020: 179-205 200 tenga validez jurídica . En el segundo caso, se ha optado por una traducción funcional, “‫”بالطريقة االستثنائية المنصوص عليها‬, para desambiguar el significado. Texto original: INTERVIENEN: En su propio nombre e interés. --------------------------------------JUICIO DE CAPACIDAD: A los efectos prevenidos en el artículo 98.1 de la Ley 24/2001 de 27 de diciembre de Medidas Fiscales, Administrativas y de Orden Social, yo, el Notario, hago constar, manifestando ambos contrayentes que ninguno de ellos está afectado por anomalías o deficiencias psíquicas o sensoriales que afectan a sus capacidades cognitivas, volitivas o sensoriales necesarias para prestar el consentimiento matrimonial, que tienen, a mi juicio, bajo mi fe y responsabilidad, en el concepto en que intervienen, capacidad legal, legitimación y facultades suficientes para otorgar la presente ESCRITURA DE CELEBRACIÓN ----------------------------------------- Propuesta de traducción: -------------------------------------------------------- .‫ باسمهم ولحسابهم الخاص‬:‫وحيث يتصرفون‬ ---‫ ديسمبر‬27 ‫ الصادر بتاريخ‬،24/2001 ‫ من القانون رقم‬98.1 ‫ طبقا ً ألحكام المادة‬:‫تقييم األهلية القانونية‬ ‫ تحت مسؤوليتنا وبنا ًء على تصريح‬،‫ نحن الموثق‬،‫ نشهد‬،‫ واإلدارية واالجتماعية‬،‫بشأن التدابير الضريبية‬ ،‫ على أنهما ال يعانيان من أي تشوهات أو عاهات نفسية أو حسية قد تؤثر على قدراتهما اإلدراكية‬،‫الزوجين‬ ‫ يتمتعان باألهلية‬،‫ بصفتهما المذكورة‬،‫واإلرادية والحسية الضرورية إلصدار اإليجاب والقبول وأنهما‬ --------------------------- ،‫ ولهذا الغرض‬،‫ والمشروعية والصالحيات الكافية إلبرام هذا العقد‬،‫القانونية‬ El término `intervención´ refiere a la calidad en la que intervienen las partes en el otorgamiento de la escritura; se traduce por ‫ أهلية التصرف‬y no debe confundirse con ‫( أهلية األداء‬capacidad de ejercicio) ni con ‫( أهلية الوجوب‬capacidad de goce). Se trata de conceptos jurídicos distintos, contemplados en los códigos civiles de España y Marruecos. En cuanto al término jurídico `juicio de capacidad´, se ha traducido por una perífrasis, al no existir un término equivalente funcionalmente en la cultura meta. Todo notario —sea de Marruecos o cualquier otro país— está obligado a asegurarse de la capacidad de actuación, ‫أهلية التصرف‬, de las personas que comparecen ante ellos, aunque en Marruecos no se incluya en este tipo de documentos un título explícito bajo el que el notario tiene que asegurar que según su criterio los intervinientes tienen esta capacidad. En cuanto al último término jurídico, consentimiento matrimonial, se ha traducido por un fraseologismo característico del lenguaje notarial marroquí, ‫صدور اإليجاب والقبول‬. 4.3. Exposición Texto original: ---------------------------------------- EXPONEN: ---------------------------------------I.- Que DON * y DOÑA *, han tramitado expediente matrimonial previa acreditación de la concurrencia de los requisitos legales necesarios para contraer matrimonio, en el Registro Civil Único de Madrid, y autorizado por Auto*, que es firme, del Magistrado Encargado del citado Registro Civil Único de Madrid, el día *, donde Soto Aranda, B.; Kabbour, R. Anaquel estud. árabes 31 2020: 179-205 201 resultó acreditada la concurrencia de los requisitos legales de capacidad para contraer matrimonio, así como la ausencia de impedimentos, su dispensa en su caso, o cualquier otro obstáculo para la celebración del matrimonio proyectado.Me exhiben testimonio del auto que autoriza el matrimonio, que incorporo a la presente matriz. -----------------------------------------------------------------------------II.- Que el expediente matrimonial se encuentra vigente a la fecha del otorgamiento de la presente escritura. -----------------------------------------------------------------III.- Que DON * y DOÑA * se ratifican en todo lo manifestado y acreditado en dicho expediente, Y MANIFIESTAN que, después de concluido el mismo, no ha sobrevenido causa ni circunstancia alguna modificativa de todo lo declarado. ----IV.- Los testigos instrumentales MANIFIESTAN, bajo su responsabilidad, que conocen a los contrayentes, que les consta su libertad de estado y la aptitud legal de los mismos para celebrar el matrimonio proyectado. ------------------------------V.- Que DON * y DOÑA *, MANIFIESTAN expresa y libremente ante los testigos instrumentales, su VOLUNTAD de celebrar el matrimonio proyectado, con arreglo a lo dispuesto en la ley española. ---------------------------------------VI.- Y expuesto cuanto antecede, de conformidad con lo dispuesto en el artículo 58 del Código Civil y 52 de la ley del Notarial. ---------------------------------------- Propuesta de traducción: -------------------------------------------- ‫ يصرحان‬-------------------------------------------‫ أنجزا ملف مستندات الزواج بعد اإلدالء بما يثبت استيفاءهما للشروط القانونية‬،* ‫ أن السيد * والسيدة‬-.1 ‫ الذي أصبح‬،* ‫ المأذون به بموجب القرار‬،‫ في السجل المدني الوحيد في مدريد‬،‫الضرورية إلبرام الزواج‬ ‫ والمثبت الستيفاء‬،* ‫ بتاريخ‬،‫ الصادر عن القاضي المكلف بالسجل المدني الوحيد في مدريد‬،‫غير قابل للطعن‬ ‫ أو االستثناء من أحد تلك الموانع إن‬،‫ وكذا انتفاء موانعه‬،‫الشروط القانونية المتعلقة باألهلية إلبرام الزواج‬ ---------------------------------- .‫ وعدم وجود أي عائق آخر إلبرام الزواج موضوع هذا العقد‬،‫وجدت‬ ------------------ .‫ الذي نرفقه بأصل العقد‬،‫وأدلى الزوجان بنسخة مصادق عليها من قرار اإلذن بالزواج‬ ------------------------- .‫ أن ملف مستندات الزواج ما زال ساري المفعول عند تاريخ تلقي هذا العقد‬-.2 ‫ بعد االنتهاء من‬،‫ ويصرحان أنه‬،‫ أن السيد * والسيدة * أكدا على تصريحاتهما الثابتة في الملف المذكور‬-.3 ------------------------------------ .‫ لم يقع أي سبب أو ظرف يؤدي إلى تعديل ما صرحا به‬،‫إجراءاته‬ ‫ وأنهما على علم بعدم ارتباطهما‬،‫ أنهما يعرفان الزوجين‬،‫ تحت مسؤوليتهما‬،‫ أن شاهدي العقد يصرحان‬-.4 ------------- .‫بآصرة زواج أخرى في الوقت الحالي وبأهليتهما القانونية إلبرام الزواج موضوع هذا العقد‬ ‫ على‬،‫ عن رغبتهما في الزواج‬،‫ صراحة وبكل حرية أمام شاهدي العقد‬،‫ أن السيد * والسيدة * يعبران‬-.5 ---------------------------------------------------------------------- .‫ضوء أحكام القانون اإلسباني‬ -------- .‫ من قانون التوثيق‬25 ‫ من القانون المدني والمادة‬85 ‫ وطبقا ً ألحكام المادة‬،‫ وبنا ًء على ما سبق‬-.6 En este apartado se observan una serie de términos jurídicos como `expediente matrimonial´, `Registro Civil Único´, `Auto´, `Magistrado Encargado del Registro Civil´, `capacidad para contraer matrimonio´, `impedimentos´, `dispensa´, `Exhibición´, `testimonio de auto´, `ratificación´, `libertad de estado´ y `aptitud legal´, que varían en su clasificación entre administrativos y jurídicos, y en el grado de equivalencia, total o parcial, en la lengua meta. Asimismo, aparecen algunos términos ambiguos, como es el caso de testimonio de auto y libertad de estado, que se refieren a “una copia de la sentencia que ha sido sellada y autenticada por el Letrado de la Administración de Justicia” y a “si el/la contrayente es soltero/a, divorciado/a o viudo/a”, respectivamente. Como excepción, hemos optado por traducir literalmente el término “Registro Civil Único” literalmente por ‫السجل المدني الوحيد‬, en vez de utilizar el término usado en Marruecos, ‫مكتب الحالة المدنية‬, para evitar una posible confusión. Soto Aranda, B.; Kabbour, R. Anaquel estud. árabes 31 2020: 179-205 202 Otro término que puede plantear dudas es el de dispensa. Según el DRAE, se entiende como “excepción graciosa de lo ordenado por las leyes generales”. El artículo 48 del Código Civil español establece que: el Juez podrá dispensar, con justa causa y a instancia de parte, mediante resolución previa dictada en expediente de jurisdicción voluntaria, los impedimentos de muerte dolosa del cónyuge o persona con la que hubiera estado unida por análoga relación de afectividad a la conyugal y de parentesco de grado tercero entre colaterales. […]. Al entender que la dispensa es una situación excepcional en la que se autoriza judicialmente la celebración de matrimonio, a pesar de la existencia de un impedimento para ello, se ha optado por traducirlo mediante la paráfrasis ‫االستثناء من أحد تلك‬ ‫ الموانع إن وجدت‬que da cuenta del sentido del término. Por su parte, el término `Auto´ puede plantear un problema de traducción, porque a pesar de ser un término jurídico, aquí no tiene aquí el significado de ‫ قرار‬o ‫حكم‬, sino más bien el sentido de ‫إذن‬, que es el término utilizado en las actas adulares marroquíes. 4.4. Estipulaciones y otorgamiento Texto original: ---------------------------------------- OTORGAN ---------------------------------------PRIMERO.- Yo, Notario procedo en alta voz a la lectura de los artículos 66, 67 y 68 del Código Civil que dicen lo siguiente: ---------------------------------------------“Artículo 66: --------------------------------------------------------------------------------Los cónyuges son iguales en derechos y deberes. -------------------------------------Artículo 67: ---------------------------------------------------------------------------------Los cónyuges deben respetarse y ayudarse MUTUAMENTE y actuar en interés de la familia. ------------------------------------------------------------------------------------Artículo 68: ---------------------------------------------------------------------------------Los cónyuges están obligados a vivir juntos, guardarse fidelidad y socorrerse mutuamente. Deberán, además, compartir las responsabilidades domésticas y el cuidado y atención de ascendientes y descendientes y otras personas dependientes a su cargo.” ------------------------------------------------------------------------------------SEGUNDO.- Preguntados por mí, el Notario, a cada uno de los contrayentes si consiente en contraer matrimonio con el otro y si efectivamente lo contraen en este acto, contestan ambos AFIRMATIVAMENTE. ----------------------------------------Así lo dicen y otorgan los comparecientes, habiendo realizado yo, Notario, las reservas y advertencias legales. ----------------------------------------------------------- Propuesta de traducción: ----------------------------------------- :‫ يتفقان على ما يلي‬------------------------------------------ :‫ ونصها‬،‫ من القانون المدني بصوت مرتفع‬86‫ و‬76 ،66 ‫ في تالوة المواد‬،‫ نحن الموثق‬،‫ بدأنا‬-.ً‫أوال‬ ----------------------------------------------------------------------------------------- :66 ‫«المادة‬ ----------------------------------------------------------- .‫يتساوى الزوجان في الحقوق والواجبات‬ --------------------------------------------------------------------------------------- :76 ‫المادة‬ Soto Aranda, B.; Kabbour, R. Anaquel estud. árabes 31 2020: 179-205 203 ----------------- .‫يجب على الزوجين احترام ومساعدة بعضهما البعض والعمل من أجل مصلحة األسرة‬ --------------------------------------------------------------------------------------- :68 ‫المادة‬ ‫ ويجب عليهما أيضا ً تقاسم‬.‫ ويخلصا ويقدما المساعدة لبعضهما البعض‬،ً‫يتعين على الزوجين أن يعيشا معا‬ ‫المسؤوليات المنزلية والرعاية واالهتمام باألصول والفروع وغيرهم من األشخاص الذين تقع مسؤولية‬ ---------------------------------------------------------------------------- .»‫إعالتهم على عاتقهما‬ ،‫ كل واحد من الزوجين إذا كان موافقا ً على إبرام هذا الزواج وفي مجلس هذا العقد‬،‫ نحن الموثق‬،‫ سألنا‬-.ً‫ثانيا‬ --------------------------------------------------------------------- .‫فصدر منهما اإليجاب والقبول‬ .‫ بعد إشعارهما بالتحفظات والتحذيرات القانونية ذات الصلة‬،‫ نحن الموثق‬،‫وتلك إرادة الحاضرين أمامنا‬ Según el DRAE, en su tercera acepción, “otorgar” significa “Disponer, establecer, ofrecer, estipular o prometer algo […] por lo común cuando interviene solemnemente la fe notarial”. Al no encontrar una equivalencia funcional en la lengua meta, se ha optado por traducirlo teniendo en cuenta el significado de “acordar y estipular”, un concepto usado en contratos árabes, ‫اتفقا على ما يلي‬. A nivel fraseológico, se ha optado por una traducción funcional para las expresiones “así lo dicen y otorgan” y “reservas y advertencias legales”, esto es, ‫ وتلك إرادة الحاضرين أمامنا‬y‫التحفظات والتحذيرات‬ ‫القانونية‬, respectivamente. 4.5. Autorización Texto original: ------------------------------------ AUTORIZACIÓN -----------------------------------Después de haber escuchado claramente a ambos contrayentes su expresa y libre voluntad, y su determinación para contraer matrimonio ante mí, Notario, y en presencia de los testigos, manifestando que consienten y efectivamente contraen matrimonio con el otro en este acto, yo, Notario, de acuerdo con las potestades que me han sido conferidas por el Ordenamiento Jurídico DECLARO UNIDOS EN MATRIMONIO a DON * y DOÑA *. --------------------------------------------------SOLICITAN AMBOS COMPARECIENTES del Señor Encargado del Registro Civil la práctica de la inscripción correspondiente, autorizando la presentación de la presente escritura en el Registro Civil por ellos mismos o por un tercero. -------------------------------------Yo, el notario, remitiré una vez firmada esta escritura copia autorizada al Registro Civil Único d Madrid. -----Y yo, Notario, DOY FE de la identidad de los otorgantes, de que, tienen a mi juicio, en el concepto en que intervienen, capacidad legal y legitimidad necesaria para otorgar esta escritura, y de que el presente otorgamiento se adecua a la legalidad y a la voluntad debidamente informada de los otorgantes e intervinientes, prestando libremente su consentimiento. ------------------------------------------------Advierto a los señores comparecientes de su derecho a leer este instrumento por sí, y no lo usan; les realizo su íntegra lectura en alta voz; se ratifican en su total contenido; y firman conmigo. ------------------------------------------------------------------ Propuesta de traducción: -------------------------------------------- ‫ موافقة‬---------------------------------------------‫ في هذا المجلس وبحضور‬،‫ نحن الموثق‬،‫وبعد تلقي اإليجاب والقبول بشكل صريح من الزوجين أمامنا‬ ،‫ وبموجب الصالحيات التي يخولها لنا القانون‬،‫ وتعبير كل منهما عن رغبته في الزواج من اآلخر‬،‫الشاهدين‬ -------------------------------------------------- .‫نعلن أن السيد * والسيدة * مرتبطان برباط الزواج‬ Soto Aranda, B.; Kabbour, R. Anaquel estud. árabes 31 2020: 179-205 204 ‫ ويوافقان‬،‫ويلتمس الطرفان الحاضران أمامنا من المكلف بالسجل المدني إدخال القيد المناسب في السجل‬ --------------------------------‫على تقديم هذا العقد إلى السجل المدني شخصيا ً أو عن طريق طرف ثا‬ ----- .‫ نسخة مطابقة لألصل من هذا العقد إلى السجل المدني الوحيد في مدريد‬،‫ نحن الموثق‬،‫وسوف نحيل‬ ،‫ بصفتهما المذكورة أعاله‬،‫ وأنهما يتمتعان‬،‫ على تحديد هوية طرفي العقد والتحقق منها‬،‫ نحن الموثق‬،‫ونشهد‬ ‫باألهلية القانونية والمشروعية إلبرام هذا العقد وأن تلقي هذا العقد متوافق مع أحكام القانون وإرادة األطراف‬ ----------------------------------------------- .‫ المعبر عنها صراحة‬،‫المبرمة للعقد والمتدخلة في ذلك‬ ‫ فتنازال عن ممارسة هذا الحق؛‬،ً‫ الحاضرين أمامنا بحقهما في تالوة هذا العقد شخصيا‬،‫ نحن الموثق‬،‫وأشعرنا‬ ---------------------- .‫وتلونا عليهما مضمون هذا العقد بصوت مرتفع؛ فوافقا على مضمونه ووقعاه معنا‬ En este apartado, cabe mencionar la traducción de términos como `expresa y libre voluntad y determinación para contraer matrimonio´, traducidos por ‫تلقي اإليجاب والقبول‬ ‫ بشكل صريح من الزوجين‬y ‫تعبير كل منهما على رغبته في الزواج من اآلخر‬, respectivamente. Aquí resulta necesaria una paráfrasis para hacer el mensaje más legible en la lengua meta, respetando también el estilo utilizado en las actas adulares marroquíes. En el caso del término ordenamiento jurídico (se trata del conjunto de leyes que tiene un país), se entiende que el notario se refiere con ello a “la Ley”, por lo que traducido por ]...[ ‫التي يخولها القانون‬. En el caso de las expresiones DECLARO UNIDOS EN MATRIMONIO, práctica de la inscripción y copia autorizada, se han traducido por equivalencias funcionales que hacen el texto más legible en la lengua meta. La segunda parte de este apartado refiere a la Ley de Protección de Datos, y el tenor del original es el siguiente: Texto original: PROTECCIÓN DE DATOS: -----------------------------------------------------------De acuerdo con lo establecido en la Ley Orgánica 15/1999, de 13 de Diciembre, de protección de datos de carácter personal, y en el Real Decreto 1720/2007, que aprueba su Reglamento de desarrollo, los intervinientes aceptan la incorporación de sus datos y la copia del documento de identidad a los ficheros de la Notaría con la finalidad de realizar las funciones propias de la actividad notarial y efectuar las comunicaciones de datos previstas en la Ley a las Administraciones Públicas y, en su caso, al Notario que suceda al actual al actual en la plaza. Puede ejercer sus derechos de acceso, rectificación, cancelación y oposición en la notaría. En caso de que se incluyan datos de personas distintas a los intervinientes, estos deberán haberles informado, con carácter previo, del contenido de este párrafo. -----------Los comparecientes se dan por satisfactoriamente atendidos e informados por mí, Notario, sobre el contenido, efectos y consecuencias del presente instrumento. ------De todo lo contenido en este instrumento público, extendido sobre COMPOSICIÓN PAPEL TIMBRADO, DOY FE. ----- Propuesta de traducción: ----------------------------------------------------------------------------------- :‫حماية البيانات‬ ‫ والمرسوم الملكي‬،‫ المتعلق بحماية البيانات الشخصية‬،15/1999 ‫طبقا ً ألحكام القانون التنظيمي‬ ‫ يوافق الطرفان الموقعان على هذا العقد على إدراج‬،‫ الذي يصادق على نظامه التنفيذي‬،1720/2007 ‫بياناتهم الشخصية ونسخة من بطاقة تعريفهم في الملفات التي يمسكها هذا الديوان ألغراض القيام بالمهام‬ ‫ للموثق الذي‬،‫ وعند االقتضاء‬،‫الخاصة بنشاط الموثقين وتبليغ البيانات بمقتضى القانون لإلدارات العامة‬ ‫ وإلغائها‬،‫ والموافقة عليها‬،‫ ويحق لهم ممارسة حقوقهم في الولوج إلى بياناتهم‬.‫سيحل محل هذا الموثق‬ Soto Aranda, B.; Kabbour, R. Anaquel estud. árabes 31 2020: 179-205 205 ‫ وعند إدراج بيانات تخص أشخاص غير األشخاص المتدخلين في هذا‬.‫واالعتراض عليها أمام هذا الديوان‬ .‫ ينبغي اطالعهم مسبقا ً بمحتوى هذه الفقرة‬،‫العقد‬ ‫ واآلثار المترتبة‬،‫ أنهم تلقوا المعلومات الكافية حول مضمون هذا العقد‬،‫ نحن الموثق‬،‫وأكد الحاضرون أمامنا‬ .‫عنه‬ ---------------------------- .‫ وبه اإلشهاد‬.‫وحرر هذا العقد الرسمي في * صفحات من الورق المدموغ‬ El acta también incluye una mención a la protección de datos de carácter personal. Para la traducción de términos relacionados con la Ley Orgánica 15/1999, de Protección de Datos de Carácter Personal, se ha optado por llevar a cabo una labor de documentación en las leyes marroquíes, y precisamente la denominada ‫القانون رقم‬ ‫ المتعلق بحماية األشخاص الذاتيين تجاه معالجة المعطيات ذات الطابع الشخصي‬80.90, puede servir de referencia para realizar una traducción acorde con la terminología utilizada en la lengua meta. 4. Conclusiones La documentación relacionada con el derecho privado como los documentos concernientes al matrimonio, al divorcio, o la herencia, están notoriamente marcados por el contexto cultural que los produce, y su traducción es puente entre dos ordenamientos jurídicos que responden a diferentes concepciones del mundo y de las relaciones sociales76. En el caso de la traducción en la combinación lingüística árabe-español-árabe, el futuro traductor ha de tener unos conocimientos básicos de los respectivos marcos legales, prestando especial atención a las variedades diatópicas que singularizan los documentos árabes según su procedencia geográfica. Junto a ello, el traductor debe tener presente que la traducción de documentos jurídicos está destinada a surtir los mismos efectos legales que los originales, de ahí que deba cuidarse la correspondencia formal y fehaciente de los mismos. Para llevar a cabo satisfactoriamente esta tarea, requiere de una competencia intercultural, en tanto en cuanto debe trasladar el discurso a un contexto meta con una función equivalente. Desde el punto de vista didáctico, la enseñanza de la traducción de este tipo de textos debe estructurarse a partir del sistema referencial de dichos textos, los aspectos culturales, junto con sus anisomorfismos. 76 GUTIÉRREZ ARCONES, David (2015) “Estudio sobre el texto jurídico y su traducción: características de la traducción jurídica, jurada y judicial” en Miscelánea Comillas [en línea]. Nº 73, disponible en https://revistas. upcomillas.es/index.php/miscelaneacomillas/article/viewFile/5493/5303 [Último acceso el 21 de noviembre de 2019], p. 142.
https://openalex.org/W2920243230
https://europepmc.org/articles/pmc6473468?pdf=render
English
null
Role of Nitrate Reductase in NO Production in Photosynthetic Eukaryotes
Plants
2,019
cc-by
9,306
Received: 16 January 2019; Accepted: 8 February 2019; Published: 6 March 2019 Abstract: Nitric oxide is a gaseous secondary messenger that is critical for proper cell signaling and plant survival when exposed to stress. Nitric oxide (NO) synthesis in plants, under standard phototrophic oxygenic conditions, has long been a very controversial issue. A few algal strains contain NO synthase (NOS), which appears to be absent in all other algae and land plants. The experimental data have led to the hypothesis that molybdoenzyme nitrate reductase (NR) is the main enzyme responsible for NO production in most plants. Recently, NR was found to be a necessary partner in a dual system that also includes another molybdoenzyme, which was renamed NO-forming nitrite reductase (NOFNiR). This enzyme produces NO independently of the molybdenum center of NR and depends on the NR electron transport chain from NAD(P)H to heme. Under the circumstances in which NR is not present or active, the existence of another NO-forming system that is similar to the NOS system would account for NO production and NO effects. PII protein, which senses and integrates the signals of the C–N balance in the cell, likely has an important role in organizing cell responses. Here, we critically analyze these topics. Keywords: nitric oxide; nitrate reductase; NOFNiR; nitrogen metabolism Keywords: nitric oxide; nitrate reductase; NOFNiR; nitrogen metabolism Role of Nitrate Reductase in NO Production in Photosynthetic Eukaryotes Manuel Tejada-Jimenez , Angel Llamas , Aurora Galván and Emilio Fernández * Departamento de Bioquímica y Biología Molecular, Campus de Rabanales y Campus Internacional de Excelencia Agroalimentario (CeiA3), Edif. Severo Ochoa, Universidad de Córdoba, 14071 Córdoba, Spain; manuel.tejada@uco.es (M.T.-J.); bb2llaza@uco.es (A.L.); bb1gacea@uco.es (A.G.) * Correspondence: bb1feree@uco.es; Tel.: +34-957-218-591 Manuel Tejada-Jimenez , Angel Llamas , Aurora Galván and Emilio Fernández * Departamento de Bioquímica y Biología Molecular, Campus de Rabanales y Campus Internacional de Excelencia Agroalimentario (CeiA3), Edif. Severo Ochoa, Universidad de Córdoba, 14071 Córdoba, Spain; manuel.tejada@uco.es (M.T.-J.); bb2llaza@uco.es (A.L.); bb1gacea@uco.es (A.G.) * Correspondence: bb1feree@uco.es; Tel.: +34-957-218-591 Manuel Tejada-Jimenez , Angel Llamas , Aurora Galván and Emilio Fernández * Departamento de Bioquímica y Biología Molecular, Campus de Rabanales y Campus Internacional de Excelencia Agroalimentario (CeiA3), Edif. Severo Ochoa, Universidad de Córdoba, 14071 Córdoba, Spain; manuel.tejada@uco.es (M.T.-J.); bb2llaza@uco.es (A.L.); bb1gacea@uco.es (A.G.) * Correspondence: bb1feree@uco.es; Tel.: +34-957-218-591 Plants 2019, 8, 56; doi:10.3390/plants8030056 plants plants www.mdpi.com/journal/plants 1. Introduction Nitric oxide (NO) is a gaseous secondary messenger in humans, animals, plants, fungi, and bacteria. In plants, NO is involved in important physiological processes, such as growth, development, metabolism, leaf senescence, biotic and abiotic stress, defense processes, and plant–pathogen interactions, which have been extensively reviewed [1–5]. In particular, in algae, such as the green alga Chlamydomonas reinhardtii, NO also participates in fundamental cell functions, such as the regulation of N-metabolism, N- and S-starvation stress, chloroplast biogenesis, programmed cell death, and responses to darkness, hypoxia, or salt stress [6–11]. In the last two decades, it has been clarified that NO is a signaling molecule in plant defense during plant–pathogen interactions [12,13]. Since then, different strategies have been used to understand NO biosynthesis in plant cells, and this subject has not been short of controversies [5,14], with some aspects yet to be understood. Two main pathways, reductive and oxidative, appear to explain NO synthesis in plants. One is based on the reduction of nitrite, and the other involves the oxidation of aminated molecules, such as the amino acid arginine [15]. In spite of the seminal work of Foresi and collaborators, who identified the first NO synthase (NOS) from the plant kingdom in the green alga Ostreococus taurii [16], the existence of a plant NOS that has the characteristics of the animal NOS has been puzzling [14,17] since no plant genome contains such a conserved gene. In fact, Jeandrof and collaborators analyzed over 1000 species of land plants and algae and found no typical NOS sequences in the 1087 sequenced transcriptomes of land plants, www.mdpi.com/journal/plants 2 of 13 Plants 2019, 8, 56 but they did find said sequences in 15 of the 265 algal species. Thus, it was concluded that land plants had evolved a mechanism to synthesize NO in a manner that is different from that used in animals [18]. In this review, the different biosynthetic processes of nitric oxide formation are critically analyzed, together with their physiological relevance. 2. Nitrite: The Substrate for Reductive NO Production Nitrite is a product of nitrate reductase (NR)-catalyzed nitrate reduction within the nitrate assimilation pathway. Nitrogen acquisition is a fundamental process for living beings, including plants in crops, in which N is usually a limiting factor that determines crop productivity [19]. Nitrate used to be the preferred form of inorganic N that was available in soils and, thus, was used in fertilizers [20]. The incorporation of nitrogen from nitrate first requires its acquisition from the medium by specific transporters, which are responsible for the sensing, uptake, storage, and distribution of nitrate among plant tissues. Plant nitrate transporters belong to several families: the nitrate transporter 1/peptide transporter/nitrate peptide transporter family (NRT1/PTR/NPF), NRT2/nitrate nitrite porter (NRT2/NNP), chloride channels (CLC), slow anion channel-associated 1 homolog 3 (SLAC1/SLAH), and aluminum-activated malate transporters (ALMT). These have all been reviewed in detail [21–25]. In the Chlamydomonas alga, this complexity is less but still significant, reflecting the importance of this step. Here, we highlight the three families of transporter proteins found in Chlamydomonas: NRT1/NPF, NRT2, and NAR1 (Figure 1). NRT1 has been described in Arabidopsis as a dual-affinity nitrate/nitrite transporter, and NRT2 (with the accessory protein NAR2) mediates the high-affinity transport (HAT) of nitrate and nitrite. Plants 2019, 8, x FOR PEER REVIEW 3 of 13 Figure 1. The schematic model for the coordinated regulation of Nitric Oxide (NO) synthesis and N metabolism. Blue arrows indicate activation and red lines indicate inhibition by trans-nitrosylation. Dashed lines represent hypothetical steps. The NOS-like component represents the L-Arg-dependent NOS activity reported in different plant species. Figure 1. The schematic model for the coordinated regulation of Nitric Oxide (NO) synthesis and N metabolism. Blue arrows indicate activation and red lines indicate inhibition by trans-nitrosylation. Dashed lines represent hypothetical steps. The NOS-like component represents the L-Arg-dependent NOS activity reported in different plant species. Figure 1. The schematic model for the coordinated regulation of Nitric Oxide (NO) synthesis and N metabolism. Blue arrows indicate activation and red lines indicate inhibition by trans-nitrosylation. Dashed lines represent hypothetical steps. The NOS-like component represents the L-Arg-dependent NOS activity reported in different plant species. Figure 1. The schematic model for the coordinated regulation of Nitric Oxide (NO) synthesis and N metabolism. Blue arrows indicate activation and red lines indicate inhibition by trans-nitrosylation. Dashed lines represent hypothetical steps. The NOS-like component represents the L-Arg-dependent NOS activity reported in different plant species. Figure 1. 2. Nitrite: The Substrate for Reductive NO Production The schematic model for the coordinated regulation of Nitric Oxide (NO) synthesis and N metabolism. Blue arrows indicate activation and red lines indicate inhibition by trans-nitrosylation. Dashed lines represent hypothetical steps. The NOS-like component represents the L-Arg-dependent NOS activity reported in different plant species. Figure 1. The schematic model for the coordinated regulation of Nitric Oxide (NO) synthesis and N metabolism. Blue arrows indicate activation and red lines indicate inhibition by trans-nitrosylation. Dashed lines represent hypothetical steps. The NOS-like component represents the L-Arg-dependent NOS activity reported in different plant species. 3. Nitrate Reductase is a Multidomain Protein NR reduces nitrate to nitrite using electrons from NAD(P)H. The plant enzyme is about 200 KDa and contains two subunits, each bearing three prosthetic groups: FAD, heme b557, and molybdenum. In an NR subunit, molybdenum is bound to a tricyclic pyranopterin and chelated by a dithiolene, which is named the molybdenum cofactor (Moco). These domains are joined by two Nitrite in the cytosol, either produced from nitrate or absorbed from the medium, has to be transported to the chloroplast by a HAT system. In Chlamydomonas, HAT of nitrite is facilitated by NAR1, which belongs to the FNT family and is absent in land plants. In higher plants, nitrite transport to chloroplasts is typically mediated by members of the CLC family [26], and it is exported from the chloroplast by a transporter from the NRT1 family, as is found in cucumber, 3 of 13 Plants 2019, 8, 56 Cucumis sativus [27,28]. Regardless, nitrite concentrations in the cytosol are maintained at very low levels (micromolar range) [29] to prevent nitrite toxicity in the cell [30]. Once in the chloroplast, nitrite is reduced to ammonium in a reaction catalyzed by nitrite reductase (NiR). All of these genes (NRT2/NAR2/NR/NAR1/NiR) in Chlamydomonas are controlled by the master regulatory gene for nitrate assimilation: NIT2 [31]. Orthologous regulatory genes in land plants—NLP genes—show a similar structural organization and signaling for nitrate [32–34]. Finally, ammonium is incorporated into C-skeletons in the form of glutamate by the glutamine synthetase/glutamate synthase (GS/GOGAT) cycle [35] (Figure 1). It is important to point out here that in contrast to the low cytosolic concentrations of nitrite, those of nitrate are high (1–6 mM). Cytosolic nitrate levels are also more stably maintained than vacuolar concentrations (5–75 mM), considering that external nitrate concentrations may change by about 10,000-fold [36–38]. 3. Nitrate Reductase Is a Multidomain Protein NR reduces nitrate to nitrite using electrons from NAD(P)H. The plant enzyme is about 200 KDa and contains two subunits, each bearing three prosthetic groups: FAD, heme b557, and molybdenum. In an NR subunit, molybdenum is bound to a tricyclic pyranopterin and chelated by a dithiolene, which is named the molybdenum cofactor (Moco). These domains are joined by two protease-sensitive hinge regions. The domains are redox centers, and electrons flow from NAD(P)H→FAD→heme →Moco, which is within the active site for nitrate reduction [43,44]. Interestingly, the enzyme has two partial activities, which can be assayed in vitro: diaphorase, which catalyzes the reduction of artificial acceptors (ferricyanide or cytochrome c with NAD(P)H), and terminal-NR, which catalyzes nitrate reduction using electrons supplied by FAD, FMN, viologens, or bromophenol blue, chemically reduced by dithionite [43,44]. The crystal structure of the dimerized form of NR and Moco domains was solved [45]. NR reduces nitrate to nitrite using electrons from NAD(P)H. The plant enzyme is about 200 KDa and contains two subunits, each bearing three prosthetic groups: FAD, heme b557, and molybdenum. In an NR subunit, molybdenum is bound to a tricyclic pyranopterin and chelated by a dithiolene, which is named the molybdenum cofactor (Moco). These domains are joined by two protease-sensitive hinge regions. The domains are redox centers, and electrons flow from NAD(P)H→FAD→heme →Moco, which is within the active site for nitrate reduction [43,44]. Interestingly, the enzyme has two partial activities, which can be assayed in vitro: diaphorase, which catalyzes the reduction of artificial acceptors (ferricyanide or cytochrome c with NAD(P)H), and terminal-NR, which catalyzes nitrate reduction using electrons supplied by FAD, FMN, viologens, or bromophenol blue, chemically reduced by dithionite [43,44]. The crystal structure of the dimerized form of NR and Moco domains was solved [45]. →Moco, which is within the active site for nitrate reduction [43,44]. Interestingly, the enzyme has two partial activities, which can be assayed in vitro: diaphorase, which catalyzes the reduction of artificial acceptors (ferricyanide or cytochrome c with NAD(P)H), and terminal-NR, which catalyzes nitrate reduction using electrons supplied by FAD, FMN, viologens, or bromophenol blue, chemically reduced by dithionite [43,44]. The crystal structure of the dimerized form of NR and Moco domains was solved [45]. 2. Nitrite: The Substrate for Reductive NO Production This is important for ensuring efficient nitrate assimilation, together with proper nitrate signaling in the tissues [38,39]. Nitrate homeostasis is the result of the membrane transporter-mediated supply of nitrate from vacuoles and the outer medium, as well as by nitrate efflux transporters such as NAXT1 [40]. NAXT1 belongs to the NRT1 family. In addition, the NR-catalyzed reaction has an effect, facilitating the conversion of nitrate into nitrite [37,38,41]. In the yeast Hansenula polymorpha, the sulfite transporter SSU2 and the nitrite transporter NAR1 have been characterized as essential components of the nitrate/nitrite efflux system [42]. 4. Does NR Catalyze Nitrite Reduction to NO? In agreement with this, Moco-deficient mutants are deficient in molybdoenzymes, as described in barley with a thermo-sensitive, wilty phenotype [53]. The most used Arabidopsis NR mutant to support the involvement of NR in NO production is the double mutant deficient in NIA1 and NIA2 genes. This nia1/nia2 double mutant has only 0.5% of the activity of wild-type NR and grows very poorly on medium with nitrate as the only nitrogen source [54]. As described below, NR is in fact involved in NO production but not through its Moco-dependent activity. All five molybdoenzymes in plants (nitrate reductase, xanthine oxidase reductase (XOR), aldehyde oxidase (AO), sulfite oxidase (SO), and mARC) are able to catalyze the one-electron reduction of nitrite to NO. The molybdoenzymes are classified depending on how Moco binds to the enzyme’s active site: either covalently through an enzyme cysteine thiol group (NR, SO, and mARC) or with inorganic sulfur (XOR and AO) [55,56]. All of these enzymes show nitrite reductase activity to produce NO in vitro and in anaerobic conditions [44]. The four known human molybdenum-containing enzymes are the same as those in plants, except for NR, and they can also function as nitrite reductases under hypoxic conditions [56]. In mammals, two known pathways for NO formation are known: arginine oxidation under normoxic or aerobic conditions and nitrite reduction during hypoxia or anaerobiosis [56]. Plant SO seems to have a less potent nitrite reductase activity than human XOR and AO [57]. Whereas plant AO participates in the synthesis of phytohormones and contributes to reactive oxygen species (ROS) production, there is no information about its in vivo NO-producing activity [5]. Other processes that might produce NO from nitrite are associated with the plasma membrane-bound NR, which appears to be related to the mycorrhizal colonization of tobacco roots [58] and the mitochondrial electron transport chain (mETC), as demonstrated in several plants [59–62]. The mitochondrial complex III and IV are primarily implicated in the nitrite reaction (Km of 175 µM), which requires anaerobic conditions since oxygen is a strong inhibitor. Therefore, this reaction can occur in plant tissues exposed to hypoxia, such as roots, and its occurrence might be important to the plant by protecting the respiratory chain and mitochondrial metabolism when oxygen is lacking [63]. Recently, a role for Alternative Oxidase (AOX) in the production of large amounts of NO, observed under hypoxia, has been shown. 4. Does NR Catalyze Nitrite Reduction to NO? The experimental data have led to the proposal that the molybdoenzyme NR is the main enzyme responsible for NO production in most plants. This proposal was based on the experimental findings described below. The first evidence linking NR and NO production was their co-elution by NADH from Blue Sepharose columns loaded with Soybean extracts with both NR and NO(X) evolution activities. In their main conclusion, the authors inferred their linkage from the fact that inhibiting the partial activities of NR and NO(X) evolution activities led to the same pattern. However, they went further by indicating that the terminal molybdenum-containing portion of NR is involved in the reduction of nitrite to NO(X) [46]. Other studies showed that several plant species emitted in vivo NO when there was nitrate in the soil, and the function was abolished in all plants in the study when they were grown on ammonium-containing soil, indicating a role for NR [47]. In addition, isotopically labeled 15N-nitrate resulted in the emission of 15NO [48]. Moreover, using NR from corn led to the production of significant amounts of NO from both nitrite and nitrate [49]. Those findings reinforced the idea that NR reduces nitrate to nitrite and further converts de novo-generated nitrite into NO. The proposal would be feasible if it could account for several facts. First, the nitrite-reducing activity of NR is very low (only 1% of the nitrate-reducing activity), and the Km of nitrite is about 10 times higher than that of nitrate, in agreement with the competitive character of nitrite (Ki = 50 µM) in nitrate reduction [50,51]. Plants 2019, 8, 56 4 of 13 Taking into consideration the intracellular concentrations of nitrite (in the micromolar range) and nitrate (in the millimolar range), this nitrite-reducing activity would be irrelevant. Another piece of evidence indicating the involvement of NR in NO production originated from the use of tungstate—an NR inhibitor—and NR-deficient mutants. In addition to its uncontrolled effects, such as metal toxicity, tungstate is a very unspecific inhibitor of Mo-enzymes. When exchanging Moco for the inactive tungsten cofactor (Wco) [52], all molybdoenzymes are inactivated, including mitochondrial amidoxime-reducing component (mARC), which is presented below as the most important enzyme in NO production under phototrophic conditions. Thus, tungstate is a clear inhibitor of NO production because of the resulting mARC inhibition and not because of NR inhibition. 4. Does NR Catalyze Nitrite Reduction to NO? In this pathway of NO production, AOX has a role in scavenging the NO and ROS linked to the hemoglobin–NO cycle, thus increasing energy efficiency without contributing to the formation of toxic peroxynitrite [64]. The implication of mitochondria in NO production from nitrite has also been shown in Chlamydomonas in the presence of high concentrations of nitrite [65]. In light of the biological importance of NO production, one would expect this process to be efficiently and finely regulated. Some of the Moco-dependent enzymes described above can mediate NO production under certain conditions, though it is difficult to currently envisage how to control this diversity of processes; even mitochondrial NO production requires anaerobiosis, suggesting a burst of NO synthesis from nitrite when this condition appears. 5. NR Does Not Catalyze In Vivo Nitrite Reduction to NO but Provides the Needed Electrons Recently, NR was shown to be a necessary partner for NO production in a dual system, which, besides NR, includes another molybdoenzyme, mARC, since renamed NO-forming nitrite reductase (NOFNiR). 5 of 13 Plants 2019, 8, 56 Plants 2019, 8, 56 mARC has been extensively characterized in prokaryotic and eukaryotic organisms [66–69]. mARC proteins are about 35 KDa and require two electron transport proteins—NADH-cytochrome b5 reductase and cytochrome b5—to be functional. mARC is a very efficient reductase [70] for a number of N-oxygenated compounds, some of which are toxic or mutagenic [71–73]. This is why mARC has been related to cell detoxification processes. Both human mARC isoforms are associated with mitochondria, but mARC could also be located in peroxisomes because its two partners, NADH-cytochrome b5 reductase and cytochrome b5, were found in this organelle using a proteomic approach [74]. mARC was also connected to NO metabolism because of its activity on Nω-hydroxy-Nδ-methyl-L-arginine, an intermediate in the L-arginine-dependent biosynthesis of NO using NADH-cytochrome b5 reductase and cytochrome b5 [75]. In addition, human mARCs have nitric oxide synthase activity from nitrite with NADH and its two above-indicated partners [76]. In the eukaryotic alga Chlamydomonas, NO synthesis is carried out by a dual system comprising NR and NOFNiR (mARC). These two components are closely connected at both the transcriptional and activity levels, so mutants lacking one of them overexpress the other [77]. NR supplies NADH electrons to NOFNiR for the reduction of nitrite to NO much more efficiently than NADH-cytochrome b5 reductase and cytochrome b5. Both NR and NOFNiR are located in the cytosol [77]. NR mutants were found to be unable to provide electrons both in vitro and in vivo for NO synthesis. Some exceptions are NR mutants such as Chlamydomonas strain 301, whose NR lacks nitrate-reducing activity since its Moco domain is affected; however, it has functional diaphorase activity with intact FAD and heme domains [77]. So, the dual complex NR:NOFNiR produces NO independently of the molybdenum center of NR and depends on the NR electron transport chain from NAD(P)H to heme. NR:NOFNiR has been proposed to be the main system producing NO during standard phototrophic, oxygenic plant growth, which corresponds to most of its aerial part [77]. In tissues exposed to hypoxia, such as roots, other molybdoenzymes or mETCs could be involved in the synthesis of NO, as discussed above. 6. NO Levels in the Cells Are Regulated The first line of control of NO levels is the regulation of its synthesis and degradation within the NO cycle (Figure 1). Members of the hemoglobin (HB) superfamily can oxygenate NO to nitrate, as was shown for Chlamydomonas THB1 [78], a class 3 truncated hemoglobin (THB) [79]. In different plant species, nitrate, nitrite, and NO upregulate HB expression [80,81]. In maize roots, the coordinated expression of both NR and HB also occurs [82]. Similarly, in Chlamydomonas, the expression of two truncated HBs, THB1 and THB2, respond selectively to N signals (nitrate, nitrite, and NO) and, interestingly, also to NIT2, the major regulatory gene of the nitrate assimilation pathway [78,83]. This regulatory gene is also essential for NR upregulation by nitrate [19]. The activity of THB1 requires electrons to be supplied by the NADH-diaphorase of NR, and the electron flow is likely from NADH to FAD [78]. Similarly, the activity of NOFNiR also requires the NADH-diaphorase of NR, but the electron flow is now from NADH to heme b [77]. Thus, nitrate through NIT2 would stimulate NO production because of NR’s increased expression (NOFNiR is not under NIT2 control), and in turn, NO degradation would occur as a result of stimulating both THB1 and NR. The homeostasis of NO is controlled by the activities of NR, NOFNiR, THB1, and THB2, which, in turn, depend on the relative concentrations of nitrate, nitrite, and NO, as well as NIT2. , , , So, just-synthesized NO, which is highly reactive, can react with different targets. Glutathione (γ-glutamylcysteinylglycine, GSH) is an essential metabolite in plants that participates in important functions, such as primary metabolism, redox signaling, and defense and detoxification processes [84]. GSH can react with NO to produce S-nitrosylated glutathione (GSNO). As a result, the half-life of NO in tissues available as a free radical gas changes from seconds to a few minutes. Thus, there are very sensitive mechanisms for regulating cellular processes. GSNO, which is considered the main reservoir for NO, provides NO signals to proteins. Protein S-nitrosation is considered the most important mode So, just-synthesized NO, which is highly reactive, can react with different targets. Glutathione (γ-glutamylcysteinylglycine, GSH) is an essential metabolite in plants that participates in important functions, such as primary metabolism, redox signaling, and defense and detoxification processes [84]. GSH can react with NO to produce S-nitrosylated glutathione (GSNO). 6. NO Levels in the Cells Are Regulated As a result, the half-life of NO in tissues available as a free radical gas changes from seconds to a few minutes. Thus, there are very So, just-synthesized NO, which is highly reactive, can react with different targets. Glutathione (γ-glutamylcysteinylglycine, GSH) is an essential metabolite in plants that participates in important functions, such as primary metabolism, redox signaling, and defense and detoxification processes [84]. GSH can react with NO to produce S-nitrosylated glutathione (GSNO). As a result, the half-life of NO in tissues available as a free radical gas changes from seconds to a few minutes. Thus, there are very sensitive mechanisms for regulating cellular processes. GSNO, which is considered the main reservoir for NO, provides NO signals to proteins. Protein S-nitrosation is considered the most important mode 6 of 13 Plants 2019, 8, 56 of action of NO. This is the covalent binding of NO to the thiol group of protein cysteine residues, and the creation of these bonds modifies the protein and can alter gene expression and/or lead to metabolic changes, all of which ultimately translate into physiological responses. Thousands of nitrosation sites have been identified in proteins [85]. GSNO is metabolized with GSNO reductase (GSNOR1) to convert it to glutathione disulfide (GSSG) and ammonia (NH3) [86,87]. GSNOR1 is a cytosolic enzyme that controls GSNO levels and, in this way, the nitrosation of proteins. GSNOR1 seems to be inhibited by NO, in which case, the scavenging of GSNO is prevented. Thus, NO controls its production and scavenging by regulating both nitrate assimilation efficiency and GSNOR1 activity [87,88] (Figure 1). Other posttranslational modifications of proteins mediated by NO are the nitration of tyrosine and amine groups and the oxidation of thiols and tyrosine [89]. NO can react with superoxide (O2−) to form peroxinitrite (ONOO−), which is a powerful oxidant contributing to the nitration of protein tyrosine residues to form 3-nitrotyrosine, which results in regulatory responses. Nitration seems to be a reversible process that might occur at specific tyrosine residues depending on the local environment and the secondary and tertiary structure of the protein. A putative specific denitrase removes the nitro group without degrading the protein, as has been shown in animal systems [89,90]. 6. NO Levels in the Cells Are Regulated Reactive oxygen species (ROS), such as oxygen, singlet oxygen, hydroxyl radical, hydrogen peroxide, and superoxide anion, all of which are important signaling compounds produced under several environmental conditions, interact with NO and other reactive nitrogen species (RNS). They mediate the responses to different environmental situations, even promoting the systemic adaptation of plants to stress situations [2,91,92]. 7. The NO Synthesis Systems Are Coordinated with Nitrogen Metabolism Under circumstances in which NR is not present or active, another NO-forming system accounts for NO production and NO effects. Analysis of this topic and possible future directions are presented here. With an ammonium medium as the sole N-source or in null NR-deficient mutants, NR is absent, and thus, the dual system NR–NOFNiR is not functional. Nevertheless, NO is being synthesized, probably due to the existence of a NOS-like activity in algae and plants. This activity can be inhibited by some compounds, which are primarily arginine analogs acting on the animal-type NOS [6,17]. Plant peroxisomes are single-membrane-bound organelles with an oxidative metabolism and a simple morphology, but they also have a complex composition of enzymes involved in the metabolism of oxygen free-radicals. Peroxisomes can generate ROS and nitric oxide and thus important signal molecules with implications for cellular metabolism in plants [91,93]. Many studies of different plant species have shown the presence of L-Arg-dependent NO synthase-like enzyme activity, which has biochemical requirements similar to animal NOS (L-Arg, NADPH, FMN, FAD, Calmodulin, and Ca2+) [94,95]. Corpas and Barroso (2017) postulated that the L-Arg-dependent NO synthesis that occurs in plants could correspond to cooperation among discrete proteins, resulting in the formation of a protein complex with requirements for enzyme activity that are similar to animal NOS [96]. This would explain the lack of success in finding canonical NOS proteins at the molecular level. On the other hand, coordination between nitrogen assimilation and the nutritional status within plant metabolism is a critical issue for plant viability. A very abundant family of N- and C-signaling proteins, widely distributed in nature, is the PII protein family from bacteria, archaea, and plants [97]. In plants, these nuclear-encoded PII proteins localize in the chloroplast and are not subject to the covalent modification reported for bacterial PII [98]. PII senses and integrates the signals of the C–N balance in the cells using 2-ketoglutarate as an indicator, together with the energy status by competitive ADP/ATP binding [99,100]. Plant PII proteins are conserved throughout the evolutionary history of the Chloroplastida—green algae and land plants [97]—and participate in a complex signal-transduction network that mediates nitrogen regulation [101]. In Arabidopsis, the PII protein controls arginine biosynthesis [102], and PII mutants show an enhanced nitrite uptake by the 7 of 13 Plants 2019, 8, 56 chloroplast [103]. 7. The NO Synthesis Systems Are Coordinated with Nitrogen Metabolism By binding effector molecules, PII interacts with and signals to other chloroplast proteins, such as N-acetyl-L-glutamate kinase (NAGK), which catalyzes the committed step in the arginine biosynthesis pathway [102] (Figure 1). Among the effectors binding plant PII protein is glutamine, which was shown to bind to the termed Q-loop of the C-terminus crystalized plant PII protein [104]. The binding of glutamine to PII changes its conformation to one that interacts and activates NAGK for the synthesis of arginine and polyamines. PII–glutamine sensing is a widespread mechanism in the plant kingdom [104]. In Chlamydomonas, arginine is a component of the same repressive pathway as ammonium and CYG56, which is a guanylate cyclase activated by NO and needed to repress nitrogen assimilation genes [105]. Interestingly, plant PII seems to be connected to the regulation of nitrite metabolism, so PII knockout mutants show an increased sensitivity to nitrite and a decrease in total amino acids, especially glutamine. Lack of PII seems to increase the C/N balance, as previously shown in cyanobacteria [30]. Recently, Chlamydomonas PII levels have been shown to be tightly controlled by the nitrogen source and the physiological status of the cells [106]. In fact, PII expression is subject to positive (nitrate and nitrite) signaling and is downregulated by ammonium via an NO-mediated process that involves an NO-dependent guanylate cyclase, similar to the negative effect of ammonium on NR expression [6]. PII expression is very similar to that of nitrate assimilation genes [25], so an interaction/coordination between PII and nitrate assimilation pathways has been suggested [106]. Under standard phototropic conditions, nitrate assimilation takes place under the positive control of nitrate, mediated by the regulatory gene NIT2 [25]. When both nitrate and ammonium are present, there is a balance between the positive and negative signals, and NR expression follows that balance; the NR transcript is detectable even in the presence of ammonium, provided that nitrate is also present [107]. The negative signal of ammonium on Chlamydomonas NIA1 gene expression depends on NO and its mediation by an NO-dependent guanylate cyclase (CYG56). So, NO concentrations in the cells increase with ammonium concentration, leading to complete NR repression [107] by means of a mechanism that is dependent on a possible NOS, which is inhibited by L-NAME. In different plants, NO production is also sensitive to the mammalian NO synthase inhibitor L-NAME [2,5]. 7. The NO Synthesis Systems Are Coordinated with Nitrogen Metabolism L-NAME was also shown to affect NO production by interfering with NR activity [108], which seems to question the mechanisms of action of this compound. However, it has to be considered that in the double nia1/nia2 Arabidopsis mutant commonly used to study NO effects [54], the content of nitrite, as expected from the NR deficiency, and of free amino acids, particularly L-arginine, are much lower than in wild-type plants [109]. Thus, both substrates for NO synthesis would be compromised. yp p y p In conclusion, PII proteins mediate the signaling of the N-source with respect to the carbon status (2-ketoglutarate). This N can be either oxidized (nitrate, nitrite) with a generally high C/N balance, or it can be reduced (ammonium) with a generally low C/N balance. With a high C/N balance, the nitrate assimilation pathway is operative, and NR expression would favor the increase in N capture and its incorporation into C-skeletons (2-ketoglutarate), which would be abundant. When the C/N balance is low, NR would be repressed, and the biosynthesis of arginine would be stimulated. So, under these two extreme conditions, the substrates to produce NO would change. In conditions of high C/N, nitrite would be efficiently produced; however, with low C/N, arginine biosynthesis would predominate with low nitrite production. So, it is proposed that the dual NR:NOFNiR system will preponderate at high C/N, whereas the NOS-like system will be mostly operative at low C/N. PII protein expression will follow a pattern similar to that of NR [110] to enhance N acquisition and balance the C/N ratio. These two extreme situations might be changed to intermediate ones, depending on the C/N balance of the cells. References 1. Wendehenne, D.; Hancock, J.T. 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Some of the biosynthetic mechanisms are starting to be disentangled, while others still require additional useful information for their elucidation. Part of the confusion regarding the Plants 2019, 8, 56 8 of 13 primary source of NO might come from the fact that several pathways might function simultaneously to different extents, depending on nutritional and environmental conditions. Many points still have to be clarified for plants. primary source of NO might come from the fact that several pathways might function simultaneously to different extents, depending on nutritional and environmental conditions. Many points still have to be clarified for plants. Author Contributions: Conceptualization, E.F.; writing-original draft preparation, E.F.; writing-review and editing M.T.-J., A.L., A.G. and E.F.; funding acquisition, E.F. Funding: This work was funded by MINECO (Grant BFU2015-70649-P), the European FEDER program, Junta de Andalucía (BIO-502), the Plan Propio de la Universidad de Córdoba, and the U.E.INTERREG VA POCTEP-055_ALGARED_PLUS5_E. Funding: This work was funded by MINECO (Grant BFU2015-70649-P), the European FEDER program, Junta de Andalucía (BIO-502), the Plan Propio de la Universidad de Córdoba, and the U.E.INTERREG VA POCTEP-055_ALGARED_PLUS5_E. Acknowledgments: We thank María Isabel Macías and Aitor Gómez for technical assistance. Acknowledgments: We thank María Isabel Macías and Aitor Gómez for technical assistance. Conflicts of Interest: The authors declare no conflict of interest. References Delledonne, M.; Xia, Y.; Dixon, R.A.; Lamb, C. Nitric oxide functions as a signal in plant disease resistance. Nature 1998, 394, 585–588. [PubMed] 13. Durner, J.; Wendehenne, D.; Klessig, D.F. Defense gene induction in tobacco by nitric oxide, cyclic GMP, and cyclic ADP-ribose. Proc. Natl. Acad. Sci. 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Challenges and improvement needs in the care of patients with central diabetes insipidus
Orphanet journal of rare diseases
2,022
cc-by
10,727
Abstract Central diabetes insipidus (CDI) is a rare condition, with significant impact on patient health and well-being. It is a chronic condition which usually requires meticulous long-term care. It can affect both children and adults. There is limited literature considering the needs and challenges inherent in providing high quality care to patients with CDI, across the care pathway. This paper seeks to address this gap by providing a unique and well-rounded understand‑ ing of clinical and healthcare systems-related challenges. It draws on insights from the literature, from direct clinical experience contributed by five clinicians as co-authors (providing insights from France, Ireland, Italy, Spain and the United Kingdom), and from patient perspectives provided through interviews with patient representatives from three patient organisations. We identify clinical challenges related to the diagnosis of CDI, including differentiating between other similar conditions and determining the underlying aetiology. Treatment is challenging, given the need to tailor medication to each patient’s needs and ongoing management is required to ensure that patients continue to respond adequately to treatment. Ongoing support is required when patients switch between formulations. We also identify healthcare systems challenges related to limited awareness of CDI amongst primary care physicians and general paediatricians, and the need for highly skilled specialist care and appropriate workforce capacity. There is also a significant need for raising awareness and for the education of both healthcare professionals and patients about different aspects of CDI, with the aim of supporting improved care and effective patient engagement with healthcare professionals. We reflect on this information and highlight improvement opportunities. These relate to developing guidance to support patients, carers, primary care physicians and general paediatricians to identify clinical features earlier, and to consider CDI as a possible diagnosis when a patient presents with suggestive symptoms. Keywords: Central diabetes insipidus, Disease burden, Healthcare improvement, Healthcare systems, Rare disease, Keywords:  Central diabetes insipidus, Disease burden, Healthcare improvement, Healthcare systems, Rare disease, DDAVP Keywords:  Central diabetes insipidus, Disease burden, Healthcare improvement, Healthcare systems, Rare disease, DDAVP Central diabetes insipidus: a rare disease with significant impacts on patient health and well‑being and a limited evidence base on how to improve patient care Central diabetes insipidus (CDI) is a rare disease affecting approximately 1 in 25,000 individuals [4]. It has signifi- cant impact on patient health and well-being and usu- ally requires long term care. The evidence base on how to care for patients with this condition is fragmented. © 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://​creat​iveco​mmons.​org/​licen​ses/​by/4.​0/. The Creative Commons Public Domain Dedication waiver (http://​creat​iveco​ mmons.​org/​publi​cdoma​in/​zero/1.​0/) applies to the data made available in this article, unless otherwise stated in a credit line to the data. Challenges and improvement needs in the care of patients with central diabetes insipidus H. Teare1, J. Argente2,3,4, M. Dattani5,6, J. Leger7, M. Maghnie8,9, M. Sherlock10, G.‑C. Ali1, J. Francombe1 and S. Marjanovic1* S. Marjanovic1* Abstract There is a need to advance knowledge on the diversity of challenges to patient care and to identify scope for improvement. *Correspondence: smarjano@randeurope.org 1 RAND Europe, Westbrook Centre Milton Road, Cambridge CB4 1YG, UK Full list of author information is available at the end of the article In this paper, we provide an overview of the diagno- sis, treatment and management of patients with CDI to Teare et al. Orphanet Journal of Rare Diseases (2022) 17:58 https://doi.org/10.1186/s13023-022-02191-2 Teare et al. Orphanet Journal of Rare Diseases (2022) 17:58 https://doi.org/10.1186/s13023-022-02191-2 (2022) 17:58 Open Access © 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://​creat​iveco​mmons.​org/​licen​ses/​by/4.​0/. The Creative Commons Public Domain Dedication waiver (http://​creat​iveco​ mmons.​org/​publi​cdoma​in/​zero/1.​0/) applies to the data made available in this article, unless otherwise stated in a credit line to the data. Teare et al. Orphanet Journal of Rare Diseases (2022) 17:58 Page 2 of 12 consider the diversity of factors that play a role in deter- mining the quality of patient care. We identify challenges and improvement needs in both adult and paediatric populations. We draw on insights from a narrative litera- ture review, complemented with the experiential knowl- edge of clinical experts and the views of representatives from patient associations. treatment over time, including when managing CDI alongside other concurrent conditions, particularly adrenocorticotropic hormone (ACTH) deficiency. Fur- thermore, the lifelong treatment that patients with CDI often require [7] places significant demand on healthcare systems for ongoing management and monitoring. The aim of this review is to expand on the key features of the care pathway and the associated factors and chal- lenges that influence patient care, and to reflect on areas in need of improvement. CDI is characterised by hypotonic polyuria and poly- dipsia resulting from decreased concentrations of argi- nine vasopressin (AVP), also known as antidiuretic hormone (ADH) [4, 11]. Other symptoms can include fatigue, dizziness, hypotension and tachycardia related to dehydration, and hypernatremia which is a direct conse- quence of CDI [15, 24]. Severe dehydration is a particular challenge in young children, who may not as yet be able to self-regulate thirst. Children can also experience vom- iting, constipation, fever, irritability, sleep disturbance, retardation of growth, failure to thrive and potentially developmental disability caused by repeated dehydration and hypernatremia [9, 15, 18, 20]. Methodsh The paper combines findings from a narrative literature review, insights based on experiential knowledge from five leading clinical experts from the United Kingdom (UK), Ireland, Italy, Spain and France who are also co- authors of the paper, and the views of patient representa- tives from three associations active in the CDI area (the Pituitary Foundation, UK; the Pituitary Foundation, Ire- land; and the Italian Family Association of Septo-optic Dysplasia and Pituitary Abnormalities, Italy). The disease is caused by damage to AVP-producing magnocellular neurons in the hypothalamus. The degree of deficiency of AVP is the main factor that determines the severity of CDI symptoms. Damage to this region of the brain can occur either due to traumatic injury [3, 11] or non-traumatically, for example in response to a tumour, infiltration, infection or inflammation, or less frequently as a result of genetic mutations [1, 21]. Hereditary forms of CDI present early in life, whereas acquired forms can occur at any age. CDI has a similar prevalence in men and women. A patient’s quality of life is often impeded as a result of the condition, for exam- ple from sleep being disturbed by nocturia [25]. There is also a risk of fatality in the most severe cases, which can happen because of chronic or severe dehydration, hyper- natremia, fever and cardiovascular failure due to a com- promised ability to regulate blood pressure [5, 7]. Adipsia can increase risk of death [12]. In patients with CDI, an intact thirst mechanism is critical for protecting against hypernatremia and dehydration. Narrative review Th i li The narrative literature review was conducted using a systematic search strategy, with the search conducted in October 2020. It included papers published between October 2015 and October 2020, with specified crite- ria for prioritising papers which were then subjected to thematic analysis. It focused on understanding the care pathway for patients with CDI, challenges and wider influences on patient care, and improvement needs. A total of 25 papers were included in the review; further detail of their selection is included in the Additional file 1. Interviews with patient representatives Perspectives were gathered from three patient repre- sentatives from associations which support patients with CDI in Ireland, Italy and the UK (unfortunately this was not feasible in Spain or France). Their per- spectives helped to better understand how patients experience CDI and their views on future priorities to improve the provision of care. Patient representative views are referenced in brackets with PR-INT X, with X being the code number for an individual interviewee. In some instances, where there is a risk of identity disclo- sure or linkage of information to a country context, or in light of informed consent, we withhold a reference to preserve anonymity. Both adults and children follow a similar diagnostic pathway. A water deprivation test (WDT), during which the patient is not allowed to drink, has been the gold standard for diagnosing CDI [20]. It involves meticulous and regular measurements of urine production, blood electrolyte concentrations, plasma and urine osmolal- ity and weight for approximately 7  h (and occasionally for shorter durations in children, if cessation of the test is necessary due to weight loss or hypernatremia) [20]. This usually takes place in a specialist hospital setting supported by an endocrinologist and specialist nurses, as water deprivation requires careful monitoring [24]. Patients can find the WDT test extremely unpleasant and challenging [PR-INT2, PR-INT3]. The water depriva- tion test is sometimes done in modified versions and for shorter periods of water deprivation in some paediatric patients and may be avoided in infancy if hypernatremia with low urine osmolality is present. Parents of children with CDI symptoms may also be asked to measure their child’s fluid balance [PR-INT3]. A WDT may not always be necessary for pregnant women, for whom such a test could be overly cumbersome [22]. There is potential for increased use of alternative diagnostic tests in the future [PR-INT2], for example measurement of copeptin and the use of dynamic tests such as arginine or hypertonic saline infusions. However, the current use of such tests across different countries remains unclear. We have thematically cross-analysed and triangulated insights from these various sources to develop a multi- faceted understanding of care pathways, associated chal- lenges and improvement needs. There are some caveats to consider when interpreting our findings. The literature review covered high income countries and additional insights from low- and middle- income countries would help to enrich these findings. Focused discussion with clinical experts fi d b ld To refine, nuance and build on insights from the lit- erature, the paper co-authors—representing health- care services research experts from the not-for-profit research institute RAND Europe (SM, HT, JF, DRR, GCA) and clinicians working with patients with CDI in France (JL), Ireland (MS), Italy (MM), Spain (JA) and UK (MD)—engaged in focused discussion. This included individual discussions between a RAND Europe health services researcher (either SM, HT, DRR, or JF) and each individual clinician, a total of five one-hour discussions. It also included group dialogue through two online workshops bringing together the clinical experts and RAND Europe researchers (work- shops were held on 23 and 27 November 2020). The workshops were opportunities for collective discus- sion about care-related improvement needs. They also served to explore healthcare system related influences CDI is one of four conditions associated with polyuria and polydipsia, along with nephrogenic diabetes insipi- dus (NDI), primary polydipsia and gestational diabetes insipidus [7]. Once diagnosed, treatment of CDI is gen- erally effective, with desmopressin (DDAVP, D-amino D-arginine vasopressin- an AVP analogue) used as the mainstream treatment to help regulate fluid in the body. However, it can be challenging to identify and accurately diagnose patients and to get them onto appropriate treat- ment [7, 8, 14, 15, 20, 23, 24]. Optimising dosages and treatment formulations and ensuring personalised care is also not straightforward [1, 22, 23]. In addition, it can be challenging to ensure that patients remain stable on Page 3 of 12 Teare et al. Orphanet Journal of Rare Diseases (2022) 17:58 Teare et al. Orphanet Journal of Rare Diseases (2022) 17:58 Teare et al. Orphanet Journal of Rare Diseases (2022) 17:58 The care pathway and associated challenges related to diagnosis, treatment and ongoing management Diagnosing CDI on quality of care and access to care in more depth, given that these aspects are under-explored in the lit- erature. Clinical co-authors from four countries also gathered estimations of the costs of care for a patient in their local healthcare setting (i.e. hospital) to provide a high-level indication of the estimated costs related to hospital care that are incurred during the patient pathway. Although these are just estimates, and do not cover primary care costs for example, they provide ini- tial insights on the resource implications of caring for patients with CDI which can be built on in future stud- ies, and that extend beyond medicine costs alone. on quality of care and access to care in more depth, given that these aspects are under-explored in the lit- erature. Clinical co-authors from four countries also gathered estimations of the costs of care for a patient in their local healthcare setting (i.e. hospital) to provide a high-level indication of the estimated costs related to hospital care that are incurred during the patient pathway. Although these are just estimates, and do not cover primary care costs for example, they provide ini- tial insights on the resource implications of caring for patients with CDI which can be built on in future stud- ies, and that extend beyond medicine costs alone. In children and adults, CDI is diagnosed using a series of tests to confirm polyuria and polydipsia and to try to understand the underlying cause. Investigations tend to follow a logical progression of clinical history and exami- nation followed by biochemical and endocrinological assessment, followed by radiology. The approach to diag- nosis is similar across the different healthcare settings considered in this paper [workshop insights]. In coun- tries where primary care is generally the first point of access to healthcare, patients with CDI will usually first present to a primary care physician (or general practi- tioner) having experienced symptoms such as dehydra- tion, thirst and frequent urination [PR-INT1, PR-INT2, PR-INT3]. Following basic tests by a primary care physi- cian, in most cases patients will be referred to a specialist endocrinologist for further diagnostic testing [PR-INT1, PR-INT2, PR-INT3]. Interviews with patient representatives Absent neurohypophysis can be diagnosed by MRI if the posterior pituitary bright spot is missing.f Differentiating between the various conditions which share symptoms of polyuria and polydipsia can be chal- lenging and can take considerable time [7, 8, 12]. Exist- ing diagnostic tests can risk false diagnoses, both positive and negative, particularly in patients with mild or partial forms of CDI [23 PR-INT2]. Understanding the potential cause of symptoms can help in the diagnostic process, as some events, such as traumatic brain injury are associ- ated with increased likelihood of CDI and can, together with other tests, help to confirm whether a patient has CDI or another condition. Therefore, a full patient his- tory is an important part of the diagnostic process [11]. Timely diagnosis is impeded if symptoms are not noticed promptly [10], or if other aetiologies for polyu- ria and polydipsia are investigated first [13]. A lack of awareness among primary care physicians regarding the symptoms of CDI [PR-INT1, PR-INT2] can also impede timely diagnosis and referral. The time to diagnosis can vary across countries. Differentiating between the various conditions which share symptoms of polyuria and polydipsia can be chal- lenging and can take considerable time [7, 8, 12]. Exist- ing diagnostic tests can risk false diagnoses, both positive and negative, particularly in patients with mild or partial forms of CDI [23 PR-INT2]. Understanding the potential cause of symptoms can help in the diagnostic process, as some events, such as traumatic brain injury are associ- ated with increased likelihood of CDI and can, together with other tests, help to confirm whether a patient has CDI or another condition. Therefore, a full patient his- tory is an important part of the diagnostic process [11]. Timely diagnosis is impeded if symptoms are not noticed promptly [10], or if other aetiologies for polyu- ria and polydipsia are investigated first [13]. A lack of awareness among primary care physicians regarding the symptoms of CDI [PR-INT1, PR-INT2] can also impede timely diagnosis and referral. The time to diagnosis can vary across countries. Given desmopressin’s mode of action, ongoing patient monitoring during treatment dose optimisation is impor- tant. This is because of the risk of developing hypona- tremia if too little water is excreted or too much fluid ingested [13], or hypernatremia [8, 12, 16, 17] if too much water is lost or too little fluid ingested. Interviews with patient representatives It is impor- tant to allow water offload in this process, to prevent hyponatraemia. Optimisation therefore involves a spe- cialist endocrinology clinic to determine patient response to treatment and whether the dose needs to be further adjusted. It usually takes 2–3  days, or up to a week as an inpatient, but can take longer for infants [workshop insights]. Treatment optimisation occurs during the same admission as the diagnostic stage. Costs associated with diagnosis can vary a great deal depending on the speed of diagnosis and the complexity of the underlying aetiology, and if additional support is needed, for example intensive care support in instances of traumatic brain injury, or genetic testing to confirm cause (see the cost section below). Interviews with patient representatives The workshops and one-to-one discussions with clinical experts are limited to the countries they work in and are based on their personal experience—however these are all leading experts in their country contexts. The inter- views are with patient representatives of a limited num- ber of patient organisations who could engage with the study, in part related to the small number of existing patient organisations active in CDI. The cost estimations in particular are limited by the availability of data and detail that could be provided, and thus can only provide an initial indication of cost rather than a comprehensive overview. Despite these caveats, the insights from the literature review and complementary views from clini- cal experts and patient representatives provide a valu- able contribution to enriching our understanding of how care for CDI is provided and what the challenges and improvement needs are. f Magnetic Resonance Imaging (MRI) is essential to determine potential causes of CDI—for example it can show thickening of the pituitary stalk which might indi- cate inflammation/autoimmune conditions, tumours or other disorders, or a neoplastic process in the sellar Teare et al. Orphanet Journal of Rare Diseases (2022) 17:58 Teare et al. Orphanet Journal of Rare Diseases (2022) 17:58 Page 4 of 12 Infants and young children will require much lower doses than adults, which will need to be adjusted over time, as the children grow and develop. Young infants are also dependent on fluid such as breast milk or formula for nutrition, and this can present additional challenges to fluid regulation and dose optimisation. This is because the primary therapeutic goals of treating CDI in young children are to reduce polyuria and decrease excessive thirst to support appropriate levels of fluid uptake, and to ensure appropriate growth. Children require adequate fluid intake and various doses of DDAVP ranging from a low dose of 10 µg/day for infants in cases of neonatal DI to 60 µg/day three times a day during later childhood, although higher doses could be necessary for individu- alised patients. Careful exact dosing by cutting the available oral disintegrating tablet DDAVP formulation should be performed, particularly for young paediatric patients. Starting with low doses and titration of the dose is important. region [7]. However, there are challenges associated with using MRI to identify etiological markers of CDI [20]. Treating CDI: treatment initiation and optimisation d h b h d Such clinic appointments are the main healthcare service-related cost associated with the longer-term management of CDI, as outlined in the cost section below. should only be administered in intensive care or post- operatively. According to one patient representative and in their country context, paediatric patients are gener- ally initiated on a nasal spray due to ease of administra- tion, but the formulation may be changed depending on the patient’s response [interview reference withheld to preserve anonymity]. In mild cases of CDI in some coun- tries, clinicians may consider not prescribing DDAVP and instead suggest strategies for managing fluid balance and using fluid replacement [interview reference withheld to preserve anonymity]. Different formulations also take dif- ferent times to act and hence some patients may prefer formulations which provide relief quicker [PR-INT2]. As part of patient management, it is sometimes neces- sary to not only adapt dosage but also to switch between different formulations of desmopressin. This can happen if patients find a specific formulation easier to adminis- ter [15] or due to potential supply issues, or differences in the effectiveness of a formulation [two interviewees, interview references withheld to preserve anonymity]. Switching between formulations requires dose titration to optimise the dose for each patient, as it is not possible to predict how patients will respond to new formulations based on their previous treatment protocols [14, 15]. This is related to the absence of conversion factors between different formulations. Switching between formulations will require further in-clinic support, which can add to the cost of treatment. Establishing conversion factors between different formulations is challenged by the fact that different formulations have different bioavailabil- ity and that there is diversity in how individuals respond to any specific formulation and dose. The UK electronic medicines compendium1 provides a correlation table for oral tablet and melt formulations for adults, but this is based on bioavailability correlation research which tends to take place in healthy volunteers, rather than patients with CDI. Whereas such information may help orient clinicians, it cannot guide clinical decision-making given the highly personalised nature of dose optimisation for CDI, and the frequent need for starting with lower doses as part of the dose optimisation process. Treating CDI with desmopressin is generally safe and effective and shown to improve patient quality of life [11], but can come with side effects. Side effects, while rare, vary slightly between formulation. Treating CDI: treatment initiation and optimisation d h b h d Once a diagnosis has been reached, it is necessary to tai- lor treatment to the individual; given the marked varia- bility in patient response to treatment, each patient needs a personalised protocol [2, 9, 20]. Desmopressin is available in several different formu- lations. Decisions relating to which formulation is most appropriate depend on the patient being treated. It can be administered orally as a tablet or as an oral disintegrating tablet (ODT), as a buccal preparation, as a nasal spray, or parenterally (usually intramuscularly or intravenously). The latter may be required in patients that are required to fast pre-operatively or for patients needing intensive care [1, 6, 8, 10, 24, PR-INT1, PR-INT2]. As introduced previously, CDI is most often treated by replacing AVP with a synthetic vasopressin analogue called desmopressin, in both adult and paediatric popula- tions [1, 6, 8, 10 PR-INT1, PR-INT2, PR-INT3, workshop insights]. In all patients with chronic CDI, a common starting point for treatment is to first address nocturia, by administering the first dose of desmopressin before bed [22]. The patient’s response and dose will be measured and adjusted over time until their symptoms are effec- tively controlled. Initial treatment optimisation generally involves starting with a low dose and gradually increas- ing if needed [10]. Some adult patients may only need a single dose per day to control their symptoms overnight (although the strength of the single dose can vary signifi- cantly between patients), while severe cases might need up to 200 μg twice or three times a day (occasionally up to 1 mg in total) [11]. Desmopressin lyophilizate sublingual tablets are a valuable option for treating CDI in infants and young children, with evidence of more stable absorption than intranasal formulations and oral tablets [9, PR-INT1 and PR-INT2]. However, it can be difficult to split these sublingual tablets into small enough doses for infants. Subcutaneous DDAVP administration enables the administration of small doses to infants but this can lead to more variable sodium concentrations than other for- mulations [9, and the experience of an expert involved with this research suggests that subcutaneous DDAVP Teare et al. Orphanet Journal of Rare Diseases (2022) 17:58 Teare et al. Orphanet Journal of Rare Diseases (2022) 17:58 Page 5 of 12 impact fluid requirements [workshop insights]. Annual or biannual clinic appointments with an endocrinologist are often required to ensure treatment doses and formu- lations remain effective. 1  Electronic Medicines Compendium. 2012. ‘DDVAP Melt 60mcg oral lyoph- ilisate’. Emc. As of 22 December 2021: https://​www.​medic​ines.​org.​uk/​emc/​ produ​ct/​170/​smpc#​gref Treating CDI: treatment initiation and optimisation d h b h d The intranasal spray, for example, may lead to dizziness, eye irritation, head- ache, flushing, nausea, vomiting, rhinitis or epistaxis and tachycardia. Evidence suggests that oral DDAVP is bet- ter tolerated by some patients [20]. Dose adjustment can help to mitigate side effects [workshop insights]. f Although desmopressin is the main treatment for CDI across different types of patient groups at present, some other treatments are used in some countries (even if not specific for use in CDI) with slightly different modes of action and side effects. These include thiazide diuretics, carbamazepine, chlorpropamide, clofibrate and indapa- mide [9, 10, 13, 15]. However, desmopressin remains by far the most commonly used form of treatment [10].h There is a lack of data on the costs associated with treatment optimisation and these costs are likely, at least in part, to depend on the complexity and length of the optimisation process. Managing patients with CDI over time the dose for paediatric patients without discussion with a specialist. In some countries, repeat prescriptions tend to be organised by primary care without specialist involve- ment, although the specialist centre would still direct dosage and monitor the patient as needed. Long term management decisions other than those related to medication will also be dependent on whether patients are able to regulate thirst themselves, or whether they are adipsic. Patients who are able to experience thirst are generally encouraged to avoid excess fluid intake, and drink to thirst rather than habit. For adipsic patients, it is more difficult to keep track of how much water intake is needed [11]. Therefore, a routine approach to drinking with a daily fluid regimen based on strictly regulated fluid intake with fixed amounts of water, is important to help mitigate the risk of hyper- or hyponatremia [9]. This can be particularly challenging if patients contract other ill- nesses, for example if they are vomiting or eating less or have diminished consciousness [11]. Fasting for a surgi- cal procedure can also introduce risk, and must be super- vised by a specialist, particularly if it requires a change in treatment formulation, for example if the patient usu- ally administers ODT [11]. According to experts con- sulted for this research, physicians should be aware of associated, treatable hypothalamic abnormalities such as obesity, sleep apnoea, seizures and thermoregulatory disorders when managing patients with adipsic diabetes insipidus. From a fluid balance perspective, these patients require regular DDAVP to treat their CDI but also need a daily fluid prescription in order to maintain euvolaemia (an appropriate blood fluid volume within the body) and eunatraemia (an appropriate concentration of sodium in blood plasma). Adjusting the desmopressin dosage and fluid intake for adipsic patients is generally done in a hos- pital setting and regular weighing and checking of serum sodium levels is necessary. Both for adults and children, management of the con- dition at home is also an important part of the patient and/or carer journey [PR-INT1, PR-INT3]. Although patients will tend to have regular six-monthly or annual follow-up appointments, ensuring appropriate medica- tion adherence and administration at home is key [PR- INT2]. Lifestyle issues also require patients to engage with ongoing management or monitoring through their own behaviours. Managing patients with CDI over time Specialists—generally endocrinologists—determine which brand (or generic) and formulation to use, depend- ing on what they think is best for the patient or what they are most familiar with [workshop insights]. In the countries included in this research, the use of branded versions appears more common at the point of prescrip- tion, possibly because generic versions are not available or because of clinician preference [workshop insights]. Whether there is therapeutic equivalence between branded or generic products for CDI has not been researched. If patients are followed up in primary care, primary care physicians can make decisions to switch between branded or generic versions, if generic versions are available. However, they generally would not change Given that CDI is usually a chronic condition, lifelong management and ongoing monitoring are necessary to ensure that patients respond well to their specific treat- ment over time. Ongoing care is also necessary to ensure that any changes to treatment are carefully managed [7]. It is difficult to predict how a patient will respond to treatment. Patient response can be influenced by their ability to adhere to treatment regimens, for example to control fluid intake or regularly take medication. It can also be influenced by other factors such as other ill- nesses—for example a cold which might affect how well a patient absorbs desmopressin nasal spray, or vomiting which will influence the intake of an oral tablet [2, 11, 15, 16]. Patient response to a specific treatment dose and for- mulation can also be influenced by lifestyle factors such as foreign travel to a warm country due to changing water intake behaviours or participating in sport which might Teare et al. Orphanet Journal of Rare Diseases (2022) 17:58 Page 6 of 12 Page 6 of 12 dose adjustment mean that it is important to involve the relevant experts early in this process to support any required changes to dose [10]. In paediatric inpatients who are unable to tolerate oral desmopressin (for exam- ple while they are unwell with other conditions such as gastro-intestinal issues) a switch to parenteral therapy may be required, although such cases are rare. This would typically be delivered intramuscularly, but intra- venous pitressin infusion (vasopressin injection) can also be used. If this is different from the patient’s usual desmopressin formulation, particularly close monitoring is required as it is not possible to predict how the patient will respond [10]. Managing patients with CDI over time For example, stress, menstruation or unusual consumption of a lot of salty/sweet foods may require desmopressin to be taken at specific times in the day [PR-INT2]. Influences on patient care and the care pathway within healthcare systemsh The diagnosis of patients with CDI, decisions about treatment dosage and formulation, and ongoing patient monitoring and management are strongly influenced by features of the wider healthcare system in which patients are treated, as discussed below [4, 5, 7–11, 13, 16, 20–23, 25]. Influences related to skills and workforce capacity Awareness raising amongst other professions such as teachers, university lecturers and air stewards may also be needed so that patients can manage their condition with as much ease as possible [PR-INT2].h f Patient education can help equip patients with knowl- edge about how to manage their disease, what to look out for and side effects [8, 12, 23], which can in turn sup- port effective communications with primary care physi- cians about treatment and monitoring needs [workshop insights]. In the UK, the Pituitary Foundation provides resources that patients can share with general practition- ers if they suspect they have CDI. There are also efforts in some countries to rename the condition so as to remove the term ‘diabetes’ to avoid confusion with diabetes melli- tus [interview reference withheld to preserve anonymity]. Adjusting treatment formulations and dosages over time also requires significant skills to make appropri- ate decisions and to support personalised care, given that different formulations have significantly differ- ent bioavailability and that patient response differs [16, 19]. Any potential changes in relation to bioavailability between branded and generic products require further research (as there are gaps in evidence on this issue), to inform policies on generic substitution and automatic dispensing.hi [ p y y] While there is a need for information and awareness raising regarding the symptoms of CDI [PR-INT1], there is also a need for more education on how families can support treatment and management. This includes a need for improved access to information about water intake, food, salt levels and measuring water balance [PR-INT3]. Beyond patients, interviewees also highlighted the need for better public understanding of CDI [PR-INT1, PR- INT2], for example to avoid patients being refused access to toilet facilities in some settings like shops [PR-INT2]. Awareness raising amongst other professions such as teachers, university lecturers and air stewards may also be needed so that patients can manage their condition with as much ease as possible [PR-INT2]. The COVID-19 pandemic has also created specific challenges for patients with CDI. These include limited access to primary care physicians to get a referral to an endocrinologist [interview reference withheld to pre- serve anonymity], challenges accessing and undertaking diagnostic testing [PR-INT1], and lack of sufficient follow up with clinicians related to ongoing management needs. Influences related to skills and workforce capacity l p y Supporting patients with CDI requires specialist skills involving endocrinologists and including paediatricians and nurses to support infants and children. Patient sup- port will also draw on other specialties depending on the cause of CDI and specific patient needs [9]. For example, patients with CDI as a result of a tumour, or as a result of tumour surgery, will require oncologist and neurosur- geon involvement in care. Patients with CDI as a result of traumatic brain injury may need intensive care teams, and patients with CDI in pregnancy will need additional support, for example from obstetrics [workshop insights]. Appropriate support requires the healthcare system to have sufficient workforce capacity and a cadre of highly skilled staff across specialties [2, 11, 15, 16]. Similarly, patients with CDI diagnosis prior to preg- nancy who are already receiving doses of exogenous vas- opressin or desmopressin may require increased doses during pregnancy and additional monitoring to ensure that there are no complications due to increased dosages [1]. Given the impact of CDI on growth and development in infants, more frequent monitoring is also particularly important in this vulnerable patient population. This will include monitoring serum sodium, weight and hydra- tion to allow doses to be modified, and home-monitor- ing including weighing wet ‘diapers’ [25]. According to an expert involved with this research, daily weighing of an infant/young child patient should make it possible to detect abnormal weight gain, and plasma sodium con- centration should be monitored frequently to reduce the risk of hyponatremia. The challenges associated with f Several of the healthcare systems included in this paper have specialist centres that support patients across a large geographic region. This allows expertise to be Teare et al. Orphanet Journal of Rare Diseases (2022) 17:58 Page 7 of 12 engaging with healthcare professionals about opti- mising treatment [17]. For example, patients may have preferences for specific formulations [workshop insights] in light of their absorption, the types of com- plications experienced or how straightforward the for- mulation is to administer. Patient engagement is also essential in the context of monitoring treatment effec- tiveness and reporting any changes in effectiveness to healthcare professionals [6, 17], as well as in the con- text of ensuring dose adherence and compliance with treatment [8, 20]. For some formulations, patient skills in administering the treatment, such as dexterity in administering intranasal formulations, play a role in treatment effectiveness [16]. Influences related to skills and workforce capacity According to one patient representative, some patient support groups and helplines saw an increase in enquir- ies as a result [interview reference withheld to preserve anonymity]. There is a need to consider how patients can best be cared for in the context of any future pandem- ics, both in terms of remote care and any essential face to face contact with clinicians. Those patients who are on both glucocorticoid (for example in the case of panhypopituitarism) and DDAVP replacements may need educational support about spe- cific sick day management. Glucocorticoids are essen- tial for the excretion of water. Some patients who have central diabetes insipidus also have anterior pituitary dysfunction and as such require glucocorticoid replace- ment. When a child taking both regular glucocorticoid and DDAVP becomes unwell, the parents may need to ensure that the glucocorticoid is doubled or trebled, and that the child is passing urine before giving further doses of DDAVP. In the event of inadequate glucocorti- coid administration, water will not be effectively excreted and the administration of DDAVP can lead to water Influences related to skills and workforce capacity concentrated but can also require patients to travel long distances to seek specialist advice and support [PR-INT1, PR-INT3]. This places significant emphasis on the first stages of patient support, often through primary care or other departments such as emergency medicine or oncology, to recognise specific patient needs [workshop insights]. In some countries, the limited number of hos- pitals with relevant clinical expertise can also create long waiting times for treatment [PR-INT1] or necessitate remote management. Healthcare professionals in primary care (both pri- mary care physicians and nurses) can sometimes lack sufficient awareness about the symptoms of CDI which can complicate timely diagnosis and referrals [PR-INT1, PR-INT2, PR-INT3]. This can sometimes lead to inap- propriate treatment [PR- INT1, PR-INT2]. Primary care physicians are also sometimes not appropriately trained on the importance of carefully managing and monitoring appropriate treatment dosing and formulations as patient conditions change [workshop insights]. In a hospital care setting, not all physicians always understand how desmo- pressin acts, which can complicate the inpatient care of patients with CDI who are admitted for other conditions [8, 25]. f Patient education can help equip patients with knowl- edge about how to manage their disease, what to look out for and side effects [8, 12, 23], which can in turn sup- port effective communications with primary care physi- cians about treatment and monitoring needs [workshop insights]. In the UK, the Pituitary Foundation provides resources that patients can share with general practition- ers if they suspect they have CDI. There are also efforts in some countries to rename the condition so as to remove the term ‘diabetes’ to avoid confusion with diabetes melli- tus [interview reference withheld to preserve anonymity]. While there is a need for information and awareness raising regarding the symptoms of CDI [PR-INT1], there is also a need for more education on how families can support treatment and management. This includes a need for improved access to information about water intake, food, salt levels and measuring water balance [PR-INT3]. Beyond patients, interviewees also highlighted the need for better public understanding of CDI [PR-INT1, PR- INT2], for example to avoid patients being refused access to toilet facilities in some settings like shops [PR-INT2]. Availability and use of appropriate treatment formulations and medicines management and the role of regulation The availability of appropriate formulations that are safe and effective as well as convenient to administer plays a role in treatment decisions and their appropriateness for users [12, 16, 20, 25]. Communication between cli- nicians and patients in relation to why some formula- tions may be available and why there may be shortages of others (or lack of availability) matters in terms of patient-centred care and good patient-physician com- munication [workshop insights]. Patient representatives raised issues regarding treatment availability and supply [interview reference withheld to preserve anonymity]. In one country, an interviewee commented that intrana- sal solution and nasal spray forms of desmopressin have been unavailable since the autumn of 2020, with patients and support groups not knowing when the supply will return [interview reference withheld to preserve ano- nymity]. Many patients using nasal formulations have subsequently transferred to sheets/melts, which created further supply issues. f Clinical experts from four countries shared cost esti- mations for their own hospital settings (see Table 1). In all four countries, the costs of healthcare service provi- sion (i.e. consultations and diagnostic tests) are covered by the public healthcare system. However, the clinical experts also noted that given the personalised nature of CDI treatment and management, costs per patient can vary substantially within any one given context [work- shop insights]. This means that healthcare services costs are not directly comparable between countries as well as between different hospital settings. In terms of medicine costs, in health systems with a mixture of public and pri- vate funding, desmopressin is usually covered as part of a long-term illness scheme, and therefore publicly funded [workshop insights]. Regulation plays an important role in ensuring the availability of appropriate treatments and in promot- ing good practice. For example, the intranasal spray for- mulation of desmopressin is no longer approved by the United States Food and Drug Administration, and this has led to a switch to the tablet or melt formulation in many countries [15]. Similarly, it is important that regula- tory officials understand reasons for certain decisions, for example in relation to medicine switching and reasons for product recalls, to support national decisions and guidance that can impact patients directly [15]. While there is literature discussing good practice for specific patient groups (e.g. Influences related to patient engagement with care related decisions and behaviours Patients and/or caregivers have an important role to play in the ongoing management of CDI and in Teare et al. Orphanet Journal of Rare Diseases (2022) 17:58 Page 8 of 12 Page 8 of 12 20162) [25]. This can have significant cost implications over a patient’s lifetime. Data from the UK3 provides costs for desmopressin acetate, with 90 oral tablets of 100 μg individual doses reported to cost £44 (based on 2016 data, equivalent to 46.62 euros in 2016) and a pack of 100 sublingual disintegrating tables of 60  μg indi- vidual doses reported to cost £51 (based on 2016 data, equivalent to 54.03 euros in 2016). A pharmacoeconomic analysis of vazomirin spray (a treatment used in Russia with desmopressin as the active ingredient) compared with other forms of administration concluded that the cost of vazomirin spray, compared to vazomirin tablets is reduced by 45–48% in patients after resection of chi- asmo-sellar region (CSR) tumours [3]. In general, cost considerations are an important factor in making treat- ment decisions. Although this research study did not focus on gathering specific medicines cost data in indi- vidual countries as part of its scope, the costs of CDI treatment are likely to vary across different geographies, as is the case with many medicines. Therefore, costs in a US context cannot be used to make inferences about costs of medicines in different European countries. intoxication and hyponatraemia with related complica- tions such as seizures. Availability and use of appropriate treatment formulations and medicines management and the role of regulation children and infants) [9], and guidance on inpatient management of CDI [4], we did not identify national level guidelines in the sample of papers reviewed relating to the countries of interest, namely: France, Ireland, Italy, Spain or the UK. Given the limitations set out above, the information in the table below is shared purely for illustrative purposes and cannot be used to infer costs for an entire system nor for other settings. It seeks only to provide some ini- tial information on what healthcare costs are like in some settings, given the paucity of any such insights in the existing literature. The data in the table can also not be aggregated into higher level categories to make inferences 2  OECD Data. 2021. ‘Purchasing power parities (PPP)’. OECD. As of 22 December 2021: https://​data.​oecd.​org/​conve​rsion/​purch​asing-​power-​parit​ies-​ ppp.​htm#​indic​ator-​chart 3  Horizon Scanning Research & Intelligence Centre. 2016. ‘Low dose desmopressin lyophilisate (Noqdirna) for nocturia in adults. NIHR Inno- vation Observatory. As of 22 December 2021: https://​www.​io.​nihr.​ac.​uk/​ wp-​conte​nt/​uploa​ds/​migra​ted/​Low-​dose-​desmo​press​in-​lyoph​ilisa​te-​Noqdi​ rna-​June16.​pdf 3  Horizon Scanning Research & Intelligence Centre. 2016. ‘Low dose desmopressin lyophilisate (Noqdirna) for nocturia in adults. NIHR Inno- vation Observatory. As of 22 December 2021: https://​www.​io.​nihr.​ac.​uk/​ wp-​conte​nt/​uploa​ds/​migra​ted/​Low-​dose-​desmo​press​in-​lyoph​ilisa​te-​Noqdi​ rna-​June16.​pdf 2  OECD Data. 2021. ‘Purchasing power parities (PPP)’. OECD. As of 22 December 2021: https://​data.​oecd.​org/​conve​rsion/​purch​asing-​power-​parit​ies-​ ppp.​htm#​indic​ator-​chart Costs and effective resourcing to ensure appropriate capacity Economic considerations will vary across healthcare systems, and economic burden may play a role for some patients [3, 25]. For example, a US-based study reported a one-month supply of intranasal DDAVP to cost USD 245.80 (based on 2016 data, equivalent to 179.43 euros in 2016), while a one-month supply of subcutane- ous DDAVP cost USD 565.25 for treating infants in the USA (based on 2016 data, equivalent to 412.63 euros in Page 9 of 12 Teare et al. Orphanet Journal of Rare Diseases (2022) 17:58 Table 1. Examples of costs for treating a patient with CDI—estimates from a sample of hospital settings (Country names withheld to preserve anonymity, all costs expressed in Euro equivalents) Stage for each country A (adults) B (paediatric) C (paediatric) D (paediatric) Diagnosis The costs presented relate to the diagnosis and management of CDI as a result of acute CDI. This will differ from adults presenting with polydipsia and polyuria, and can take over six months longer to diagnose Initial visit including blood tests: EUR 750–800 Cost of hospitalisation and first diag‑ nosis: EUR 1500–4500 Including admission in the paediatric endocrine unit for initial evaluation, diagnosis and treatment initiation (four days), and for repeated evaluation every six months during the first three years after diagnosis (two days for clinical, biological and cerebral MRI assess‑ ments and treatment optimisation): EUR 50,000 Cost estimates for paediatric patients including admission into the paediatric endocrine unit for initial evaluation, diagnosis and treatment initiation (three days), and for repeated evaluation every six months during the first three years after diagnosis: EUR 37,600 (costs of DDAVP not included) One day of testing including water deprivation test: EUR 2300–2500 In patients who need cerebrospinal fluid analysis and/or pituitary stalk biopsy, additional costs: EUR 2000–3000 MRI: EUR 400 Supporting patients (based on the imaging work-up) every six months for the first two years of treatment: EUR 400–1000 Neurosurgery including hospital stay if required: EUR 10,000–15,000 (approximately EUR 1500 per day) Idiopathic CDI may have additional costs a long time after diagnosis as a result of complications developing – specific cost data not available. Costs and effective resourcing to ensure appropriate capacity ICU stay if required: EUR 10,000 per day Genetic diagnosis may be needed in rare cases: EUR 500 Long term follow-up Outpatient visits: EUR 300 for two visits per year One clinical and biological examination every six months: EUR 3000 per year (for following seven years post diagnosis) Approximately EUR 2350 per year. Data on costs of treatments (therapeu‑ tics) is not available Desmopressin: EUR 6000 for 10 years Desmopressin treatment (on average 60 ug x 3/day): EUR 6500 for 10 years Data on costs of treatments (therapeu‑ tics) is not available If other hormonal treatment is required, such as growth hormone treatment (with about 1 mg per day), an addi‑ tional cost of up to EUR 90,000 for 10 years can be incurred Approximate total for 10 years EUR 8450–34,200 (more if longer than one day in ICU, and not including medication costs) EUR 12,100–28,500 Approximately EUR 60,000 (depending on patient complexity) (if growth hormone treatment if needed, additional costs are incurred) Approximately EUR 54,000 (depending on patient complexity) al settings (Country names withheld to preserve anonymity, all costs expressed in Long term follow-up Teare et al. Orphanet Journal of Rare Diseases (2022) 17:58 Page 10 of 12 about overall diagnosis and care costs, given that differ- ent settings collect different types of cost-related infor- mation. However, the table begins to shed light on the potential cost items implied in the care of patients with CDI and indicates that the healthcare service costs are not negligible. professionals to identify symptoms, determine optimal treatment approaches and to effectively manage changes in treatment needs over time.h The cost data drawn from four hospital settings, despite the caveats associated with the data, provides an indica- tion of the impact that time-consuming and challenging diagnostic testing and personalisation of treatment can have on healthcare system resource demands, with costs concentrated in the early stages of the patient pathway. Further research is needed to understand healthcare ser- vice-related costs across a broader range of settings. g g The data provided for each country refer to a specific patient population (paediatric or adult CDI patients), with particular mechanisms for data collection and pro- vision based on information clinicians could gather. Other caveats to bear in mind are that the figures pro- vided could be broken down by initial diagnosis, long term care and estimates of total costs for a 10-year timeframe and indicate highly variable ranges, in part dependent on patient complexity. For example, cost can be influenced by whether additional complex testing is needed beyond standard tests for CDI, such as additional cerebrospinal fluid analysis or biopsy to determine the cause of CDI, and by whether patients with CDI caused by traumatic brain injury need to be supported by neu- rosurgery. It can also be influenced by how long a patient needs to stay in hospital and whether they then need intensive care. If other hormonal treatment is required in addition to desmopressin, such as growth hormone treat- ment, there will be an additional cost. Therefore, costs of care are highly individualised depending on the specific circumstances of a patient. However, despite these fig- ures being only indicative and context-specific and illus- trating diverse cost-ranges across contexts, they indicate that the costs of caring for patients with CDI are not neg- ligible and reinforce key insights we have gained about the complexity of the process. Long term follow-up We have also identified key areas for future explora- tion, as part of a commitment to improving the diag- nosis, treatment and management of patients with CDI and supporting patient quality of life and outcomes. Our research suggests a need to focus efforts on some key areas. We acknowledge that the extent to which these apply more widely—i.e. beyond the countries represented by the clinical co-authors (France, Ireland, Italy, Spain, and the UK)—merits further research. However, these insights are also supported by the challenges highlighted in the wider global literature. More specifically, in reflecting on the challenges we have identified in this paper, there is scope to consider actions which could lead to improved guidance for pri- mary care physicians on how to identify symptoms and manage patients with CDI. There is also a need for more information, education and awareness raising for patients, carers and families on how to better manage the condition, including in light of comorbidities and changes in patient circumstances. Working with patient associations may be important in this regard. Develop- ing education and outreach for patients may also help patients engage with primary care and with specialists in secondary care. It is worth noting that there can also be additional out of pocket costs for patients, such as the cost of items nec- essary for coping with the condition, for example ice cold water, nappies and toilet rolls [PR-INT2]. Given the complexities of managing patients with CDI, further research is needed to understand whether there is potential for greater international consensus or guidance on best practice in the treatment and management of spe- cific types of patients, not dismissing the importance of personalised care. This may require research that would gather retrospective and real-world data to identify how specific practices differ across a broad range of contexts and how they relate to patient outcomes for patients with specific clinical and behavioural parameters. References 1. Ananthakrishnan S. Diabetes insipidus during pregnancy. Best Pract Res Clin Endocrinol Metab. 2016;30(2):305–15. 1. Ananthakrishnan S. Diabetes insipidus during pregnancy. Best Pract Res Clin Endocrinol Metab. 2016;30(2):305–15. 2. Arima H, Oiso Y, Juul KV, Nørgaard JP. Efficacy and safety of desmopressin orally disintegrating tablet in patients with central diabetes insipidus: results of a multicenter open-label dose-titration study. Endocr J. 2013;60(9):1085–94. 2. Arima H, Oiso Y, Juul KV, Nørgaard JP. Efficacy and safety of desmopressin orally disintegrating tablet in patients with central diabetes insipidus: results of a multicenter open-label dose-titration study. Endocr J. 2013;60(9):1085–94. 3. Astaf’eva LI. The efficacy of desmopressin in the treatment of central diabetes insipidus after resection of chiasmo-sellar region tumors. Zh Vopr Neirokhir Im N Burdenko. 2017;81(4):61–9. 4. Baldeweg SE, Ball S, Brooke A, Gleeson HK, Levy MJ, Prentice M, et al. Society for endocrinology clinical guidance: Inpatient management of cranial diabetes insipidus. Endocr Connect. 2018;7(7):G8-g11. Funding 8. Christ-Crain M, Hoorn EJ, Sherlock M, Thompson CJ, Wass JAH. Endocri‑ nology in the time of COVID-19: management of diabetes insipidus and hyponatremia. Eur J Endocrinol. 2020;183(1):G9-g15. 8. Christ-Crain M, Hoorn EJ, Sherlock M, Thompson CJ, Wass JAH. Endocri‑ nology in the time of COVID-19: management of diabetes insipidus and hyponatremia. Eur J Endocrinol. 2020;183(1):G9-g15. This research was funded by Ferring Pharmaceuticals. Additional file 1: Methodology supplement. Received: 26 October 2021 Accepted: 30 January 2022 Received: 26 October 2021 Accepted: 30 January 2022 Availability of data and materials 9. Dabrowski E, Kadakia R, Zimmerman D. Diabetes insipidus in infants and children. Best Pract Res Clin Endocrinol Metab. 2016;30(2):317–28. 9. Dabrowski E, Kadakia R, Zimmerman D. Diabetes insipidus in infants and children. Best Pract Res Clin Endocrinol Metab. 2016;30(2):317–28. The data generated or analysed during this study are included in this published article and its Additional file 1. In line with informed consent, an exception is individual transcripts of interviews and workshop discussions due to risks of disclosing linkage of individual statements to individuals even when data is de-identified and a commitment to preserve anonymity. Further information outlining the methods used in this study is provided in the sup‑ porting information. 10. Elder C, Dimitri P. Diabetes insipidus and the use of desmopressin in hos‑ pitalised children. Arch Dis Childh Educ Pract Ed 2017;102:edpract-2016. 10. Elder C, Dimitri P. Diabetes insipidus and the use of desmopressin in hos‑ pitalised children. Arch Dis Childh Educ Pract Ed 2017;102:edpract-2016. 11. Garrahy A, Moran C, Thompson CJ. Diagnosis and management of centra diabetes insipidus in adults. Clin Endocrinol (Oxf). 2019;90(1):23–30. 11. Garrahy A, Moran C, Thompson CJ. Diagnosis and management of centra diabetes insipidus in adults. Clin Endocrinol (Oxf). 2019;90(1):23–30. 12. Garrahy A, Thompson CJ. Management of central diabetes insipidus. Best Pract Res Clin Endocrinol Metab. 2020:101385. 13. Hunter JD, Calikoglu AS. Etiological and clinical characteristics of central diabetes insipidus in children: a single center experience. Int J Pediatr Endocrinol. 2016;2016:3. Discussion and conclusion: towards improved care pathways Within this paper we have considered current practice in the diagnosis, treatment and management of CDI, and discussed the associated challenges relating to dif- ferential diagnosis and highly personalised treatment and management. We have shown that CDI requires complex patient support. This means care delivery needs to be flexible to meet individual patient needs. The fact that patient response to the mainstream treatment desmopressin can change over time makes long term management needs difficult to predict. This highlights the need for patients to be well-informed in order to effectively engage with healthcare professionals in primary and specialist care. It also highlights the need for highly skilled healthcare i Finally, the COVID-19 pandemic introduced further challenges for patients with CDI, given high needs for inpatient care during diagnosis and treatment optimi- sation and there is scope for the clinical community to jointly consider how to approach patient care in the con- text of future pandemic preparedness. We hope that the insights and reflections we have shared in this research help to raise awareness of the Teare et al. Orphanet Journal of Rare Diseases (2022) 17:58 Page 11 of 12 Page 11 of 12 complexity of managing patients with CDI and support future efforts of clinicians, patient associations, policy- makers and the community of patients and carers com- mitted to improving the care of patients with this rare but life-impacting condition. Universitario Niño Jesús, Instituto de Investigación La Princesa, Universidad Autónoma de Madrid, Madrid, Spain. 3 Centro de Investigación Biomédica en Red (CIBER) de Fisiopatología de la Obesidad y Nutriciόn (CIBEROBN), Instituto de Salud Carlos III, Madrid, Spain. 4 IMDEA, Food Institute, CEIUAM+CSI, Madrid, Spain. 5 UCL Great Ormond Street (GOS) Institute of Child Health, University College London, London, UK. 6 Great Ormond Street Hospital for Children, London, UK. 7 Assistance Publique‑Hôptaux de Paris, Pediatric Endocrinol‑ ogy‑Diabetology Department, Reference Center for Growth and Development Endocrine Diseases, Robert Debré University Hospital, Université de Paris, NeuroDiderot INSERM UMR 1141, 75019 Paris, France. 8 Department of Pedi‑ atrics, IRCCS Istituto Giannina Gaslini, Genoa, Italy. 9 Department of Neurosci‑ ence, Rehabilitation, Ophthalmology, Genetics, Maternal and Child Health, University of Genova, Genoa, Italy. 10 Department of Endocrinology, Beaumont Hospital and Royal College of Surgeons in Ireland, Dublin, Ireland. Acknowledgements We would like to thank Pat MacBride from the Pituitary Foundation, UK, Robert Brady from the Pituitary Foundation, Ireland, and Diana Vitali from the Italian Family Association of Septo-optic Dysplasia and Pituitary Abnormalities, Italy, for their insightful contributions to this study, providing an overview of the patient experience across different health systems. We would also like to thank Dr. Camilla D’Angelo and Giulia Maistrello, analysts at RAND Europe, for their help in translation, as part of the recruitment of patient representa‑ tives, and Dr. Daniela Rodriguez Rincon for her involvement in early stages of the research. This research study was funded by Ferring Pharmaceuticals, and we thank Teodor Burtea, MD and Lena Maislisch, M.Sc., both from Global Medical Affairs at Ferring, for their contributions to the concept for this study at ideation stages. The research was conducted and data analysed entirely independently, in line with RAND Europe’s role as an impartial, not-for-profit research organisation. Competing interests The authors have no competing interests to declare. 18. Mavinkurve M, McGrath N, Johnston N, Moloney S, Murphy NP, Hawkes CP. Oral administration of diluted nasal desmopressin in manag‑ ing neonatal central diabetes insipidus. J Pediatr Endocrinol Metab. 2017;30(6):623–8. Authors’ contributions 5. Barbot M, Ceccato F, Zilio M, Albiger N, Sigon R, Rolma G, et al. Cardio‑ vascular autonomic dysfunction in patients with idiopathic diabetes insipidus. Pituitary. 2018;21(1):50–5. G-CA and JF were responsible for conducting interviews with patient rep‑ resentatives, all authors were involved in the workshop discussions. SM and HT contributed to the design of the study. All authors (HT, JA, MD, JL, MM, MS, G-CA, JF, SM) contributed to drafting and developing the manuscript. All authors read and approved the final manuscript. 6. Chanson P, Salenave S. Diabetes insipidus and pregnancy. Ann Endocrino (Paris). 2016;77(2):135–8. 7. Christ-Crain M, Bichet DG, Fenske WK, Goldman MB, Rittig S, Verbalis JG, et al. Diabetes insipidus. Nat Rev Dis Primers. 2019;5(1):54. 7. Christ-Crain M, Bichet DG, Fenske WK, Goldman MB, Rittig S, Verbalis JG, et al. Diabetes insipidus. Nat Rev Dis Primers. 2019;5(1):54. Ethics approval and consent to participate 14. Ito A, Nozaki A, Horie I, Ando T, Kawakami A. Relation between change in treatment for central diabetes insipidus and body weight loss. Minerva Endocrinol. 2019;44(1):85–90. This research took the form of a literature review, supplemented with a small number of interviews and workshops with clinical experts and representatives from patient organisations. It was judged to pose minimal risks to participants and ethical approval was not sought. The research was conducted in accord‑ ance with the Declaration of Helsinki. All participants gave informed consent and were provided with participant information sheets as part of this process. 15. Kalra S, Zargar AH, Jain SM, Sethi B, Chowdhury S, Singh AK, et al. Diabetes insipidus: the other diabetes. Indian J Endocrinol Metab. 2016;20(1):9–21. 16. Kataoka Y, Nishida S, Hirakawa A, Oiso Y, Arima H. Comparison of inci‑ dence of hyponatremia between intranasal and oral desmopressin in patients with central diabetes insipidus. Endocr J. 2015;62(2):195–200. Supplementary Information The online version contains supplementary material available at https://​doi.​ org/​10.​1186/​s13023-​022-​02191-2. Consent for publication p All authors have consented to the publication of this manuscript. 17. Masri-Iraqi H, Hirsch D, Herzberg D, Lifshitz A, Tsvetov G, Benbassat C, et al. Central diabetes insipidus: clinical characteristics and long-term course in a large cohort of adults. Endocr Pract. 2017;23(5):600–4. Author details 1 1 RAND Europe, Westbrook Centre Milton Road, Cambridge CB4 1YG, UK. 2 Department of Pediatrics and Pediatric Endocrinology, Hospital Infantil Page 12 of 12 Teare et al. Orphanet Journal of Rare Diseases (2022) 17:58 Teare et al. Orphanet Journal of Rare Diseases (2022) 17:58 19. Nozaki A, Ando T, Akazawa S, Satoh T, Sagara I, Horie I, et al. Quality of life in the patients with central diabetes insipidus assessed by Nagasaki Diabetes Insipidus Questionnaire. Endocrine. 2016;51(1):140–7. 20. Patti G, Ibba A, Morana G, Napoli F, Fava D, di Iorgi N, et al. Central diabe‑ tes insipidus in children: Diagnosis and management. Best Pract Res Clin Endocrinol Metab. 2020:101440. 20. Patti G, Ibba A, Morana G, Napoli F, Fava D, di Iorgi N, et al. Central diabe‑ tes insipidus in children: Diagnosis and management. Best Pract Res Clin Endocrinol Metab. 2020:101440. 21. Patti G, Scianguetta S, Roberti D, Di Mascio A, Balsamo A, Brugnara M, et al. Familial neurohypophyseal diabetes insipidus in 13 kindreds and 2 novel mutations in the vasopressin gene. Eur J Endocrinol. 2019;181(3):233–44. 22. Refardt J, Christ-Crain M. Diabetes insipidus in pregnancy: how to advice the patient? Minerva Endocrinol. 2018;43(4):458–64. 22. Refardt J, Christ-Crain M. Diabetes insipidus in pregnancy: how to advice the patient? Minerva Endocrinol. 2018;43(4):458–64. 3. Refardt J, Winzeler B, Christ-Crain M. Diabetes Insipidus: An Upda 23. Refardt J, Winzeler B, Christ-Crain M. Diabetes Insipidus: Endocrinol Metab Clin North Am. 2020;49(3):517–31. 23. Refardt J, Winzeler B, Christ-Crain M. Diabetes Insipidu Endocrinol Metab Clin North Am. 2020;49(3):517–31. 24. Sbardella E, Pozza C, Isidori AM, Grossman AB. Endocrinology and adoles‑ cence: dealing with transition in young patients with pituitary disorders. Eur J Endocrinol. 2019;181(4):R155–71. 25. Smego AR, Backeljauw P, Gutmark-Little I. Buccally administered intra‑ nasal desmopressin acetate for the treatment of neurogenic diabetes insipidus in infancy. J Clin Endocrinol Metab. 2016;101(5):2084–8. Teare et al. Orphanet Journal of Rare Diseases (2022) 17:58 Publisher’s Note Springer Nature remains neutral with regard to jurisdictional claims in pub‑ lished maps and institutional affiliations. Springer Nature remains neutral with regard to jurisdictional claims in pub‑ lished maps and institutional affiliations. • fast, convenient online submission • thorough peer review by experienced researchers in your field • rapid publication on acceptance • support for research data, including large and complex data types • gold Open Access which fosters wider collaboration and increased citations maximum visibility for your research: over 100M website views per year • At BMC, research is always in progress. Learn more biomedcentral.com/submissions Ready to submit your research Ready to submit your research ? 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W4394815528.txt
https://www.emerald.com/insight/content/doi/10.1108/IJDI-12-2023-0287/full/pdf?title=efficiency-of-brics-countries-in-sustainable-development-a-comparative-data-envelopment-analysis
en
Efficiency of BRICS countries in sustainable development: a comparative data envelopment analysis
International journal of development issues
2,024
cc-by
8,611
The current issue and full text archive of this journal is available on Emerald Insight at: https://www.emerald.com/insight/1446-8956.htm Efficiency of BRICS countries in sustainable development: a comparative data envelopment analysis Isabella Melissa Gebert Iscte-Instituto Universitário de Lisboa, Lisbon, Portugal, and Efficiency of BRICS countries Received 12 December 2023 Revised 27 February 2024 Accepted 8 March 2024 Felipa de Mello-Sampayo Iscte-Instituto Universitário de Lisboa and Business Research Unit (BRU-IUL), Lisbon, Portugal Abstract Purpose – This study aims to assess the efficiency of Brazil, Russia, India, China, South Africa (BRICS) countries in achieving sustainable development by analyzing their ability to convert resources and technological innovations into sustainable outcomes. Design/methodology/approach – Using data envelopment analysis (DEA), the study evaluates the economic, environmental and social efficiency of BRICS countries over the period 2010–2018. It ranks these countries based on their sustainable development performance and compares them to the period 2000–2007. Findings – The study reveals varied efficiency levels among BRICS countries. Russia and South Africa lead in certain sustainable development aspects. South Africa excels in environmental sustainability, whereas Brazil is efficient in resource utilization for sustainable growth. China and India, despite economic growth, face challenges such as pollution and lower quality of life. Research limitations/implications – The study’s findings are constrained by the DEA methodology and the selection of variables. It highlights the need for more nuanced research incorporating recent global events such as the COVID-19 pandemic and geopolitical shifts. Practical implications – Insights from this study can inform targeted and effective sustainability strategies in BRICS nations, focusing on areas such as industrial quality improvement, employment conditions and environmental policies. Social implications – The study underscores the importance of balancing economic growth with social and environmental considerations, highlighting the need for policies addressing inequality, poverty and environmental degradation. Originality/value – This research provides a unique comparative analysis of BRICS countries’ sustainable development efficiency, challenging conventional perceptions and offering a new perspective on their progress. Keywords Sustainable development, Data envelopment analysis, Efficiency, BRICS Paper type Research paper © Isabella Melissa Gebert and Felipa de Mello-Sampayo. Published by Emerald Publishing Limited. This article is published under the Creative Commons Attribution (CC BY 4.0) licence. Anyone may reproduce, distribute, translate and create derivative works of this article (for both commercial and non-commercial purposes), subject to full attribution to the original publication and authors. The full terms of this licence may be seen at http://creativecommons.org/licences/by/4.0/legalcode Fundação para a Ciência e Tecnologia under UIDB/00315/2020 grant is gratefully acknowledged. The funding body had no influence in the design of the study, collection, analysis or interpretation of data or in writing the manuscript. International Journal of Development Issues Emerald Publishing Limited 1446-8956 DOI 10.1108/IJDI-12-2023-0287 IJDI 1. Introduction Economic growth theories have evolved from emphasizing capital accumulation, as in classical and neoclassical theories, to highlighting the importance of human capital in more contemporary frameworks such as the Endogenous Growth theory (Choudhry et al., 2020). Dore and Teixeira (2023) synthesized these factors into seven categories: human capital, labor and demographic conditions, technology and innovation, macroeconomic conditions, international trade and FDI, natural resources and institutional conditions. However, the relative influence of these factors varies significantly between and within countries, as noted by Chirwa and Odhiambo (2016). In developed countries, key factors include physical capital, fiscal policy and human capital, among others, while developing countries focus more on external elements such as FDI and foreign aid. The complex interplay of these factors is further complicated by issues such as income inequality, labor exploitation and environmental degradation, underscoring the need for quality growth that reflects true wellbeing (Lopez et al., 2008; Ranis et al., 2000). This recognition has led to the development of new indicators such as the Human Development Index, the Gini Coefficient and life expectancy at birth, which include aspects of life quality, income distribution and health, but often overlook the environmental costs of economic growth (Santana et al., 2014; Chen et al., 2021; Hasell, 2023). The concept of Sustainable Development emerged as a response to these challenges, aiming to harmonize economic growth with environmental conservation (Almeida et al., 2017). Its roots can be traced back to classical economists such as Malthus, Smith, Ricardo and Mill, who expressed concerns about the sustainability of economic growth and its implications for future generations (Purvis et al., 2019). The concept gained prominence after the 1972 Stockholm Conference, which led to the formation of the United Nations Environmental Program. However, the conference highlighted the tension between environmental protection and the development needs of different countries (Chasek, 1994; Prizzia, 2017). Sustainable development was formally defined as meeting current needs without compromising future generations’ ability to meet their own (Brundtland, 1987). The term “sustainability” has since been interpreted in various ways, often encompassing social, economic and environmental dimensions. This three-pillar model, while not explicitly outlined in foundational documents such as the Brundtland Report or Agenda 21, has become a widely accepted framework for understanding sustainability (Purvis et al., 2019; Moldan et al., 2012). Brown et al. (1987) and Pope et al. (2004) further explored these dimensions, emphasizing the integration of social needs, environmental protection and economic growth as key to implementing sustainable development. Brazil, Russia, India, China and South Africa (BRICS) countries account for approximately 40% of the global population, 30% of the land area, 18% of international trade and a quarter of the world’s GDP, surpassing the economies of both the USA and the European Union, as highlighted by O’Neill (2021). Moreover, their substantial role in fossil fuel production and consumption ranks them among the largest emitters of greenhouse gases (GHGs), significantly impacting global climate governance, a challenge discussed by Downie and Williams (2018). The BRICS face the complex task of maintaining rapid economic growth while controlling carbon emissions. Exploring their historical growth determinants, environmental impacts and population welfare offers crucial insights for their future development paths. Following Santana et al. (2014), this study examines how well the BRICS countries are doing in terms of sustainable development, using the data envelopment analysis (DEA) method that allows to rank the BRICS countries based on their economic, environmental and social efficiency. We specifically look at how efficiently these countries have been using their productive resources and technological innovations to achieve sustainable development over an eight-year period (2010–2018). The motivation for extending the data for Santana et al.’s (2014) paper stems from the dynamic nature of economic, social and environmental development across the BRICS. Santana et al.’s (2014) study provided critical insights into the efficiency of these countries in converting inputs into sustainable development outcomes over the period 2001–2007. However, the global economic landscape, environmental challenges and social dynamics have undergone significant changes since then. Extending the data set beyond 2007 allows for an updated analysis that reflects recent developments, including technological advancements, policy reforms and shifts in global economic power dynamics. This extension aims to capture the evolving nature of sustainability efforts within the BRICS, offering a more current understanding of their progress and challenges in achieving economic growth, environmental protection and social welfare. It acknowledges the importance of continuous monitoring and evaluation in the context of sustainable development, providing stakeholders with relevant data to inform policymaking and strategic planning in an ever-changing global environment. The results of this study show the different challenges the BRICS countries have in achieving sustainable development. Brazil is efficient in using its resources and foreign investment for sustainable growth but needs to work on issues such as deforestation, social inequality and political instability. Russia is the most efficient of the group but relies heavily on oil and gas, which affects its sustainability. It could improve by investing more in human resources. India and China have grown economically but this has led to more pollution and a lower quality of life. They need to focus on cleaner industrial growth and better working conditions and use foreign investment and research to reduce pollution and improve living standards. South Africa has low pollution levels and is doing well in addressing social and health issues, but it needs to keep working on these areas to improve people’s lives. This paper is organized as follows. Section 2 starts discussing BRICS countries’ economic development. The methodology section given in Section 3 explains the research design and analysis techniques used. The findings and analysis along with the results are presented in Section 4. The discussion is presented in Section 5. The paper ends with a conclusion given in Section 6 which summarizes the main points and suggests areas for future research. 2. Economic growth and sustainable development of the Brazil, Russia, India, China, South Africa Since their formal recognition as a cohesive group in 2009, although they did not initially collaborate closely, as noted by Lowe (2016), BRICS leaders have convened regularly to formulate cooperative policies and strategies aimed at bolstering their joint economic growth. Despite attracting global attention for their economic prowess, growth within the BRICS has seen a period of stagnation and inconsistency since 2011, as observed by Fisher (2022). The BRICS consist of a diverse set of countries, each with unique political and economic frameworks (Chatterjee and Naka, 2022). Brazil and Russia, for instance, are primarily exporters of raw materials, dependent on their rich mineral reserves. In contrast, China and India have leveraged their vast labor forces, with China emerging as a major manufacturer and India as a significant service provider (Streltsov et al., 2021). South Africa stands out as the most industrialized nation in Africa, with substantial mineral wealth and a reputation as a stable investment destination (Lowe, 2016). Despite their differences, these countries share certain common economic traits (Basu et al., 2013). For instance, a large proportion of trade in intermediates occurs among BRICS countries (De Mello-Sampayo, 2017a, 2017b). Efficiency of BRICS countries IJDI Key macroeconomic factors shaping the BRICS’ economic growth include fiscal policy, exchange rates, trade openness, FDI inflows and inflation rates (Bezerra and Silva, 2021). The role of FDI in economic development is debated; some studies, such as those of Khalid and Marasco (2019), suggest it is beneficial, while others present a more nuanced view. Choudhry et al. (2020) and Saini and Singhania (2018) indicate that the impact of FDI can be significant, especially when coupled with technology transfer and capital accumulation. Borensztein et al. (1998) found FDI’s impact to be positive but contingent on the host country’s absorptive capacity. Further, Khan and Nawaz (2019) observed a positive correlation between trade openness, FDI and income distribution. Long-term benefits of FDI in BRICS, aligned with technical cooperation, were noted by Prabhakar et al. (2015) and Agrawal (2015). Joshua et al. (2020) identified FDI as a crucial element for South Africa’s economic growth, noting that in 2017, BRICS attracted 19% of global FDI inflows (UNCTAD, 2018). Human capital is another pivotal factor for economic development in these countries. It enhances trade openness and fosters knowledge transfer, as noted by Nakabashi and Figueiredo (2005). Barro (1991) posited that human capital facilitates growth by disseminating technology from more developed countries, allowing less wealthy nations to accelerate their economic expansion. Fisher (2022) confirmed a strong positive relationship between labor force participation and GDP per capita growth rate in BRICS from 2009 to 2019. Hartman and Kwon (2005) found that human capital significantly reduces environmental pollution in China. Nakabashi and Figueiredo (2005) further argued that human capital indirectly bolsters income growth per worker through the acceleration of technological diffusion, intensified by imports and FDI. Hu (2021) echoed these findings, emphasizing the role of human capital in enhancing the benefits of imported technology and innovation. Infrastructure investment, encompassing transportation, energy, telecommunications and other critical facilities, also plays a crucial role in economic performance. Calderon and Serven (2015) and Kodongo and Ojah (2016) identified a strong link between infrastructure investment and economic growth. Meidayati (2017) highlighted the impact of telecommunications infrastructure and market size on developing countries’ economies. However, Apurv and Uzma (2020) found mixed results regarding the significance of infrastructure investment in economic growth, particularly in Brazil and South Africa compared to Russia, India and China. Technological advancements, R&D expenditure and knowledge spillovers are key contributors to the BRICS’ productivity and competitiveness, as outlined by Franco and Oliveira (2017) and Hu (2021). Gyedu et al. (2021) analyzed the influence of R&D, trademarks and patents on economic growth, concluding that innovation investments significantly bolster growth. Ndlovu and Inglesi-Lotz (2020) explored variations in the impact of R&D expenditure on economic growth across BRICS countries. The BRICS’ rapid economic expansion has raised concerns about their seemingly limitless exploitation of natural resources, as discussed by Gomes and Silva (2017). Their significant environmental footprint and GHG emissions underscore the urgency of implementing strategies to mitigate global pollution. While the BRICS are active participants in international environmental conventions, they lack a unified approach to sustainable policy transition, operating under the principle of “common but different responsibilities”. Brazil and South Africa emphasize sustainable development, whereas China and India prioritize economic growth, as noted by Cavalcanti (2018). Regarding climate change, Brütsch and Papa (2013) argued that the BRICS lack sufficient common interests to form a lasting coalition. Although the group has convened environment and energy minister meetings (BRICS, 2015), they have yet to establish specific cooperation mechanisms. Each nation faces unique challenges related to climate change. Russia continues to rely on traditional energy sources, addressing issues such as inequality and poverty. Brazil, India and China are moving towards renewable energy through legislative changes (Basile and Cecchi, 2019). South Africa, while transitioning to a low-carbon society, has implemented strategies to promote social inclusion and address climate change (Cavalcanti, 2018). Each BRICS country provides detailed reports on their commitments to the UN’s Sustainable Development Goals and Millennium Development Goals. However, Basile and Cecchi (2019) noted inconsistencies in their engagement with these goals and the Paris Agreement, with a continued dependence on traditional energy sources in some countries. Efficiency of BRICS countries 3. Methodology In this study, we aim to evaluate the efficiency of BRICS countries in channeling their productive capacities and innovative capabilities into sustainable development. The DEA model is used to measure and compare the efficiency of BRICS countries over the period from 2010 to 2018. We conducted three separate DEA analyses, each focusing on one of the sustainable development pillars: economic, social and environmental sustainability. The source of the data is the World Data Bank. The selection of input variables was grounded in existing literature. The variable entitled Gross Fixed Capital Formation (GFCF) was chosen to represent the capital variable, reflecting its established correlation with economic growth as discussed in the studies by Meyer and Sanusi (2019) and Topcu et al. (2020). Following Santana et al. (2014) and Bekun et al. (2019), the unemployed population was used as a proxy for human capital. Research and development (R&D) expenditure was used to assess the effect of technological innovation on sustainable development (Bayarçelik and Tas el, 2012; Costantini et al., 2023). The variable foreign direct investment (FDI) inflows are included in our analysis acknowledging its significant role in innovation and sustainable development in developing countries, as evidenced in studies by Lee et al. (2021), Chai et al. (2021), Sarkodie and Strezov (2019) and Sunde (2016). For output variables, gross domestic product (GDP) was selected to represent economic growth (Apergis and Payne, 2011; Sanz-Díaz et al., 2017). The variable life expectancy was chosen to reflect the social dimension, aligning with its use as a general health indicator in studies by Luy et al. (2020), Mariano and Rebelatto (2014) and Magombeyi and Odhiambo (2018). To proxy environmental sustainability, CO2 emissions were used as a standard metric for assessing environmental impact (Maryam et al., 2017; Shikwambana et al., 2021; Lee et al., 2021). The stepwise method was applied to validate the chosen variables for DEA applications. This process starts with selecting the most statistically significant variable, followed by a sequential addition or removal of variables based on set criteria. The variables used in the three DEA applications are detailed in Table 1, categorized by the type of efficiency they represent (economic, environmental, social), their inputs and outputs. For the first and second applications, the economic and environmental efficiency was tested, using GDP and CO2 as the outputs, respectively, and GFCF, R&D, FDI and unemployed population as inputs for both applications. The third application concerns the Application Type of efficiency Inputs Output 1 2 3 Economic Environmental Social GFCF, R&D, FDI, unemployed population GFCF, R&D, FDI, unemployed population GFCF, R&D, FDI GDP CO2 emissions Life expectancy Source: Table by authors Table 1. Variables used IJDI social pillar. Its output is the life expectancy, and the analysis was made considering the inputs GFCF, R&D and FDI. The DEA is a non-parametric linear programming method used to measure the efficiency of decision making units (DMUs) when facing multiple inputs and multiple outputs (Charnes et al., 1978). One of the most commonly used models is the BCC model (Banker et al., 1984). The BCC model allows for variable returns to scale (VRS), meaning that as inputs are increased, outputs do not necessarily increase in a fixed proportion. As the BRICS countries aim to increase the outputs, i.e. aim for their sustainable development, the BCC–output-oriented model is used to analyze the economic, social and environmental applications. The output-oriented DEA-BCC model is characterized by its objective function and constraints, with a focus on maximizing output efficiency given a set of inputs and outputs, defined as follows: n X Min vj  xj0 – w; j¼1 Subject to m X ui  yi0 ¼ 1; i ¼ 1 m X i ¼ 1 ui  yik  n X vj  xjk þ w # 0; for k ¼ 1; 2; . . . ; h: j¼1 Where n and m are the number of inputs and outputs analyzed, respectively; h is the number of decision-making units (DMUs) analyzed; w is the scale factor; vj is the weight of input j for the DMU; ui is the weight of output i for the DMU; xj0 is the amount of input j of the DMU; yi0 is the amount of output i of the DMU; xjk is the amount of input j of DMU k; and yik is the amount of input i of DMU k. The nature of the output CO2 emissions, which is undesirable was transformed to fit the DEA framework. This transformation involved multiplying the emissions data by “1” and adding a translation vector to ensure the transformed values remained positive without altering their relational dynamics. To enhance discrimination between DMU efficiency scores, we used the inverted frontier method (Angulo Meza et al., 2003). This approach involves reversing the roles of inputs and outputs in the DEA model and then creating a composite index to rank the units. This index is computed by averaging the classic frontier efficiency score and the inverted efficiency score, normalized against the highest value obtained (Leta et al., 2005). Finally, the study includes an analysis of efficiency trends over the years using the “window analysis” technique as described by Cooper et al. (2007). This approach treats each unit per year as a distinct DMU, with a moving average calculated as new units are added and old ones removed. The number of windows and their respective amplitudes are determined based on the total number of years analyzed, in this case, resulting in five windows with a five-year amplitude, as follows: w¼kpþ1 p¼ kþ1 2 In which, w represents the number of windows; p represents the window amplitude and k represents the number of years. As the years taken into consideration for this analysis are 9, the number of windows corresponded to 5 and the amplitude corresponded to 5 years. 4. Results The results of the DEA across three sustainability dimensions: economic, social and environmental are presented in Tables 2, 3 and 4, respectively. Efficiency scores for each country were computed for every analysis window, leading to a comprehensive average index for each sustainability aspect. Table 5 ranks the countries according to the average index. Finally, Table 6 provides data on gross domestic product (GDP), CO2 emissions, life expectancy, GFCF, research and development (R&D) expenditure, foreign direct investment (FDI) inflows and the unemployed population of BRICS countries, offering insights into their development trajectories and policy focuses. The economic dimension results are outlined in Table 2, showing efficiency trends across five-year windows from 2010 to 2018 for each BRICS country, alongside their average total Country Brazil Russia India China South Africa 1 (2010/14) (%) 2 (2011–15) (%) Window 3 (2012–16) (%) 4 (2013–17) (%) 5 (2014–18) (%) Mean total (%) 79.62 90.18 68.26 72.42 74.03 78.16 92.16 68.45 70.57 72.52 77.80 91.69 70.76 70.57 71.77 78.60 91.79 71.68 70.75 71.14 72.58 92.14 70.03 68.27 68.23 77.35 91.60 69.84 70.52 71.54 Source: Table by authors Country Brazil Russia India China South Africa 1 (2010/2014) (%) 2 (2011–2015) (%) Window 3 (2012–2016) (%) 4 (2013–2017) (%) 5 (2014–2018) (%) Mean total (%) 90.36 93.49 89.63 52.91 89.54 89.09 93.50 88.14 52.63 97.81 89.31 93.95 88.44 53.87 98.49 89.53 94.13 87.98 53.18 98.40 89.21 95.70 87.76 53.23 99.03 89.50 94.16 88.39 53.16 96.65 1 (2010/2014) (%) 2 (2011–2015) (%) Window 3 (2012–2016) (%) 4 (2013–2017) (%) 5 (2014–2018) (%) Mean total (%) 94.54 95.76 47.58 16.45 99.65 94.20 96.57 47.46 15.21 99.47 93.33 97.48 47.51 14.77 99.40 92.11 98.19 47.75 14.20 99.41 89.04 97.69 47.13 13.51 97.63 92.65 97.14 47.49 14.83 99.11 Source: Table by authors Country Brazil Russia India China South Africa Source: Table by authors Efficiency of BRICS countries Table 2. Economic application Table 3. Social application Table 4. Environmental application IJDI efficiency. Russia emerges as the leader in this category, with Brazil, South Africa and China following and India trailing. Notably, India and China displayed increasing efficiency scores in the initial windows, but most countries except Russia experienced a decline in the subsequent window. Table 3 details the social sustainability findings, positioning South Africa at the forefront, closely followed by Russia. Both these countries improved their efficiency scores over the study period. Brazil, occupying the third rank, showed a declining trend, with India and China at the lower end, China recording the least efficiency at 53.16%. Environmental efficiency, as per Table 4, places South Africa at the top with near-perfect efficiency, closely followed by Russia. Brazil, India and China round out the rankings, with China significantly lagging behind others with a mere 14.83% efficiency. To synthesize these results, Table 5 was constructed, offering a consolidated view of the efficiency rankings across all three applications. Russia and South Africa consistently rank high, while India and China are positioned at the bottom of the efficiency scale. Table 6 provides a breakdown of the input and output values for the variables used in the DEA, averaged over the period 2010–2018. Table 6 serves as a quantitative foundation, underpinning the DEA analysis and offering deeper insights into the variables influencing each country’s sustainable development efficiency. Notably, China leads in GDP, CO2 emissions and life expectancy, reflecting its rapid industrial growth and large population. India, with significantly high CO2 emissions, also has a high unemployed population, suggesting economic growth may not be equitably translating into job creation. Russia and Brazil present moderate figures in most categories, though Brazil’s relatively high Application Social Economic Table 5. Mean efficiency rankings of BRICS from 2010 to 2018 Brazil Russia India China South Africa 3rd 2nd 4th 5th 1st 3rd 2nd 4th 5th 1st Source: Table by authors Country Brazil Russia India China South Africa Table 6. Descriptive statistics 2nd 1st 5th 4th 3rd Environmental GDPa 1,790,640.45 1,359,389.57 2,002,225.282 10,413,780.02 338,802.96 Mean total (2010–2018) CO2 Life emissionsb expectancyc GFCFd R&De 454540.79 1628548.22 2062884.15 9741657.54 430355.03 74.1 71.9 68.9 76.7 63.0 FDIf 329,921,331.99 21,565.3 65,892,09 291,458,527.51 14,454,58 25,851.68 609,207,470.49 14,053.51 33,945.33 4,483,614.96 208,971.73 249,339.79 59,412.47 2,376.60 3,561.31 Unmployed populationg 9.21 4.29 37.85 35.36 5.10 Notes: World Data Bank; agross domestic product (constant 2015, million US$); CO2 emissions (kt); clife expectancy at birth (years); dgross fixed capital formation (constant, million 2015, US$); eR&D expenditure (constant 2015, million US$); fFDI inflows (constant 2015, million US$); gunmployed population (million) Source: Table by authors unemployment rate stands out. China’s substantial R&D expenditure and FDI inflows underline its position as a global economic powerhouse with a strong emphasis on innovation. In comparison, there are relatively low R&D investments in Russia and South Africa. The comparative analysis shown in Table 7 underscores the diverse trajectories of BRICS nations in balancing economic growth with social welfare and environmental sustainability. Brazil has seen a shift in its sustainability landscape, with its economic efficiency slightly declining from the top position, indicating slight decline in its economic management effectiveness. Socially, Brazil experienced a significant drop in efficiency, moving from first to third place, suggesting challenges in sustaining its previously achieved social welfare gains. Environmentally, Brazil improved its efficiency score but fell in rankings, implying that while it has intensified its environmental conservation efforts, other BRICS nations have made more substantial strides in this domain. Russia has demonstrated remarkable progress, especially in economic terms, where it leaped from the lowest to the highest efficiency score, showcasing significant improvements in economic management and output efficiency. Socially, Russia maintained its second-place ranking with an enhanced efficiency score, indicating steady advances in social welfare. The country also made notable gains in environmental efficiency, moving up in both score and ranking, which underscores Russia’s effective strategies in environmental management. India has remained at the lower end of economic efficiency among the BRICS, with only a minor improvement in score, highlighting ongoing difficulties in efficiently converting inputs into economic outputs. In the social domain, despite maintaining a low ranking, India’s improved score signals progress in social development. However, India faced a sharp decline in environmental efficiency, suggesting escalating challenges in managing environmental sustainability amid its economic and demographic growth. Economically, China saw a slight improvement in its efficiency score but dropped in ranking, suggesting that while there has been some progress, other BRICS countries have outpaced China in economic efficiency. Socially, China’s efficiency score and ranking both Country Application Brazil Economic Social Environmental Economic Social Environmental Economic Social Environmental Economic Social Environmental Economic Social Environmental Russia India China South Africa Efficiency score (2001–2007)a (%) Rank (2001–2007)a Efficiency score (2010–2018)b (%) Rank (2010–2018)b 98 99 90 51 89 78 49 49 81 65 56 21 66 76 99 1st 1st 2nd 4th 2nd 4th 5th 5th 3rd 3rd 4th 5th 2nd 3rd 1st 77 90 93 92 94 97 70 88 48 71 53 15 72 97 99 2nd 3rd 3rd 1st 2nd 2nd 5th 4th 4th 4th 5th 5th 3rd 1st 1st Sources: aSantana et al. (2014) results; bour results; table by authors Efficiency of BRICS countries Table 7. Evolution of BRICS countries’ efficiency in economic, social and environmental: a comparative analysis IJDI declined, reflecting significant hurdles in addressing social disparities despite its rapid economic growth. Environmentally, China experienced the most considerable decline in efficiency, remaining the least efficient, which highlights the severe environmental challenges it faces, underscoring the urgent need for sustainable environmental policies. South Africa experienced a drop in its comparative economic ranking but socially, the country has made remarkable strides, a testament to its effective strategies and policies aimed at enhancing social welfare and addressing societal challenges. Environmentally, South Africa has upheld its premier status, demonstrating a consistent dedication and effective approach to environmental management and sustainability initiatives. 5. Discussion In Brazil, median input levels, coupled with the second-highest foreign direct investment (FDI) inflows, have resulted in a paradox of high-efficiency scores and a mid-range ranking. This dichotomy points to a potential for Brazil to enhance its output maximization. Economic growth data from the Brazilian Institute of Geography and Statistics highlight uneven GDP growth, notably a decline from an average of 3.6% annually between 2001 and 2010 to just 0.7% between 2011 and 2018. This fluctuation reflects the impact of fiscal policies aimed at stimulating private consumption and demand, which, despite initial success, led to economic downturns post-2013 (Costa et al., 2017). Economically, Brazil’s efficient conversion of median inputs into high outputs places it second in economic efficiency among the BRICS. This suggests that maintaining input levels while refining fiscal policies could elevate GDP output. Environmentally, Brazil ranks third with a 92.68% efficiency score, reflecting its substantial renewable energy sector, primarily hydropower (Udemba and Tosun, 2022). However, high GHG emissions because of deforestation counter this achievement (Timperley, 2018). The correlation between rising FDI and decreasing CO2 emissions (Khatoon et al., 2022), amidst increasing deforestation, underscores the need for focused environmental policies. Socially, Brazil’s life expectancy of around 75 years (2018) and significant strides in reducing inequality during the Worker’s Party (PT) governance (Oliveira, 2023) contrast with its third-place social efficiency ranking. Programs such as Bolsa Família have substantially improved living conditions (Campoli et al., 2019), yet political shifts and reduced focus on such initiatives (Costa, 2019) have stalled progress, emphasizing the imperative of sustained social policies. Considering the comparative analysis with Santana et al.’s (2014) findings, Brazil’s trajectory in sustainability shows a moderate downturn in economic efficiency from its previously top position, a significant drop in social welfare efficiency from first to third place and an enhancement in its environmental endeavors. However, this progress is shadowed by more substantial advancements made by other BRICS nations in environmental management during the same period. Russia’s median Global Fixed Capital Formation (GFCF) and research and development (R&D) inputs, combined with the lowest unemployment, have yielded the highest efficiency rates in the group. Post-USSR, the Russian economy’s transition to a market-based system saw concentrated economic power and reliance on oil, accounting for 20% of GDP (Orazalin and Mahmood, 2018; Dabrowski, 2023). Despite a modest GDP, Russia’s efficient use of inputs, especially in using human capital and infrastructure development (Serbian et al., 2023), has led to a top economic efficiency ranking. Environmentally, Russia stands second with a 97.14% efficiency rate, managing low CO2 outputs from median inputs. However, as the world’s fourth-largest GHG emitter (Zagoruichyk, 2022), optimizing R&D and FDI towards reducing emissions is crucial. Socially, Russia’s advancements in poverty reduction, despite a lower life expectancy of 72.7 years, reflect a 94.16% efficiency rating, underscoring the role of government initiatives in social development (Rudenko and Satre, 2018). In comparison to Santana et al.’s (2014) earlier analysis, Russia demonstrates remarkable progress, moving from the lowest to the highest in economic efficiency, steadfastly holding its rank in social welfare and making notable strides in environmental efficiency. This upward trajectory signifies Russia’s successful implementation of effective management strategies and policy advancements, distinguishing its comprehensive growth and sustainability efforts among the BRICS nations. India’s high unemployment and R&D inputs, juxtaposed with median FDI inflows, have not translated into proportional outputs, placing it last in economic efficiency. Post-1991 reforms propelled growth, shifting the economic structure towards services over industry (The World Bank, 2018a, 2018b, 2018c; Anand, 2014). However, this premature deindustrialization might explain India’s low economic efficiency, as the advantages of industrialization remain unexploited. Environmentally, India ranks fourth with a 47.49% score, reflecting increased CO2 emissions alongside economic growth (Zameer et al., 2020; Timperley, 2019). The primary emission sources, including energy and agriculture, suggest inefficiencies in input utilization. Socially, India’s rapid growth has not inclusively benefitted its population, evident in its fourth-place social efficiency ranking and life expectancy of around 69 years. Initiatives aimed at reducing poverty and promoting non-farm employment (Pattayat et al., 2022) indicate potential areas for improvement. Relative to Santana et al.’s (2014) results, India’s journey reflects ongoing challenges in economic efficiency and environmental sustainability. Despite slight enhancements in economic performance and social development, India has experienced a discernible deterioration in environmental efficiency. This contrast highlights the persistent difficulties India faces in aligning its rapid economic and demographic expansion with effective environmental governance and sustainability practices. China, despite its high inputs and outputs, demonstrates suboptimal efficiency scores. As the world’s second-largest economy, its fourth-place economic efficiency ranking might stem from its shift to a service-oriented economy and rising unemployment influenced by technological advancements (Du and Wei, 2022; The World Bank, 2018a, 2018b, 2018c). In environmental terms, China ranks last with a 14.83% efficiency score, exacerbated by a vertically managed administrative system that prioritizes economic growth over environmental standards (Chai et al., 2021). Addressing FDI’s role in pollution (Azam et al., 2019; Chai et al., 2021) is essential for environmental improvement. Socially, China’s challenges in poverty, inequality and labor market distortions (Ebenstein et al., 2015) contribute to its last-place social ranking. When juxtaposed with Santana et al.’s (2014) findings, China’s path reflects incremental progress in economic efficiency and a decline in social welfare efficiency, alongside facing pronounced environmental hurdles. These dynamics underscore the pressing necessity for China to devise and implement more robust policies that reconcile its swift economic ascent with the imperatives of environmental protection and social equity. South Africa, with the lowest inputs and outputs, has attained notable efficiency across all domains. Its third-place economic ranking, with a 70.52% score, suggests potential for growth through enhanced FDI and R&D (Makhoba et al., 2019; Sunde, 2016; Quaynor et al., 2022). Environmental leadership is evident in its top ranking, reflecting the lowest CO2 outputs among BRICS and the impact of optimized inputs on environmental quality (Joshua et al., 2020). Socially, despite low life expectancy, South Africa’s high efficiency score is Efficiency of BRICS countries IJDI attributed to significant improvements in health and a need for policies promoting inclusive growth (WHO Africa, 2022; Francis and Webster, 2019; The World Bank, 2018a, 2018b, 2018c). Comparing South Africa’s sustainable development from 2000–2007 to the latest data, the country shows a nuanced evolution. Economically, there is a slight improvement in efficiency, but its ranking among BRICS nations has fallen, indicating faster progress by others. Socially, South Africa has moved to the top in efficiency, reflecting strong efforts in addressing social issues. Environmentally, it continues to lead, showing consistent commitment to sustainability. This comparison reveals South Africa’s progress in social and environmental areas, despite economic challenges relative to other BRICS countries. 6. Conclusions This study delves beyond traditional gross domestic product metrics to understand economic development, emphasizing the importance of sustainable growth that harmonizes economic expansion with quality-of-life improvements. Emerging countries, particularly the BRICS countries, have gathered global attention because of their rapid economic growth, substantial populations and rich natural resources. Hence, analyzing their growth bases and impacts on future generations is crucial, especially regarding how endogenous growth drivers can bolster sustainable development. Focusing on the BRICS countries’ resource conversion efficiency into sustainable development, this research used DEA across economic, social and environmental facets from 2010 to 2018. Compared to the previous study that used the 2000–2007 period, Russia and South Africa emerge as leaders, with Brazil also performing well. Russia’s success highlights the impact of strategic investments in human capital and green technologies. South Africa’s leading environmental efforts and top social ranking demonstrate progress in overcoming social challenges. Brazil shows effective resource and FDI conversion but faces challenges such as deforestation and social inequality. India needs to address CO2 emissions and improve working conditions, leveraging FDI and R&D for environmental and social gains. China’s low efficiency underscores the need for better industrial practices, employment conditions and targeted technological investments. Despite differing time frames, the studies collectively illustrate the ongoing efforts of BRICS nations to balance economic advancement with environmental protection and social inclusion, highlighting both achievements and areas requiring further attention. While insightful, this study misses recent global events, notably the COVID-19 pandemic and the Russia–Ukraine conflict, that have likely impacted BRICS’ sustainable development trajectories. Thus, future research must incorporate these developments, expanding variables and methodologies to offer a more complete understanding of the BRICS’ sustainable development paths. References Agrawal, G. (2015), “Foreign direct investment and economic growth in BRICS economies: a panel data analysis”, Journal of Economics Business and Management, Vol. 3 No. 4, pp. 421-424. 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(2022), “The carbon brief profile: Russia”, September 22, available at: www. carbonbrief.org/the-carbon-brief-profile-russia/ Zameer, H., Yasmeen, H., Zafar, W.M., Waheed, A. and Sinha, A. (2020), “Analyzing the association between innovation, economic growth, and environment: divulging the importance of FDI and trade openness in India”, Environmental Science and Pollution Research, Vol. 27 No. 23, pp. 29539-29553. Corresponding author Felipa de Mello-Sampayo can be contacted at: fdmso@iscte-iul.pt For instructions on how to order reprints of this article, please visit our website: www.emeraldgrouppublishing.com/licensing/reprints.htm Or contact us for further details: permissions@emeraldinsight.com Efficiency of BRICS countries
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Deep-learning-based Automatic Detection and Classification of Traffic Signs Using Images Collected by Mobile Mapping Systems
Sensors and materials
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*Corresponding author: e-mail: kemyoung@nsu.ac.kr https://doi.org/10.18494/SAM3956 Keywords: high-definition maps, traffic sign, mask R-CNN, Inception-v3, autonomous driving Keywords: high-definition maps, traffic sign, mask R-CNN, Inception-v3, autonomous driving As interest in autonomous driving has increased in recent years, various sensors have been developed for use in vehicles to detect and classify traffic signs. When road traffic facilities are not recognized owing to the malfunction of sensors, point cloud data and images collected by mobile mapping systems (MMSs) are used to construct high-definition maps containing road traffic facility information. However, when traffic signs, among the targets of high-definition map construction, are constructed using point cloud data, it becomes difficult to detect and classify traffic signs because they are highly reflective. In this study, we detected and sub- classified traffic signs by combining Mask Regions with Convolutional Neuron Network (Mask R-CNN) and Inception-v3 models based on image data obtained using MMSs. Image data obtained by various types of MMS were used to detect traffic signs and classification results were verified. The detection accuracy of traffic signs was 87.6% and the classification accuracy was 77.5%; thus, the method proposed in this study can be used to automatically construct traffic signs for high-definition maps. 4801 4801 Sensors and Materials, Vol. 34, No. 12 (2022) 4801–4812 MYU Tokyo Sensors and Materials, Vol. 34, No. 12 (2022) 4801–4812 MYU Tokyo S & M 3144 Sensors and Materials, Vol. 34, No. 12 (2022) 4801–4812 MYU Tokyo Deep-learning-based Automatic Detection and Classification of Traffic Signs Using Images Collected by Mobile Mapping Systems Hyeong-Yoon So and Eui-Myoung Kim* Graduate School of Spatial Information Engineering, Namseoul University, 91, Daehak-ro, Seonghwan-eup, Seobuk-gu, Cheonan-si, Chungcheongnam-do, 31020 Republic of Korea (Received April 27, 2022; accepted August 3, 2022; online published August 15, 2022) 1. Introduction As interest in autonomous driving has increased, studies are actively underway to detect and classify road traffic facilities on roads using various sensors, such as LiDAR, RADAR, and cameras. However, when sensors are used, there are cases when a malfunction occurs or objects of interest are not detected under adverse circumstances, including long distance, blind spots, or bad weather conditions.(1) Accordingly, the need to construct high-definition maps is imperative so that this information can compensate for the malfunction of the sensors. In South Korea, mobile mapping systems (MMSs) are currently used to collect various data, such as horizontal images, panoramic images, point clouds, and positional information to construct high-definition maps.(2) The process of producing high-definition maps is as follows: the operator uses the collected point cloud to detect and classify objects of interest manually with the naked eye, and then digitizes them to construct maps. However, in the process of detecting and classifying objects of interest, those that are highly reflective to laser beams, such as traffic ISSN 0914-4935 © MYU K.K. https://myukk.org/ ISSN 0914-4935 © MYU K.K. https://myukk.org/ ISSN 0914-4935 © MYU K.K. https://myukk.org/ 4802 Sensors and Materials, Vol. 34, No. 12 (2022) signs, have an insufficient point density in the point cloud, as shown in Figs. 1(b) and 1(d), making it difficult to classify and locate traffic signs with the naked eye. In contrast, classifying traffic signs and determining their locations are improved using images from MMS cameras, as shown in Figs. 1(a) and 1(c). In the case of traffic signs, using images from MMS cameras, rather than using point clouds, is effective when detecting and classifying objects of interest. In studies on the detection of traffic signs using point clouds collected by MMSs, after classifying the ground and non-ground surfaces, methods such as Euclidean distance clustering, filtering and clustering using reflection intensity, and bag of visual phrases (BoVP) can be used to detect traffic signs.(3,4) However, there are limitations, namely, the reflection intensity must be normalized for traffic sign detection, the process of preprocessing the point clouds consumes much time, and the results of traffic signs vary depending on the threshold setting determined by the user experience. For the efficient construction of high-definition maps, research is required to detect and classify traffic sign objects using images. 1. Introduction Studies on the detection of traffic signs using images have used image processing algorithms and convolutional neural network (CNN)-based deep learning models. Studies using image processing algorithms include a study using a FAST feature point extraction method and a study using the Viola-Jones algorithm to determine the edge of a traffic sign and then detect the traffic sign region using support vector machine (SVM).(5,6) These methods can be used to detect the edge of the traffic sign, but their limitation is that the region cannot be detected if no edge is determined for the traffic sign. Another study used color, shape, and both color and shape to detect traffic signs.(7) In the case of the color-based method, the process was fast and simple, but it was sensitive to light. The shape-based method was limited in that the detection result varied depending on the type of edge detector used. When color and shape were used simultaneously, traffic signs could be detected effectively using a maximally stable extremal region (MSER) and high-contrast region extraction (HCRE), but the limitation in using color and shape together is that the detection accuracy of the traffic signs varied depending on the color enhancement method. In a study that detected objects using a machine learning or CNN-based deep learning algorithm, the You Only Look Once (YOLO) V4-Tiny model was trained using approximately 9000 images to detect and classify 45 (a) (b) (c) (d) Fig. 1. (Color online) (a), (c) Traffic signs in images from MMS. (b), (d) Traffic signs in point cloud from MMS. (a) (c) (d) (b) (d) (a) (b) (c) Fig. 1. (Color online) (a), (c) Traffic signs in images from MMS. (b), (d) Traffic signs in point cloud from MMS. Sensors and Materials, Vol. 34, No. 12 (2022) 4803 types of traffic sign, but it showed a low accuracy of approximately 52%.(8) After training a Mask R-CNN model using 6000 images, a high accuracy of 95.2% was obtained after detecting and classifying ten types of traffic sign.(9,10) Through these research cases, it was confirmed that the Mask R-CNN model can be used to detect and classify traffic signs, and the outlines of the traffic signs can be found through segmentation, a characteristic of the Mask R-CNN model. 1. Introduction The studies on object classification include the use of the MSER method to detect traffic signs in images and Inception-v3 to classify them into three main categories, namely, warning, regulatory, and mandatory signs, and the Belgian traffic sign dataset (BTSD) was used to train an Inception-v3 model and classify 62 traffic signs.(11–14) Through these studies, it can be seen that in order to classify traffic signs, it is necessary to find the areas of traffic signs to be extracted from images first. Therefore, the purpose of this study is to propose a methodology for automatically detecting and classifying traffic signs by combining a Mask R-CNN model and an Inception-v3 model in images acquired using MMSs. 2.1 Methodology In this study, we propose a methodology that combines a Mask R-CNN model and an Inception-v3 model to detect and sub-classify traffic signs in the images obtained using MMSs (Fig. 5). The first step in automatically constructing traffic signs on high-definition maps is to detect the region of the traffic sign in each image and define its main classification as a warning, regulatory, or mandatory sign. The Mask R-CNN model was applied to the traffic sign regions in the original images collected using MMSs in order to define their respective main classifications. Then, the image coordinates (x, y) for the edge of the traffic sign region were obtained and recorded in a JavaScript object notation (JSON) format file. The JSON format file includes records of information, such as the main classification code, main classification name, and upper left and lower right image coordinates of the object region. After automatically detecting the region of the traffic sign in the image and providing a main classification as either a warning, regulatory, or mandatory sign, the Inception-v3 model was required to sub-classify the traffic sign type in the second step. To this end, the Inception-v3 model was applied to read the JSON format file (the output of the first step), and after creating cropped images using the upper left and lower right image coordinates of the object region, the sub-classification class was assigned, attendant to the main classification. 2. Materials and Methods The traffic signs defined to construct high-definition maps in South Korea are classified into three main categories (Fig. 2): warning signs (B1_1), regulatory signs (B1_2), and mandatory signs (B1_3), which consist of 47, 27, and 35 classes, respectively, comprising a total of 103 classes as sub-classifications.(2) The warning signs have a triangular shape with a red edge and the regulatory signs have a circular shape with a red edge. The mandatory signs are circular, triangular, or square with a blue edge. Unlike Korean traffic signs, U.S. warning signs have a yellow diamond shape as shown in Fig. 3(a), and regulatory and mandatory signs include squares and letters on a white background as shown in Figs. 3(b) and 3(c).(15) Unlike the Korean warning signs shown in Fig. 4(a), the Czech warning signs in Europe are triangular signs with white backgrounds as shown in Figs. 4(b) and 4(c). In addition, comparing the Korean and Austrian regulatory signs shows that the shapes are similar, but the background color or symbols are different.(16) (a) (b) (c) Fig. 2. (Color online) (a) Warning signs defined in high-definition maps, (b) regulatory sings, and (c) mandatory signs. (a) (c) (b) (c) (a) Fig. 2. (Color online) (a) Warning signs defined in high-definition maps, (b) regulatory sings, and (c) mandatory signs. Sensors and Materials, Vol. 34, No. 12 (2022) 4804 Fig. 3. (Color online) (a) Warning, (b) regulatory, and (c) mandatory signs in the United States. (a) (b) (c) (b) (c) (a) (a) (a) (b) (c) Fig. 3. (Color online) (a) Warning, (b) regulatory, and (c) mandatory signs in the United States. Fig. 4. (Color online) (a) Korean warning sign, (b) Czech warning sign, (c) French warning sign, (d) Korean no overtaking regulatory sign, (e) Austrian no overtaking regulatory sign, (f) Korean one-way mandatory sign, and (g) Austrian one-way mandatory sign. (a) (b) (c) (d) (e) (f) (g) (e) (f) (g) (d) (d) (b) (e) Fig. 4. (Color online) (a) Korean warning sign, (b) Czech warning sign, (c) French warning sign, (d) Korean no overtaking regulatory sign, (e) Austrian no overtaking regulatory sign, (f) Korean one-way mandatory sign, and (g) Austrian one-way mandatory sign. 2.2 Construction of training data In this study, the AI-Hub data managed by the National Information Society Agency (NIA) was used to train the Mask R-CNN and Inception-v3 models.(17) The “Panorama Image Set of Sensors and Materials, Vol. 34, No. 12 (2022) 4805 Fig. 5. (Color online) Methodology for detecting and classifying traffic signs. Fig. 5. (Color online) Methodology for detecting and classifying traffic signs. Road Environments (v. 1.0)” provided by AI-Hub is a dataset that provides eight-direction images (.jpg) captured in the daytime using a horizontal camera mounted on an MMS and the label information (.json), such as traffic lights, traffic signs, and road surface marking (Table 1). The label information has records of the image name, class code and name, and upper left and lower right image coordinates. AI-Hub provides approximately 330000 images of training data, but in some cases, the label was missing as shown in Fig. 6(a), or the labeling standard was not met as shown in Fig. 6(b). Therefore, we manually labeled 10000 images to develop the training data for traffic signs. To proceed with the training of the Mask R-CNN model, we divided the 10000 manually constructed images in a ratio of 8:2 to create training and validation data. Table 2 details the construction of the training and validation data. To proceed with the training of the Inception-v3 model, we used 86400 images provided by the AI-Hub and the object region information in the label file (.json) to create cropped images as shown in Fig. 7. The created cropped images were separated into three folders [warning (B1_1), regulatory (B1_2), and mandatory (B1_3)] through the information provided in the label file (.json). Then, three training datasets were constructed so that each folder would have sub-folders consisting of sub-classification codes. 3.1 Training and validation of Mask R-CNN model The training was performed for 600 epochs using the constructed training data, and 100 steps were performed in one epoch. Among the 600 weighted models created through the epochs, a weighted model that had a low loss rate indicating the difference between the values predicted using the model and the true values of the problem and a high mean average precision (mAP) using the validation data was used to detect the traffic sign. The mAP represents an index that can consider both detection rate (recall) and accuracy (precision) at the same time. Table 3 shows the loss and mAP for 200, 400, 568, and 600 epochs performed to detect the traffic signs, and Fig. 8 shows a graph of the loss rate for the validation data. Using Table 3 and 4806 Sensors and Materials, Vol. 34, No. 12 (2022) Table 1 (Color online) Sample images and label information provided by AI-Hub. Category Description Format Example Planar split images 8 split images .jpg Annotation information Object location and type information .json { "interface": { "id": "028770", "filename": "028770_Panorama_crop_2.jpg", "path": "02/crop_image/", "resolution": [1000, 1000], "location": "Gangnam-gu", "datetime": "2020-01-09 11:28:18.0", "annotations": [ { "annotation_id": 161305, "annotation_type": "bbox", "class_code": "0201022600000", "class_name": "Traffic sign/Regulatory sign/Slow", "memo": "", "coord_xy": [[863, 940], [373, 443]] }, { "annotation_id": 161314, "annotation_type": "polygon", "class_code": "0204001300000", "class_name": "Road sign/Crosswalk warning", "memo": "", "coord_xy": [[613, 660, 700, 646], [562, 555, 591, 599] ] } ] } } Table 1 (Color online) Sample images and label information provided by AI-Hub. Category Description Format Example Planar split images 8 split images .jpg Annotation information Object location and type information .json { "interface": { "id": "028770", "filename": "028770_Panorama_crop_2.jpg", "path": "02/crop_image/", "resolution": [1000, 1000], "location": "Gangnam-gu", "datetime": "2020-01-09 11:28:18.0", "annotations": [ { "annotation_id": 161305, "annotation_type": "bbox", "class_code": "0201022600000", "class_name": "Traffic sign/Regulatory sign/Slow", "memo": "", "coord_xy": [[863, 940], [373, 443]] }, { "annotation_id": 161314, "annotation_type": "polygon", "class_code": "0204001300000", "class_name": "Road sign/Crosswalk warning", "memo": "", "coord_xy": [[613, 660, 700, 646], [562, 555, 591, 599] ] } ] } } { (a) (b) Fig. 6. (Color online) (a) Missing label contained in AI-hub dataset and (b) non-standard label. (a) (b) (a) (b) Fig. 6. (Color online) (a) Missing label contained in AI-hub dataset and (b) non-standard label. Sensors and Materials, Vol. 34, No. 12 (2022) 4807 (a) (b) Fig. 7. (Color online) (a) AI-Hub original image (.jpg), (b) AI-Hub labeling data (.json), and (c) cropped images. 3.2 Assessment of Mask R-CNN model Three types of MMS test set were used to objectively assess the performance of the training results of the Mask R-CNN model, which was intended to detect and classify traffic signs into either warning, regulatory, or mandatory signs. In test set 1, we used the images not used in the training of the Mask R-CNN model from the data provided by AI-Hub. For test set 2, we used ‘Pegasus: Two Ultimate’, Leica’s MMS. Lastly, for test set 3, we used R1, an MMS of NaverLabs. Table 4 provides detailed information and shows the mAP results for the test sets. In assessing the accuracy of detecting the traffic signs and determining their main classification, high accuracies of 91.3, 81.2, and 90.2% were obtained for the test datasets of test sets 1, 2, and 3, respectively. Figure 9 shows sample results of the detection of the traffic signs and their main classification in the three test sets using the weighted Mast R-CNN model. 3.1 Training and validation of Mask R-CNN model Table 2 Detailed information of training and validation data. Training set (8000 images) Validation set (2000 images) ID CODE Description Number of labels Ratio (%) Number of labels Ratio (%) 1 B1_1 Warning 446 9.7 116 10.0 2 B1_2 Regulatory 2162 46.9 535 46.2 3 B1_3 Mandatory 2000 43.4 508 43.8 Total 4608 1159 (c) Table 2 Detailed information of training and validation data. Table 2 Detailed information of training and validation data. (a) (b) (c) (c) (b) (a) Fig. 7. (Color online) (a) AI-Hub original image (.jpg), (b) AI-Hub labeling data (.json), and (c) cropped images Fig. 8. (Color online) Loss graph for validation data. Table 3 Mean average precision and loss by number of epochs observed during training. Epoch 200 400 568 600 Train_mAP (%) 72.5 93.8 95.5 95.4 Train_loss (%) 30.3 19.8 12.7 13.3 Val_mAP (%) 88.6 89.9 90.7 90.1 Val_loss (%) 47.8 47.3 37.9 46.0 Fig. 8. (Color online) Loss graph for validation data. 4808 Sensors and Materials, Vol. 34, No. 12 (2022) Fig. 8, we found that in the case of 568 epochs, the training accuracy (95.5%) and the validation accuracy (90.7%) were the highest, and the loss was the lowest. On the basis of these results, a weighted model of 568 epochs was used for the detection of traffic signs to classify the traffic signs into three main categories, namely, warning, regulatory, and mandatory signs. Table 5 Dataset AI-Hub Dataset IMAGE 86400 Epoch 100000 Learning rate 0.001 Batch size 100 Warning Regulatory Mandatory Original Using_IDG Original Using_IDG Original Using_IDG mAP (%) 83.1 90.0 88.9 95.3 86.2 91.5 Loss (%) 84.9 21.1 38.1 20.4 56.3 17.5 3.3 Training and validation of Inception-v3 model The Inception-v3 model was used to sub-classify the traffic signs, which were already classified as warning, regulatory, or mandatory signs. We created sub-classifiers based on the main categories to train the Inception-v3 model. The three sub-classifiers were trained by setting the epochs to 100000 times, the learning rate to 0.001, and the batch size to 100. As a result of the training, we found that the training accuracy was 83.1% for the warning sign sub-classifier, 88.9% for the regulatory sign sub-classifier, and 86.2% for the mandatory sign sub-classifier. That the training accuracy was not higher than 90% was determined to be due to the imbalance in the sub-classifier data used for the training. Therefore, the amount of the training data was increased by repeating the processes of rotating the image, changing the image brightness, and changing the image size using the Image Data Generate (IDG) function provided by TensorFlow version 2.4.(18,19) As a result, the training accuracy improved to 90.0% for the warning signs, 95.3% for the regulatory signs, and 91.5% for the mandatory signs (Table 5). Table 4 Detailed information on the test sets and the accuracy of their classification using Mask R-CNN model. Test set 1 (1000 images) Test set 2 (1000 images) Test set 3 (175 images) ID Description Number of labels Ratio (%) Average precision AP (%) Number of labels Ratio (%) Average precision AP (%) Number of labels Ratio (%) Average precision AP (%) 1 Warning 74 12.1 95.0 156 24.6 80.6 3 1.4 100.0 2 Regulatory 330 54.0 92.3 358 56.4 86.5 134 63.2 94.7 3 Mandatory 207 33.9 86.7 121 19.1 76.3 75 35.4 76.0 Total 611 mAP 91.3 635 mAP 81.2 212 mAP 90.2 Detailed information on the test sets and the accuracy of their classification using Mask R-CNN model. Sensors and Materials, Vol. 34, No. 12 (2022) 4809 Fig. 9. (Color online) (a) Sample results of detection and classification using Mask R-CNN model in test sets 1, (b) 2, and (c) 3. (a) (b) (c) (c) (a) (b) (a) (b) (c) Fig. 9. (Color online) (a) Sample results of detection and classification using Mask R-CNN model in test sets 1, (b) 2, and (c) 3. 3.4 Assessment of Inception-v3 model To assess the performance of the trained warning, regulatory, and mandatory sign classifier models, we created and used cropped images based on the label data of the test datasets used for the performance assessment of the Mask R-CNN model. According to the performance assessment results of the three sub-classifiers, the accuracies (mAP) were 77.3, 78.6, and 76.7% shown in Table 6. All three datasets were able to partially increase the evaluation accuracy by increasing the diversity of training data using IDG, but the Sensors and Materials, Vol. 34, No. 12 (2022) 4810 Table 6 (Color online) Classification results of symmetric traffic signs. (Color online) Classification results of symmetric traffic signs. Images Code Keep Left (313) Keep Right (314) Classification result Code Amount Ratio (%) Code Amount Ratio (%) 313 232 96.27 314 34 75.56 309 1 0.41 313 11 24.44 306 1 0.41 308 1 0.41 307 1 0.41 314 4 1.66 311 1 0.41 Total 241 Total 45 high evaluation accuracy was not recorded. This means that the classification weight model calculated from the training data constructed using IDG has not been generalized. To solve this problem, it can be seen that it is necessary to increase the diversity of forms for training traffic signs by additionally constructing new data in training data. Table 6 shows the classification results of symmetrical traffic signs. It was found that left-side traffic was misclassified as right-side traffic and vice versa. For this reason, it can be seen that the accuracy of classification is rather low in the case of symmetrical traffic signs. 4. Conclusions In this study, a methodology that combines Mask R-CNN and Inception-v3 models to detect and sub-classify 103 traffic signs to produce high-definition maps of South Korea using images obtained by MMSs is described. The Mask R-CNN model was used to determine the regions of the objects, and the main categories were defined as warning, regulatory, or mandatory signs. These categories were then used in the Inception-v3 model to sub-classify the traffic signs into 103 classes. The weighted Mask R-CNN model was trained to detect the traffic signs defined in the high- definition maps using the images captured by MMSs and to classify them into three main categories. The trained and weighted model was assessed using the test datasets obtained from various MMSs, and an average accuracy of 87.6% was obtained. The weighted model was trained using Inception-v3 to create sub-classification categories for the traffic signs. The imbalance of the data between the classification targets in the training process of the weighted model could be partially solved by using the IDG function. Combining these models enabled the classification of 103 types of traffic sign in high-definition maps with an average accuracy of 77.5%. Sensors and Materials, Vol. 34, No. 12 (2022) 4811 The methodology proposed in this study can be used to detect and classify traffic signs systematically to produce the high-definition maps required for autonomous driving. It will be necessary to obtain additional training data to improve the accuracy of the models. References Korean Soc. Surv. Geod. 37 (2019) 119. https://doi.org/10.7848/ksgpc.2019.37. and I. Y. Hong: J. Korean Soc. Surv. Geod. 37 (2019) 119. 19 Image Data Generator of Tensorflow Core v2.0: https://www.tensorflow.org/api_docs/python/tf/keras/ preprocessing/image/ImageDataGenerator (accessed June 2022). References 1 Y. S. Na, S. K. Kim, Y. S. Kim, J. Y. Park, J. M. Jeong, K. C. Jo, S. J. Lee, S. J. Cho, M. H. Sunwoo, and J. M. 1 Y. S. Na, S. K. Kim, Y. S. Kim, J. Y. Park, J. M. Jeong, K. C. Jo, S. J. Lee, S. J. Cho, M. H. Sunwoo, and J. M. Oh: J. Korean Soc. Automot. Eng. 28 (2020) 797. https://doi.org/10.7467/KSAE.2020.28.11.797 2 National Geographic Information: HD Map Quality Crafting Manual, https://www.ngii.go.kr/kor/contents/ view.do?sq=1241&board_code=contents_data (accessed June 2022). 3 Y. Yu, J. Li, C. Wen, H. Guan, H. Luo, and C. Wang: J. Photogramm. Remote Sens. 113 (2016) 106. https://doi. org/10.1016/j.isprsjprs.2016.01.005 4 A. Alvaro, S. Mario, A. Juan, A. Garcia, and R. Belen: Expert Syst. Appl. 89 (2017) 286. https://doi. org/10.1016/j.eswa.2017.07.042 5 M. J. Choi, J. K. Suhr, K. Choi, and H. G. Jung: IEEE 7 (2019) 149846. https://doi.org/10.1109/ ACCESS.2019.2947287 6 P. Viola and M. J. Jones: Int. J. Comput. Vis. 57 (2004) 137. https://doi.org/10.1023/B:VISI.0000013087.49260. fb 7 C. Liu, S. Li, F. Chang, and Y. Wang: IEEE 7 (2019) 86578. https://doi.org/10.1109/ACCESS.2019.2924947 7 C. Liu, S. Li, F. Chang, and Y. Wang: IEEE 7 (2019) 86578. https://doi.org/10.1109/ACCESS.2019.2924947 8 L. Wang, K. Zhou, A. Chu, G. Wang, and L. Wang: IEEE 9 (2021) 124963. https://doi.org/10.1109/ ACCESS.2021.3109798 8 L. Wang, K. Zhou, A. Chu, G. Wang, and L. Wang: IEEE 9 (2021) 124963. https://doi.org/10.1109/ ACCESS.2021.3109798 9 D. Tabernik and D. Skočaj: IEEE Trans. Intell. Transp. Syst. 21 (2020) 1427. https://doi.org/10.1109/ TITS.2019.2913588 10 K. He, G. Gkioxari, P. Dollar, and R. Girshick: Proc. IEEE Int. Conf. Comput. Vis. (2017) 2980. https://doi. org/10.48550/arXiv.1703.06870 g 11 Y. Yang, H. Luo, H. Xu, and F. Wu: IEEE Trans. Intell. Transp. Syst. 17 (2016) 2022. https://doi.org/10.1109/ TITS.2015.2482461 12 C. Lin, L. Li, W. Luo, K. Wang, and J. Guo: Period. Polytech. Transp. Eng. 47 (2018) 242. https://do org/10.3311/PPtr.11480f 13 C. Szegedy, V. Vanhoucke, S. Ioffe, J. Shlens, and Z. Wojna: Proc. IEEE Comput. Soc. Conf. Comput. Vis. Pattern Recognit. (2016) 2818. https://doi.org/10.1109/CVPR.2016.308 14 D. G. Kim, Y. G. Kang, Y. R. Park, N. Y. Kim, and J. Y. Lee: Spat. Inf. Res. 28 (2020) 241. https://do org/10.1007/s41324-019-00285-x 15 United States Road Symbol Signs: https://mutcd.fhwa.dot.gov/services/publications/fhwaop02084/index.htm (accessed June 2022). 16 Comparison of European Road Signs: https://en.wikipedia.org/wiki/Comparison_of_European_road_sig (accessed June 2022). 17 The Ministry of Science and ICT in Korea: https://aihub.or.kr (accessed June 2022). 18 J. S. Kim and I. Y. Hong: J. About the Authors Hyeong-Yoon So received his B.S. degree from Namseoul University, Korea, in 2021. He has been enrolled in the master’s program in spatial information engineering at Namseoul University, Korea, since 2021. His research interests are in photogrammetry, artificial intelligence, and GIS. (ssoss95@naver.com) Sensors and Materials, Vol. 34, No. 12 (2022) 4812 Eui-Myoung Kim received his B.S. and M.S. degrees from Gyeongsang National University, Korea, in 1994 and 1996, respectively, and his Ph.D. degree from Yonsei University, Korea, in 2000. From 2000 to 2002, he was a senior researcher at the Korea Institute of Civil Engineering and Building Technology (KICT). He worked as a postdoctoral fellow at the University of Calgary, Canada, from 2003 to 2005. He was with the Korea Geospatial Information and Communication (KSIC) from 2005 to 2006. Since 2007, he has been a professor at Namseoul University, Korea. His research interests are in photogrammetry and GIS. (kemyoung@nsu.ac.kr) Eui-Myoung Kim received his B.S. and M.S. degrees from Gyeongsang National University, Korea, in 1994 and 1996, respectively, and his Ph.D. degree from Yonsei University, Korea, in 2000. From 2000 to 2002, he was a senior researcher at the Korea Institute of Civil Engineering and Building Technology (KICT). He worked as a postdoctoral fellow at the University of Calgary, Canada, from 2003 to 2005. He was with the Korea Geospatial Information and Communication (KSIC) from 2005 to 2006. Since 2007, he has been a professor at Namseoul University, Korea. His research interests are in photogrammetry and GIS. (kemyoung@nsu.ac.kr) Eui-Myoung Kim received his B.S. and M.S. degrees from Gyeongsang National University, Korea, in 1994 and 1996, respectively, and his Ph.D. degree from Yonsei University, Korea, in 2000. From 2000 to 2002, he was a senior researcher at the Korea Institute of Civil Engineering and Building Technology (KICT). He worked as a postdoctoral fellow at the University of Calgary, Canada, from 2003 to 2005. He was with the Korea Geospatial Information and Communication (KSIC) from 2005 to 2006. Since 2007, he has been a professor at Namseoul University, Korea. His research interests are in photogrammetry and GIS. (kemyoung@nsu.ac.kr)
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Study on the mechanical properties of clayey slip zone soil considering montmorillonite content
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Research Article Keywords: cohesive slipband soils, expansion characteristics, hydrophilic minerals, montmorillonite, shear strength parameters Posted Date: January 4th, 2023 DOI: https://doi.org/10.21203/rs.3.rs-2178907/v1 License:   This work is licensed under a Creative Commons Attribution 4.0 International License. Read Full License Posted Date: January 4th, 2023 DOI: https://doi.org/10.21203/rs.3.rs-2178907/v1 License:   This work is licensed under a Creative Commons Attribution 4.0 International License. Read Full License Page 1/21 Page 1/21 Page 1/21 Abstract The strength property of slip zone soil determines the evolution mechanism and deformation damage of landslides. When the slip zone soil contains a certain amount of hydrophilic minerals, it, therefore, has swelling properties. These swelling properties cause the mechanical property of cohesive slip zone soils to differ from those of conventional-type of slip zone soils. Thus, four cases of typical landslide cohesive slip zone soils in the Shanxi Province of the Yellow River Basin were used as research objects to study the mechanism of hydrophilic minerals on the mechanical properties of cohesive slip zone soils. The mineral fractions of the cohesive slip zone soils were analyzed using X-ray diffraction tests, followed by direct shear tests to analyze the pattern of the shear strength parameters of cohesive slip zone soils under different hydrophilic mineral contents with water content. Finally, the free swelling rate test was used to verify that montmorillonite minerals are the main hydrophilic minerals affecting the swelling characteristics of cohesive slip zone soils. The results were as follows. The cohesion and internal friction angle of clayey slipband soils exhibited two patterns (negative exponential/negative correlation) with the variation of water content, and the critical values of the pattern of the variation of the shear strength parameters with water content were affected by montmorillonite content ranging from 17.65% to 34.52%. The shear strength attenuation of clayey slip zone soils showed a positive correlation with the change in montmorillonite content, and the swelling property caused by montmorillonite minerals was the intrinsic reason for the shear strength attenuation of clayey slip zone soils under different water content conditions. The research results explain the shear strength decay law of cohesive slip zone soils from the perspective of mineral composition and reveal the unique strength properties of these slip zone soils. 1 Introduction Domestic and foreign research res contain a certain amount of clay minerals(Yan C J et al,200 B L et al,2021) but are different from general clay soils, and these slip soils swelling characteristics(Chen J et al,2014;K of hydrophilic minerals in different areas of cohesive slip so mineral contents cause different swelling results. These giv characteristics, which cause different degrees of weakening attenuation(Baldermann A et al,2021). The experimental study on the strength characteristics of slipband soils containing clay particles by Chen Xiaoping (Chen X P et al,2011)showed that the shear stress–shear strain relationship was of a hardening type; with increased shear displacement, the shear stress exerted itself to the peak relatively quickly and thereafter basically stabilized. Meanwhile, Han Lei et al.(Han L et al,2018) found that gravelly clayey slipband soils also exhibit strain-hardening characteristics through triaxial tests. The study of Bo Wu et al.(Wu B et al,2018) showed that under any normal stress, loess-type slipband soils exhibit strain- softening shear properties. In another study, Yiying Zhang et al.(Zhang Y Y et al,2021) conducted ring shear tests on tuff rubble slipband soils, which also exhibited strain-softening shear characteristics. This further illustrated that the mechanical properties of cohesive slipband soils are different from those of other types of slipband soils. In addition, Wen BP et al. (Wen B P et al,2017)found that slipband soils containing clay minerals underwent two different creep patterns under residual conditions. Meanwhile, Li Jiang et al.(Li J et al,2015) studied the changes in microstructure and mechanical properties of softened interbedded slip zone soils after softening, where microstructural damage was the intrinsic cause of the decrease in mechanical strength of slip zone soils. Scaringi G et al.(Scaringi G et al,2018) found the time course pattern of the mechanical properties of cohesive slip zone soils with time. In summary, cohesive slip zone soils have mechanical properties that distinguish them from other types of slip zone soils, and their in-depth study should broaden the research field of slip zone soils. Domestic and foreign scholars have conducted studies on the physical properties and mechanical properties of slip zone soils. However, there is a lack of research on the correlation between the mineral components and mechanical properties of clayey slip zone soils, not to mention the explanation of the decay of their strength properties with water content from a mineral perspective. 1 Introduction Therefore, in this paper, four cases of clayey slip zone soils in the Shanxi Province of the Yellow River Basin, where weak layer- controlled landslides typically occur, were used as the research objects. The mineral components and mechanical properties of these slip zone soils were systematically studied using X-ray diffraction (XRD) analysis, direct shear test, and free expansion rate test. The research results can further reveal the characteristics of the strength properties of such slip zone soils and contribute to the scientific prevention and control of landslide hazards. 1 Introduction Slippery zone soil is an important research topic for determining landslide geological disaster genesis mechanisms and evolution processes. In this respect, scholars have studied the microscopic characteristics(Cheng Q G,1999;Wu B et al,2018;Sun J X et al,2021), mineral fraction(Yan C J et al,2002;Cui D S et al,2018), grain size gradation(Wang L et al,2017), resistivity properties(Long J H et al,2007), and mechanical properties(Yagi N et al,2003;Long J H et al,2010;Chen X L et al,2015;Jiang QQ et al,2019;Zhou JJ et al,2020) of slipband soils. One type of slip zone soil is controlled and gradually evolved by mudstone, shale, and clayey rock. These slip zone soils are called clayey slip zone soils because of the large amount of clay minerals in the mineral fraction. Moreover, they often have scale-like microstructure characteristics(Dimaio C et al,2010), as well as low permeability, high plasticity, and low friction angle. According to the practical experience of the subject group in landslide engineering for many years, clayey slipband soil landslides are widely distributed in the Yellow River basin in the loess, coal, and bauxite strata and in slopes controlled by soft-type strata with mainly expansive minerals, posing a great threat to the safety of people and property in the Yellow River basin every year. From the summary of the research progress on slip zone soils by Li et al(Li X et al,2010)., the classification of slip zone soils still must be further enhanced and the understanding of the essential factors of slip zone soil strength further deepened. Therefore, systematic research on cohesive slip zone Page 2/21 soils must be conducted. Domestic and foreign research results have shown that cohesive slip soils contain a certain amount of clay minerals(Yan C J et al,2001;Dai S B et al,2005;Zheng G D et al,2006;Zhu B L et al,2021) but are different from general clay soils, and the presence of hydrophilic minerals gives these slip soils swelling characteristics(Chen J et al,2014;Khan M S et al,2018). The types and contents of hydrophilic minerals in different areas of cohesive slip soils vary, and different types of hydrophilic mineral contents cause different swelling results. These give cohesive slip soils different swelling characteristics, which cause different degrees of weakening effects on the degree of shear strength attenuation(Baldermann A et al,2021). soils must be conducted. 2 Test Sample 2.1 Sampling process Page 3/21 Page 3/21 Through field investigations and engineering practice project data collection, experimental samples were obtained from boreholes, exploratory wells, and probing troughs, among others, for typical weak layer control landslides in the Shanxi Province, as shown in Fig. 1, with a total of four typical landslide cases. The weight of the experimental samples obtained for each landslide viscous slip zone soil was 10 kg. Then, the soil samples were immediately packed into containers and sealed using cling films and brought back to the laboratory. g g g g p p j , p p obtained from boreholes, exploratory wells, and probing troughs, among others, for typical weak layer control landslides in the Shanxi Province, as shown in Fig. 1, with a total of four typical landslide cases. The weight of the experimental samples obtained for each landslide viscous slip zone soil was 10 kg. Then, the soil samples were immediately packed into containers and sealed using cling films and brought back to the laboratory. 3.1 Test procedure XRD tests were conducted using a Smartlab SE03030502 model X-ray diffractometer. The soil samples were first taken from each typical landslide cohesive slip zone soil, pounded into blocks, and ground into powder using an agate mortar. The powder was then passed through a 200-mesh sieve (no granularity and flour texture to the touch), dried at a temperature of 105°C for 12 h, and then loaded into IMEC-type sample tubes for backup. The X-ray diffractometer was calibrated according to the instrument manual before use and was ready for small-angle (0.6°–5°) and wide-angle (5°–85°) diffraction test start-up with a scan step of 0.01°. The test target was a copper target (radiation source ray λ = 0.154056 nm), and the test was started using an external calculation book control system. 2.2 Determination of the saturated water content The landslide cases in Dongshan, Taiyuan; Linxian, Lvliang; Xiangning, Linfen; and Zezhou, Jincheng, were labeled as DS, LX, XN, and ZZ, respectively. The saturated water content of each sample was measured in an indoor laboratory, and the test results are shown in Table 1. Table 1 Saturated water content of the soil samples Soil sample number Protostratigraphic era Original stratigraphic lithology Saturated water content (%) DS C3t Carbonaceous mudstone 39.64 LX T2t Mudstone 30.32 XN N2b Clay 27.99 ZZ C3t Shale 31.12 Table 1 3.2 Analysis of mineral components The raw XRD data were processed using the Jade 6.5 software. The diffraction pattern was determined by taking the diffraction angle (2θ) as the horizontal coordinate and the intensity of the diffraction peaks as the vertical coordinate. Using the experimentally obtained diffraction pattern, the physical phase Page 4/21 Page 4/21 species categories of the mineral components contained in the clayey slip zone soil were determined by comparing the standard diffraction characteristics of various minerals provided by the ICDD and the JCPDS. species categories of the mineral components contained in the clayey slip zone soil were determined by comparing the standard diffraction characteristics of various minerals provided by the ICDD and the JCPDS. Mineral peaks did not appear in the small-angle test diffraction patterns, and the minerals present in the small-angle diffraction did not appear in the experimental samples. From the diffraction pattern curve of the wide-angle test, as shown in Fig. 2, the identification and analysis concluded that clayey slipband soil samples contained minerals such as montmorillonite, kaolinite, illite, chlorite, gypsum, chlorite, talc, black mica, quartz, and white mica. Moreover, there were some differences in the mineral analogs and contents among the samples consistent with the research results of Shaobin Dai et al(Dai S B et al,2005). In addition, the hydrophilic minerals in the mineral fraction of the clayey slickensides were mainly montmorillonite, kaolinite, illite, and chlorite. The mineral composition of the clayey slip zone soil was different from that of the general slip zone soil, where the usual soil does not contain a certain amount of feldspar(Yan C J et al,2002;Dai S B et al,2005), calcite(Cui D S et al,2018), and other minerals. Another explanation is that the feldspars in slip soils are rapidly transformed into montmorillonite and kaolinite when they undergo this process(Baldermann A et al,2021), which is also the reason for the high content of hydrophilic minerals in clayey slip soils. After the qualitative analysis of the phases, the exact content of each mineral was further determined using the K-value method and the adiabatic method, as shown in Fig. 4. The percentages of hydrophilic minerals contained in the clayey slip zone soils of the typical landslides were 60.10%, 42.40%, 54.14%, and 35.58%, respectively, and their common hydrophilic minerals were montmorillonite and kaolinite. 3.2 Analysis of mineral components In addition, the landslides in Dongshan, Taiyuan; Xiangning, Linfen; and Zezhou, Jincheng, also contained illite minerals, whereas the landslide in Linxian, Lvliang, contained chlorite minerals, of which montmorillonite minerals had the highest percentage of all four hydrophilic minerals. The presence of a large number of hydrophilic mineral components in the mineral fraction of clayey slip zone soils is an important basis for distinguishing them from other types of slip zone soils. 4.1 Straight shear test The slow shear test was conducted using the ZJ strain-controlled straight shear instrument produced by Nanjing Soil Instrument Factory Co., Ltd. The test was performed in strict accordance with the Standard for Geotechnical Test Methods GB/T50123-2019. Appropriate amounts of cohesive slip zone soil samples were obtained and ground through a 0.5-mm soil sieve to study the effect of different moisture contents on the shear strength of cohesive slip zone soils. As the rock and soil bodies in the Yellow River basin and other arid regions often contain crystalline salts, changes in environmental factors such as acidity and alkalinity brought about by their dissolution in aqueous solutions were avoided. Pyrolysis (500°C) and elution (deionized water)(Bing H et al,2011;Zhao Z W et al,2021) were thus adopted in sequence to wash the salts to eliminate alkaline solution ions (e.g., Na+ and Ca2+), acid solution ions Page 5/21 (e.g., SO4 2− and NO3−), and organic impurities. Then, the cohesive slip zone soil after salt washing was dried and processed, each time weighing 50 g of dry soil samples and setting the slow shear test moisture content ratio according to the saturated moisture content steps of each typical landslide cohesive slip zone soil in Table 1. Each case test sample was prepared in four groups with different water contents; 5, 7.5, 10, 15, and 20 ml of distilled water were drawn using a medical syringe, and the soil samples were evenly sprayed with 10%, 15%, 20%, 30%, and 40% of the water contents. The containers were then sealed using a cling film and left for 24 h. Straight shear specimens (61.8 mm in diameter and 20 mm in height) of the remodeled cohesive slip zone soils were prepared using the pressed sample method and then placed on the straight shear table with an impermeable film separating the upper and lower ends of the specimens from the permeable stone to avoid the effect of matrix suction during the shear test. Then, vertical pressures were applied at 100, 200, 300, and 400 kPa, respectively, with a shear volume of 6 mm and a shear rate of 0.02 mm/min. The experimental data were recorded according to the shear displacement. The variation patterns of shear stress with shear displacement for cohesive slipband soils were obtained, as shown in Fig. 3. The unified law of change was divided into three stages. 4.1 Straight shear test The first stage shows that the shear stress increases rapidly with increasing shear displacement. The second stage shows that the rate of increase of shear stress gradually slows down and the curve shows a curved arc section. Finally, the third stage shows a stable fluctuation period, the shear stress growth rate tends to stabilize, and the growth rate remains basically the same. The variation patterns of shear stress with shear displacement did not show a yielding phenomenon but showed hardening-type characteristics, consistent with the results of Chen Xiaoping(Chen X P et al,2011) and Han Lei et al(Han L et al,2018). on clay-bearing slipband soils. Because the slipband soil underwent complex processes such as shear misalignment and grinding and crushing, which led to the damage of the clay mineral grain crystal layer, it did not show the strain-softening phenomenon as what appeared in the ring shear test of slipband soil with artificially added clay grain content conducted by Cao Shichao et al(Cao S C et al,2019). In addition, from the shear stress–shear strain test curve, it can be initially seen that the peak shear stress decreases with increasing water content under the same vertical pressure condition. The shear stress corresponding to a shear displacement of 5 mm was used as the shear strength. At this time, most of the working conditions have reached the peak shear stress. Then, the shear strength of each typical landslide cohesive slip zone soil was extracted and its relationship with the vertical pressure was plotted, as shown in Fig. 4. The unified law showed that the shear strength increased as the vertical pressure increased, which exhibited the same regular characteristics as those of the general slip zone soil: that is, with higher vertical pressure, the clay slip zone soil compactness increased, and the mineral particles were in closer contact. In addition, the shear strength of the cohesive slipband soils decreased with increasing water content, which verified the preliminary conclusions drawn above. However, the degree of influence of the water content varied among the test samples. For instance, the shear strength of the clayey slip zone soil of the landslide in Dongshan, Taiyuan, was greatly influenced by the water content because hydrophilic minerals accounted for 60.10% of its mineral fraction, where the swelling Page 6/21 Page 6/21 characteristics of the extremely hydrophilic clay minerals under high water content conditions led to an increase in shear strength attenuation. 4.1 Straight shear test The next landslide cases affected by water content were the LX and XN clayey slip zone soils, which comprised 42.40% and 54.14% of hydrophilic minerals, respectively. The least affected by water content changes was the landslide clayey slip zone soil in Zechou, Jincheng City, with only 25.77% of hydrophilic minerals. Using the data on the shear strength and vertical pressure of each viscous slip zone soil above, the cohesion and internal friction angle can be calculated through linear fitting combined with the Coulomb strength formula, and the calculation results are shown in Table 2. A scatter plot of the variation of each shear strength parameter with moisture content was plotted, as shown in Fig. 5. The cohesion and internal friction angle of the cohesive slip zone soil decreased as the water content increased, which followed the general law characteristics of unsaturated soil. In addition, the shear strength parameters showed two patterns with the variation of moisture content. Table 2 Shear strength parameters of the cohesive slip zone soils Sample number Water content (%) Cohesive force (kPa) Internal friction angle (°) DS 10 49.11 21.52 20 42.35 17.65 30 35.23 7.49 40 25.16 4.35 LX 10 18.43 26.71 15 13.56 22.27 20 10.33 21.21 30 3.08 17.76 XN 10 22.46 22.59 15 15.63 21.25 20 9.34 19.32 30 5.03 17.96 ZZ 10 18.32 25.19 15 13.86 23.84 20 11.35 23.38 30 10.07 23.09 Linear and exponential functions were used to fit and analyze the relationship between cohesion and internal friction angle with the variation of moisture content. The formulas are given as follows. Linear and exponential functions were used to fit and analyze the relationship between cohesion and internal friction angle with the variation of moisture content. The formulas are given as follows. y = Aω + B 1 y = Aω + B y = C exp(−ω/D) + E 1 1 1 y = C exp(−ω/D) + E y = C exp(−ω/D) + E y = C exp(−ω/D) + E 2 where y is the cohesion or angle of internal friction; ωis the water content; and A, B, C, D, and E are the model parameters. On the basis of the test data in Table 2 to fit the linear and exponential functions, the fitted curve is shown in Fig. 5. 4.1 Straight shear test where y is the cohesion or angle of internal friction; ωis the water content; and A, B, C, D, and E are the model parameters. On the basis of the test data in Table 2 to fit the linear and exponential functions, the fitted curve is shown in Fig. 5. The results of the fitted function for the variation of cohesion with moisture content are as follows. The results of the fitted function for the variation of cohesion with moisture content are as follows. The results of the fitting function of the variation of the angle of internal friction with the moisture content are as follows. The cohesion and internal friction angle of the ZZ clay slip soils with low hydrophilic mineral content showed negative exponential relationships with water content, whereas the cohesion and internal friction angle of the DS clay slip soils with high clay mineral content showed negative linear relationships with water content. These indicated that the hydrophilic mineral content affected the shear strength parameters of the clay slip soils with water content. Meanwhile, XN with high hydrophilic mineral content and LX with low content of clayey slickensides showed opposite patterns, and the influence of montmorillonite as the main hydrophilic mineral on the decay pattern of shear strength parameters was well explained. The critical values of the patterns of shear strength parameters with water content for the XN clayey slip zone soil with lower montmorillonite content ranged from 17.65–34.52%. Further, the shear strength attenuation degree (water content 10–30%) of the clayey slip zone soils of each typical landslide was plotted with the variation of montmorillonite content, as shown in Fig. 6. The degree of cohesion decay showed a certain negative correlation with the decrease in montmorillonite content, which was because montmorillonite minerals absorb water and swell under low water content conditions to form a more compact structure between particles, and the degree of cohesion decay showed a smaller degree. However, Fig. 5 shows that with the continuous increase in water content, the decreasing cohesion trends of the DS and LX clayey slip zone soils, which had the greatest Further, the shear strength attenuation degree (water content 10–30%) of the clayey slip zone soils of each typical landslide was plotted with the variation of montmorillonite content, as shown in Fig. 6. 4.1 Straight shear test The degree of cohesion decay showed a certain negative correlation with the decrease in montmorillonite content, which was because montmorillonite minerals absorb water and swell under low water content conditions to form a more compact structure between particles, and the degree of cohesion decay showed a smaller degree. However, Fig. 5 shows that with the continuous increase in water content, the decreasing cohesion trends of the DS and LX clayey slip zone soils, which had the greatest montmorillonite contents, were greater than those of the XN and ZZ samples, and the cohesion showed a positive correlation with montmorillonite content. This was because montmorillonite minerals continued to absorb water under high water contents and swell violently, and the resulting swelling force caused the more fragile particles to break and disintegrate. Moreover, the porosity jump caused a decrease in the degree of association between particles, resulting in a continuous decrease in cohesion and showing a positive correlation characteristic trend. The degree of decay of the internal friction angle showed a good positive correlation in the range of 10–30% water content, and the trend of the degree of decay shown in Fig. 5 remained unchanged. This indicates that the cohesion of the cohesive slip zone soil has a certain hysteresis with respect to the internal friction angle. 4.2 Free expansion rate test The free expansion rate test of the cohesive slip zone soils was continued to further verify the shear strength decay law of cohesive slip zone soils. Using a WX-2000 swelling rate tester, appropriate amounts of soil samples after washing salt treatment and passing through a 0.5-mm soil sieve and drying were taken. Then, distilled water was added, and the samples were placed in a loose state where they were free to swell in air. The free swelling rate of clay slip belt soil was calculated using formula (3): δef = × 100 Vwe −V0 V0 δef = × 100 Vwe −V0 V0 3 Equation: is the free swelling rate; is the volume of the sample after swelling in water; is the initial volume of the sample. δef Vwe V0 Page 9/21 Page 9/21 The free swelling rate of each typical landslide cohesive slip zone soil specimen was obtained, and the swelling time was more than 3 d. At this time, the volume of each sample was stable after water absorption and swelling. The free expansion rate was up to 62.5%, with a minimum of 13.0%. A line graph of the free swelling rate as affected by the montmorillonite and kaolinite mineral contents is plotted in Fig. 7. There was a positive correlation between the free swelling rate of viscous slickensides and the increase in montmorillonite content, whereas the effect of kaolinite clay minerals on the free swelling rate showed a local dislocation relationship. The natural swelling rate test results verified the conclusion that montmorillonite is the main expansive clay mineral in the slow shear test, consistent with the swelling research results of single clay minerals by Bao-Long Zhu et al(Zhu B L et al,2021). The spacing before and after swelling of montmorillonite was about 1 nm, whereas the spacings before and after swelling of the other hydrophilic minerals were only 0.0006–0.008 nm. Thus, montmorillonite was the main hydrophilic mineral for water absorption and swelling. However, the free swelling rate results did not increase equiproportionally with the increase in montmorillonite content because the shear fragmentation experienced by the slipband soils led to the damage and destruction of the crystalline layer of montmorillonite clay minerals, which eventually showed different proportional degrees. 5 Discussion The microstructure of montmorillonite minerals as a composite aluminum-silicate crystal exhibits scale- like or lamellar shapes(Dimaio C et al,2010), as shown in Fig. 8. Their mechanism of hydration expansion is that the cations (Na+/Ca2+) inherent between the crystals of montmorillonite form a hydration film through the adsorption of water molecules on the surface, which continuously increases the lattice spacing and manifests as volume expansion in the macroscopic scale. The expansion force generated by the water absorption and expansion of montmorillonite minerals also makes the contact between the mineral particles closer, changing the contact mode between the mineral particles from a point–point contact to a line–line contact or even a face–face contact. The increased degree of particle association manifests in the shear stress–shear strain curve as a hardening characteristic, which is a unique feature of the mechanical properties of cohesive slip zone soils. In addition, because the vertical load was applied to the test sample in the slow shear test, the cohesive slip zone soil cannot achieve free expansion but instead underwent isovolume expansion under constant volume conditions, which produced an expansion force on the cover of the straight shear pedestal(Xiong Y F et al,2021;Zhu B L et al,2021) and caused a reduction effect on the vertical load in the axial direction. Moreover, the actual load value applied to the sample was less than the theoretical load value, causing the attenuation of cohesion with the change in moisture content to weaken and the attenuation of the angle of internal friction to increase, resulting in the correlation characteristics in Fig. 6. This effect becomes more prominent as the water content of the prepared samples increased and the mineral content of montmorillonite increased. The degree of decay of cohesion and internal friction angle was increased by the combined effect of water content and montmorillonite content, which led to the fragmentation of fragile particles inside the microstructure after water absorption and swelling. Meanwhile, the leap in porosity led to a continuous decrease in the degree of association between particles, which finally caused a continuous decrease in Page 10/21 Page 10/21 cohesion and internal friction angle, showing the two patterns in Fig. 5. Therefore, the reason for the degree of shear strength attenuation of the clayey slipband soils is the presence of the montmorillonite mineral in addition to the water content factor. 6 Conclusion In this study, relevant experimental analyses were conducted on four cases of typical landslide cohesive slip zone soils in the Shanxi Province of the Yellow River Basin to study the mechanism of the influence of hydrophilic minerals on the mechanical properties of cohesive slip zone soils. The following research conclusions were obtained: ) The hydrophilic minerals in the mineral fraction of the clay slip zone soils were mainly philic minerals in the mineral fraction of the clay slip zone soils were mainly (1) The hydrophilic minerals in the mineral fraction of the clay slip zone soils were mainly montmorillonite, kaolinite, illite, and chlorite. The percentages of the hydrophilic minerals in the clayey slip zone soils of each typical landslide were 60.10% (DS), 42.40% (LX), 54.14% (XN), and 35.58% (ZZ), and montmorillonite minerals had the highest percentages in hydrophilic minerals. (2) The stress–strain curve of the cohesive slipband soil shows a hardening-type characteristic. The variations of cohesion and internal friction angle with water content showed two patterns. The samples with low montmorillonite content showed a negative exponential relationship, whereas the samples with high montmorillonite content showed a negative correlation. Critical values for the patterns of variation of shear strength parameters with moisture content as affected by the montmorillonite content ranged from 17.65–34.52%. (3) The shear strength attenuation degree of clayey slipband soils showed a positive correlation with the change in montmorillonite content. When the range of water content was small, the attenuation degree of cohesion showed a negative correlation with the change in montmorillonite content; when the range of water content was large, the attenuation degree of cohesion showed a positive correlation with the change in montmorillonite content. Moreover, the degree of decay of the internal friction angle with the variation of montmorillonite content all showed positive correlation characteristics. (3) The shear strength attenuation degree of clayey slipband soils showed a positive correlation with the change in montmorillonite content. When the range of water content was small, the attenuation degree of cohesion showed a negative correlation with the change in montmorillonite content; when the range of water content was large, the attenuation degree of cohesion showed a positive correlation with the change in montmorillonite content. Moreover, the degree of decay of the internal friction angle with the variation of montmorillonite content all showed positive correlation characteristics. 5 Discussion When the content of intact montmorillonite clay minerals increased to a certain degree or with an artificially proportioned hydrophilic mineral content, the mechanical properties exhibited a strain-softening phenomenon(Zhang Y Y et al,2021), but the softening amplitude was small and its mechanical properties favored the nature of clay. However, the mechanical properties of clayey slip soils obtained from the test were not exactly the same as those of clay. Therefore, clayey slip zone soils are an extremely special type of slip zone soil. cohesion and internal friction angle, showing the two patte degree of shear strength attenuation of the clayey slipband mineral in addition to the water content factor. When the co increased to a certain degree or with an artificially proportio mechanical properties exhibited a strain-softening phenom amplitude was small and its mechanical properties favored properties of clayey slip soils obtained from the test were n Therefore, clayey slip zone soils are an extremely special ty The results of the water absorption and swelling of clayey slip zone soils showed plastic deformation and sharp decay of shear strength on a macroscopic scale, which verified that natural slopes and artificial slopes containing weak interlayers are prone to landslides through sliding along the weak layer under the conditions of natural rainfall and artificial irrigation, among others. Acknowledgements The authors would like to thank the Shanxi Metallurgical Geotechnical Engineering Investigation Ltd for their cooperation in conducting the experiment tests. Funding This research was partially funded by the Development Fund Project of State Key Laboratory of Deep Geotechnical Mechanics and Underground Engineering of China(SKLGDUEK2124)and Shanxi Natural Science Foundation of China(20210302124112). Abbreviations XRD:X-ray diffraction;ICDD:International Centre For Diffraction Data;JCPDS:Joint Committee on Powder Diffraction Standards 6 Conclusion (4) There was a positive correlation between the free swelling rate of clayey slip zone soils and the content of montmorillonite minerals. Montmorillonite was the main expansive clay mineral, and its (4) There was a positive correlation between the free swelling rate of clayey slip zone soils and the content of montmorillonite minerals. Montmorillonite was the main expansive clay mineral, and its Page 11/21 swelling property was the intrinsic reason for the shear strength decay of the clayey slip zone soil under different water content conditions. swelling property was the intrinsic reason for the shear strength decay of the clayey slip zone soil under different water content conditions. Author details 1College of Mining Engineering,Taiyuan University of Technology,Taiyuan,030024,China;2Architectural Design and Research Institute (Group) Co.,Ltd.,Tongji University,Shanghai,200092,China;3Shanxi Metallurgical Geotechnical Engineering Investigation Ltd,Taiyuan,030000,China Competing interests I have declare that there is no any competing interests. Availability of data and materials All the datasets that have been used and analysed during the current study is available from the corresponding author on reasonable request. 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Jiang QQ, Liu LL, Jiao YY et al (2019)Strength properties and microstructure characteristics of slip zone soil subjected to wetting-drying cycles.Rock and Soil Mechanics40(3):1005–1012 + 1022.http://doi.org/10.16285/j.rsm.2017.1706 14. Khan MS, Hossain MS, Khan MA et al (2018)Performance of Recycled Plastic Pin (RPP) for Slope Stabilization.Sustainable Civil Infrastructures,136–148 14. Khan MS, Hossain MS, Khan MA et al (2018)Performance of Recycled Plastic Pin (RPP) for Slope Stabilization.Sustainable Civil Infrastructures,136–148 Page 13/21 15. Li J, Xu Q, Hu ZM et al(2015)Experimental research on softening of undisturbed saturated slip soil i eastern of Sichuan province red bed.Chinese Journal of Rock Mechanics and Engineering34(Sup Page 13/21 15. Li J, Xu Q, Hu ZM et al(2015)Experimental research on softening of undisturbed saturated slip soil in eastern of Sichuan province red bed.Chinese Journal of Rock Mechanics and Engineering34(Sup 2):4333–4342.http://doi.org/10.13722/j.cnki.jrme.2014.1003 2):4333–4342.http://doi.org/10.13722/j.cnki.jrme.2014.1003 16. Li X, Liang SY, Zheng G D(2010)Progresses in sliding zone soil of landslides.Advances in Earth Science12(9):2639 16. Li X, Liang SY, Zheng G D(2010)Progresses in sliding zone soil of landslides.Advances in Earth Science12(9):2639 17. Page 12/21 Long JH, Guo WB, Li P et al(2010)Creep property of soil in sliding zone of loess landslide.Chinese Journal of Geotechnical Engineering32(7):1023–1028 17. Long JH, Guo WB, Li P et al(2010)Creep property of soil in sliding zone of loess landslide.Chinese Journal of Geotechnical Engineering32(7):1023–1028 18. Long JH, Li TL, Lei XF et al(2007)Study on physical properties of soil in sliding zone of loess landslip.Chinese Journal of Geotechnical Engineering(2):289–293 18. Long JH, Li TL, Lei XF et al(2007)Study on physical properties of soil in sliding zone of loess landslip.Chinese Journal of Geotechnical Engineering(2):289–293 19. Scaringi G, Hu W, Xu Q et al (2018)Shear-Rate-Dependent Behavior of Clayey Bimaterial Interfaces at Landslide Stress Levels.Geophysical Research Letters45(2):766–777. http://doi.org/10.1002/2017GL076214 19. Scaringi G, Hu W, Xu Q et al (2018)Shear-Rate-Dependent Behavior of Clayey Bimaterial Interfaces at Landslide Stress Levels.Geophysical Research Letters45(2):766–777. 19. Scaringi G, Hu W, Xu Q et al (2018)Shear-Rate-Dependent Behavior of Clayey Bimaterial Interfaces at Landslide Stress Levels.Geophysical Research Letters45(2):766–777. http://doi.org/10.1002/2017GL076214 20. Sun JX, Gu TF, Kong JX et al(2021)Comparative analysis of microstructure of slip zone soil and undisturbed soil.Journal of Arid Land Resources and Environment35(3):126– 133.http://doi.org/10.13448/j.cnki.jalre.2021.078 20. Sun JX, Gu TF, Kong JX et al(2021)Comparative analysis of microstructure of slip zone soil and undisturbed soil.Journal of Arid Land Resources and Environment35(3):126– 133.http://doi.org/10.13448/j.cnki.jalre.2021.078 21. Wang L, Zhao FS, Cheng XH et al (2017)Physical and mechanical properties and microstructures of slip soil of loess bedrock contact landslide.Journal of Earth Sciences and Environment39(3):450– 458 21. Wang L, Zhao FS, Cheng XH et al (2017)Physical and mechanical properties and microstructures of slip soil of loess bedrock contact landslide.Journal of Earth Sciences and Environment39(3):450– 458 22. Wen BP, Jiang XZ (2017) Effect of gravel content on creep behavior of clayey soil at residual state: implication for its role in slow-moving landslides. Landslides 14(2):559–576. http://doi.org/10.1007/s10346-016-0709-3 22. Wen BP, Jiang XZ (2017) Effect of gravel content on creep behavior of clayey soil at residual state: implication for its role in slow-moving landslides. Landslides 14(2):559–576. http://doi.org/10.1007/s10346-016-0709-3 23. Wu B, Zhao FS, Wang L et al (2018)Physical and mechanical properties of slip zone soil in loess- bedrock landslides and its evolutionary process.Coal Geology&Exploration46(4):129–134 + 141 23. Wu B, Zhao FS, Wang L et al (2018)Physical and mechanical properties of slip zone soil in loess- bedrock landslides and its evolutionary process.Coal Geology&Exploration46(4):129–134 + 141 24. Page 12/21 Xiong YF, Lv SH, Xiao X(2021)Study on the Stability of a Landslide Considering the Action of Expansive Force of Slip Soils.Resources Environment & Engineering35(3):369– 379.http://doi.org/10.16536/j.cnki.issn.1671-1211.2021.03.017 24. Xiong YF, Lv SH, Xiao X(2021)Study on the Stability of a Landslide Considering the Action of Expansive Force of Slip Soils.Resources Environment & Engineering35(3):369– 379.http://doi.org/10.16536/j.cnki.issn.1671-1211.2021.03.017 25. Yagi N, Yatabe R, Bhanary NP et al (2003) (2003)Shear characteristics of the reconsolidated clay remolded under low water content. Paper presented at the 3rd International Symposium on Deformation Characteristics of Geomaterials Deformation Charactistics of Geomaterials,Lyon,France 25. Yagi N, Yatabe R, Bhanary NP et al (2003) (2003)Shear characteristics of the reconsolidated clay remolded under low water content. Paper presented at the 3rd International Symposium on Deformation Characteristics of Geomaterials Deformation Charactistics of Geomaterials,Lyon,France 26. Yan CJ, Tang HM, Chen JY et al (2002)Studies of soil microstructures and compositions of slipping zone in reservior district of Three Gorges Profect. Rock and Soil Mechanics 23(Sup 1): 23– 26.http://doi.org/10.16285/j.rsm.2002.s1.007 26. Yan CJ, Tang HM, Chen JY et al (2002)Studies of soil microstructures and compositions of slipping zone in reservior district of Three Gorges Profect. Rock and Soil Mechanics 23(Sup 1): 23– 26.http://doi.org/10.16285/j.rsm.2002.s1.007 27. Yan CJ, Tang HM, Sun Y Z(2001)Study on the soil of slipping zone in landslides and its significance by scanning electron microscope and X-Ray diffractometer.Geological Science and Technology Information20(4):89–92 27. Yan CJ, Tang HM, Sun Y Z(2001)Study on the soil of slipping zone in landslides and its significance by scanning electron microscope and X-Ray diffractometer.Geological Science and Technology Information20(4):89–92 28. Zhang YY, Guo CB, Yang ZH et al(2021)Study on shear strength of deep-seated sliding zone soil of Zhouchangping landslide in Maoxian,Sichuan.Journal of Engineering Geology29(3):764- 776.http://doi.org/10.13544/j.cnki.jeg.2020-610 28. Zhang YY, Guo CB, Yang ZH et al(2021)Study on shear strength of deep-seated sliding zone soil of Zhouchangping landslide in Maoxian,Sichuan.Journal of Engineering Geology29(3):764- 776.http://doi.org/10.13544/j.cnki.jeg.2020-610 Page 14/21 29. Zhao ZW, Wang ZB, Guo XL et al(2021)Review on technologies for organic impurities removal from industrial waste salt.Environmental Protection of Chemical Industry41(6):673–677 Page 14/21 29. Zhao ZW, Wang ZB, Guo XL et al(2021)Review on technologies for organic impurities removal from industrial waste salt.Environmental Protection of Chemical Industry41(6):673–677 30. Zheng GD, Xu S, Lang YH et al(2006)Pyrite in sliding mud of the Nakataura landslide in Toyama Prefecture,Japan. Geochimica35(2):201–210.http://doi.org/10.19700/j.0379-1726.2006.02.012 30. Zheng GD, Xu S, Lang YH et al(2006)Pyrite in sliding mud of the Nakataura landslide in Toyama Prefecture,Japan. Geochimica35(2):201–210.http://doi.org/10.19700/j.0379-1726.2006.02.012 30. Page 12/21 Zheng GD, Xu S, Lang YH et al(2006)Pyrite in sliding mud of the Nakataura landslide in Toyama Prefecture,Japan. Geochimica35(2):201–210.http://doi.org/10.19700/j.0379-1726.2006.02.012 31. Zhou JJ, Zhao FS, Yuan XQ et al (2020)Creep process and the microstructural evolution of sliding- zone soil.Hydrogeology & Engineering Geology47(3):115–121. http://doi.org/10.16030/j.cnki.issn.1000-3665.2019010011 31. Zhou JJ, Zhao FS, Yuan XQ et al (2020)Creep process and the microstructural evolution of sliding- zone soil.Hydrogeology & Engineering Geology47(3):115–121. http://doi.org/10.16030/j.cnki.issn.1000-3665.2019010011 32. Zhu BL, Li Q, Wang SL et al (2021) Study on time-dependent effect and three-dimensional macro- micro swelling pressure model of black shale under influence of water. J Railway Sci Eng 18(10):2610–2619. http://doi.org/10.19713/j.cnki.43-1423/u.T20201067 32. Zhu BL, Li Q, Wang SL et al (2021) Study on time-dependent effect and three-dimensional macro- micro swelling pressure model of black shale under influence of water. J Railway Sci Eng 18(10):2610–2619. http://doi.org/10.19713/j.cnki.43-1423/u.T20201067 Figures Figures Page 15/21 Figure 1 Clay slip zone soil of each typical landslide. (a) Location of the Yellow River Basin and landslide ca China based on the 1980 Chinese National Geodetic Coordinate System. (b–e) Landslides in (b) Dongshan, Taiyuan, (c) Linxian, Lvliang, (d) Xiangning, Linfen, and (e) Zezhou, Jincheng Page 15/21 Figure 1 Clay slip zone soil of each typical landslide. (a) Location of the Yellow River Basin and landslide cases in China based on the 1980 Chinese National Geodetic Coordinate System. (b–e) Landslides in (b) Dongshan, Taiyuan, (c) Linxian, Lvliang, (d) Xiangning, Linfen, and (e) Zezhou, Jincheng Figure 1 Page 15/21 Clay slip zone soil of each typical landslide. (a) Location of the Yellow River Basin and landslide cases in China based on the 1980 Chinese National Geodetic Coordinate System. (b–e) Landslides in (b) Dongshan, Taiyuan, (c) Linxian, Lvliang, (d) Xiangning, Linfen, and (e) Zezhou, Jincheng Figure 2 XRD pattern analysis and mineral composition. (a) DS-XRD, (b) LX-XRD, (c) XN-XRD, and (d) ZZ-XRD Figure 2 XRD pattern analysis and mineral composition. (a) DS-XRD Figure 2 XRD pattern analysis and mineral composition. (a) DS-XRD, (b) LX-XRD, (c) XN-XRD, and (d) ZZ-XRD XRD pattern analysis and mineral composition. (a) DS-XRD, (b) LX-XRD, (c) XN-XRD, and (d) ZZ-XRD XRD pattern analysis and mineral composition. (a) DS-XRD, (b) LX-XRD, (c) XN-XRD, and (d) ZZ-XRD d mineral composition. (a) DS-XRD, (b) LX-XRD, (c) XN-XRD, and (d) ZZ-XRD Page 16/21 Page 16/21 ure 3 ear stress versus shear position curve (vertical pressure: 300 kPa). (a) DS, (b) LX, (c) XN, and (d) ZZ gure 3 hear stress versus shear position curve (vertical pressure: 300 kPa). (a) DS, (b) LX, (c) XN, and (d) ZZ Figure 3 Shear stress versus shear position curve (vertical pressure: 300 kPa). (a) DS, (b) LX, (c) XN, and (d) ZZ Page 17/21 Page 17/21 ure 4 ationship between the shear strength and vertical pressure. (a) DS, (b) LX, (c) XN, and (d) ZZ Figure 5 Relationship between the shear strength parameters and water content. (a) c and (b) φ Figure 6 Relationship between shear strength parameters and montmorillonite content. (a) c and (b) φ Figure 4 Relationship between the shear strength and vertical pressure. (a) DS, (b) LX, (c) XN, and (d) ZZ Relationship between the shear strength and vertical pressure. (a) DS, (b) LX, (c) XN, and (d) ZZ Page 18/21 Figure 5 Relationship between the shear strength parameters and water content. (a) c and (b) φ Figure 6 Relationship between shear strength parameters and montmorillonite content. (a) c and (b) φ Relationship between shear strength parameters and montmorillonite content. (a) c and (b) φ Page 19/21 Page 19/21 Figure 7 Free expansion rate test results Figure 7 Free expansion rate test results Figure 7 Free expansion rate test results Page 20/21 Figure 8 Mechanism of water absorption and expansion of cohesive slip zone soil Mechanism of water absorption and expansion of cohesive slip zone soil Page 21/21 Page 21/21 Page 21/21
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Vertebral cryptococcosis in an immunocompetent patient - a case report
˜The œPan African medical journal
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Abstract We report an unusual case of 70 years old, immunocompetent woman who was diagnosed with vertebral cryptococcosis. The diagnosis was made on the basis of radiological and histological findings. The outcome was favorable under antifungal treatment. &Corresponding author: Ammouri Wafa, Department of Internal medicine, Avicenne university hospital, Rabat, Morocco &Corresponding author: Ammouri Wafa, Department of Internal medicine, Avicenne university hospital, Rabat, Morocco Key words: Cryptococcosis, vertebral, immunocompetent, C neoformans, Morocco Received: 19/03/2011 - Accepted: 10/04/2011 - Published: 10/04/2011 Received: 19/03/2011 - Accepted: 10/04/2011 - Published: 10/04/2011 Open Access Bachir Houda1, Ammouri Wafa1,&, Tazi Mezalek Zoubida1, Adnaoui Mohamed1, Aouni Mohamed1, Har 1Department of Internal medicine, Avicenne University Hospital, Rabat, Morocco Pan African Medical Journal – ISSN: 1937- 8688 (www.panafrican-med-journal.com) Published in partnership with the African Field Epidemiology Network (AFENET). (www.afenet.net) Introduction Cryptococcosis is infection with Cryptococcus neoformans fungus. C neoformans is an opportunistic fungal infection that defines immunosuppression. It can infrequently be seen in immunocompetent patients. Cryptococcosis presents in two forms: pulmonary and cerebromeningeal. Skeletal cryptococcosis is uncommon. There have been only occasional case reports of thoracic vertebral cryptococcosis presenting as a cord compression in an immunocompetent patient [1].We report a case of an unusual presentation of cryptococcal infection in an immunocompetent patient. Conclusion Vertebral cryptococcosis can rarely occur in an immunocompetent person without HIV, where it usually goes undiagnosed. The clinician should know this rare and severe infectious complication because the treatment is specific and the prognosis depends of the precociousness of the diagnosis. Discussion Cryptococcus neoformans is encapsuled yeast, found in pigeon and other bird dropping. It can be inhaled by humans. Most infectious occur in immunocompromised patients, with HIV, transplants patients or patients on long-term corticosteroid therapy [1]. The disease also appears to be more frequent in diabetes [2]. It can rarely be seen in immunocompetent patients [3]. The overall incidence of crytococcosis in immunocompetent individuals has been estimated at 0.2 per million per year [4]. The lung is the portal of entry and then the dissemination is haematogenous [5]. Bones involvement in this Infection is not common. The first described case was reported by Busse and Bushke in 1894 – 1895, which demonstrated that the yeast produced osteomyelitis of the tibia [6]. A variety of bones can be involved by the fungal organism, but vertebras are the single most likely site [7]. The vertebral involvement can mimic tuberculosis of the spine [7]. Like our patient, radiological findings of cryptococcal bone lesions are non- specific, consisting of osteolytic destruction of the vertebral bodies with paraspinal abscesses; thus simulating tuberculosis, which is more prevalent in tropical areas [4]. Diagnosis of bony infection requires biopsy unless C neoformans can be is isolated from another body site; less than half of the patients will have a positive serum cryptococcal antigen test [8]. The level of antigen titre correlates with the severity of the disease [5]. Diagnosis of bony infection requires biopsy unless C neoformans can be is isolated from another body site; less t positive serum cryptococcal antigen test [8]. The level of antigen titre correlates with the severity of the disease Cryptococcal vertebral infection causing cord compression can be treated surgically through debridement and bone grafting. The combination of medical treatment is necessary including, antifungal agents such as amphotericin B, flucytosine and fluconazole [9]. Combination therapy might be needed even if patients are immunocompetent [9]. Patient and case report A 70 years old woman was admitted for paraplegia. Past medical history of the patient revealed bilateral sciatica evolving since July 2009, not alleviated by symptomatic treatment. A month prior admission, the sciatica symptoms worsened towards a gradual onset of paraplegia, prompting the current consultation. No other significant medical history was noted. On admission, body temperature was 37°, heart rate was 75b/mn, and blood pressure 130/60 mmhg. Neurological examination showed spastic paraparesis, deep tendon reflexes of the lower extremities were lively. Babinski reflex was bilaterally positive. Laboratory findings showed: erythrocyte sedimentation rate 100mm/h, white blood cell count 4800/ mm³, lymphocytes 1600/mm³ and haemoglobin level 12g/dl HIV serology was negative; complement levels and measurement of CD4/CD8 T-lymphocyte ratio were within the normal range. HIV serology was negative; complement levels and measurement of CD4/CD8 T-lymphocyte ratio were within the A lumbar magnetic resonance imaging was performed showing a lesion process extending from Th8 to Th10, with spinal cord compression. The lesion was hypo-intense on T1-weighted images and hyper-intense on T2-weighted images (Figure 1, 2). Culture of cerebrospinal fluid established the presence of C neoformans. Posterior laminectomy was performed, revealing an abnormal tissue lesion. Biopsy showed, on histological examination, numerous fungal organisms with thick capsules and a granulomatosis inflammatory process without necrosis. There were no malignant cells (Figure 3). Chest X ray examination and thoracic computed tomography were normal. The diagnosis of spinal cryptococcosis in an immunocompetent patient was established and the patient was treated by intravenous amphotericin B (0.7 mg/ kg/day) with relay by fluconazol (400mg/d). On follow-up, the patient remains well with no neurological or other sequel. Pan African Medical Journal. 2011; 8:42 This article is available online at: http://www.panafrican-med-journal.com/content/article/8/42/full/ © Bachir Houda et al. The Pan African Medical Journal - ISSN 1937-8688. This is an Open Access article distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/by/2.0), which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited. Page number not for citation purposes 1 Pan African Medical Journal – ISSN: 1937- 8688 (www.panafrican-med-journal.com) Published in partnership with the African Field Epidemiology Network (AFENET). (www.afenet.net) Page number not for citation purposes Introduction Authors’ contribution All authors have read and agreed to the final version of this manuscript and have equally contributed to its content and to the management of the case. Figures Figure 1: The lesion process extending to the right posterior hemi-arch of the eighth thoracic vertebrae (TH8), responsible for an important spinal cord compression at this level. Figure 2: The lesion process extending to the right posterior hemi-arch of the ninth (TH9), responsible for an important spinal cord compression at this level. Figure 3: Fungal organisms with thick capsules, a granulomatous inflammatory process without necrosis, an abundant eosinophile cytoplasm and cells with a dark ring in the periphery. Marking cytokeratin was negative (hematoxylin eosin, original x 100) Competing interests The authors declare no competing interests. Page number not for citation purposes Authors’ contribution Page number not for citation purposes 3 Page number not for citation purposes 4 Page number not for citation purposes Page number not for citation purposes 6 References 1. Awashti M, Patankar T, Sah P, Castillo M. Cerebral cryptococcosis: atypical appearances on CT. Br J Radiol. 2001 Jan;74(877):83-5. This article on PubMed 2. Glynn MJ, Duckworth G, Ridge JA, Grange WJ, Gibbs DD. Cryptococcal spondylitis: solitary infective bone lesions are not always tuberculosis. Br J Rheumatol. 1994 Nov;33(11):1085-6. This article on PubMed 3. Saigal M G, Donovan Post M Judith, Lolayekar S, Murtaza A. Unusual Presentation of central nervous system cryptococcal infection in an immunocompetent patient. Am J Neuroradiol. 2005; 26: 2522-2526. This article on PubMed 4. Gupta S K, Chhabra R, Sharma B S, Khosla A. Vertabral cryptococcosis simulating tuberculosis. British Journal of Neurosurgery. Br J Neurosurg. 2003 Dec;17(6):556-9. This article on PubMed 5. Mitchell TG, Perfect JR. Cryptococcosis in the era of AIDS-100 years after the discovery of Cryptococcus neoformans. Clin Microbiol Rev. 1995; 8: 515-48. This article on PubMed 5. Mitchell TG, Perfect JR. Cryptococcosis in the era of AIDS-100 years after the discovery of Cryptococcus neoformans. Clin Microbiol Rev. 1995; 8: 515-48. This article on PubMed 6. Bushke A. Ueber eine durch coicidien hemorgerufene krankhitedes menschen. Deutsch Med Wochenschr. 1895; 21:14 6. Bushke A. Ueber eine durch coicidien hemorgerufene krankhitedes menschen. Deutsch Med Wochenschr. 1895; 21:14 7. Gurevitz O, Goldschmied-Reuven A, Block C, Kopoloric J, Fecfel Z, Hassin D. Cryptococcus neoformans vertebral osteomyelitis. J Med Vet Mycol. 1994;32(4):315-8. This article on PubMed 8. Casadevall A, Perfect JR. Cryptococcus neoformans. Washington DC. ASM Press 9. Matsushita T, Suzuki K. Spastic paraparesis due to cryptococcal osteomyelitis :a case report. Clin Orthop Relat Res. 1985 Jun;(196):279- 84. This article on PubMed Page number not for citation purposes
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https://egusphere.copernicus.org/preprints/2023/egusphere-2023-3000/egusphere-2023-3000.pdf
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Comment on egusphere-2023-3000
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ERROR: type should be string, got "https://doi.org/10.5194/egusphere-2023-3000\nPreprint. Discussion started: 22 December 2023\nPub ic domain CC\n1.0.\nl\n.\n0 https://doi.org/10.5194/egusphere-2023-3000\nPreprint. Discussion started: 22 December 2023\nPub ic domain CC\n1.0. l\n. 0 https://doi.org/10.5194/egusphere-2023-3000\nPreprint. Discussion started: 22 December 2023\nPub ic domain CC\n1.0. l\n. 0 Impact of Hurricane Irma on Coral Reef Sediment Redistribution at \n5 \nLooe Key Reef, Florida, USA \nKimberly K. Yates1, Zachery Fehr2, Selena Johnson1, David Zawada1 \n1U.S. Geological Survey, St. Petersburg, FL, 33701, United States \n2Cherokee Nation System Solutions, Tulsa, OK, 74166, United States, Contractor to the U.S. Geological Survey \nCorrespondence to: Kimberly K. Yates (kyates@usgs.gov) \n10 Rates of elevation \nchange during this post-storm period were one to two orders of magnitude greater than decadal and multi-decadal rates of \nchange in the same location, and changes showed erosion of approximately 50% of sediment deposited during the storm event \nas seafloor sediment distribution began to re-equilibrate to non-storm sea state conditions. Our results suggest higher resolution \n30 \nelevation-change data collected over seasonal and annual time periods could enhance characterization and understanding of \nshort-term and long-term rates and processes of seafloor change and help guide post-storm recovery and restoration of benthic \nhabitats in topographically complex coral reef systems. Abstract. Understanding event-driven sediment transport in coral reef environments is essential to assessing impacts to reef \nspecies, habitats, restoration, and mitigation, yet there remains a global knowledge gap due to limited quantitative studies. Hurricane Irma made landfall in the Lower Florida Keys with sustained 209 km h-1 winds and greater than 8 m waves on 10 \nSeptember 2017, directly impacting the Florida Reef Tract (FRT), and providing an opportunity to perform a unique comprehensive, quantitative assessment of its impact on coral reef structure and sediment redistribution. We used lidar and \n15 \nmultibeam derived digital elevation models (DEMs) collected before and after the passing of Hurricane Irma over a 15.98 km2 \narea along the Lower FRT including Looe Key Reef to quantify changes in seafloor elevation, volume, and structure due to \nstorm impacts. Elevation change was calculated at over 4-million point-locations across 10 habitat types within this study area \nfor two time periods using data collected from 1) approximately one year before the passing of Irma and three to six months 15 comprehensive, quantitative assessment of its impact on coral reef structure and sediment redistribution. We used lidar and \n15 \nmultibeam derived digital elevation models (DEMs) collected before and after the passing of Hurricane Irma over a 15.98 km2 \narea along the Lower FRT including Looe Key Reef to quantify changes in seafloor elevation, volume, and structure due to \nstorm impacts. Elevation change was calculated at over 4-million point-locations across 10 habitat types within this study area \nfor two time periods using data collected from 1) approximately one year before the passing of Irma and three to six months following the storm’s impact, and 2) from three to six months after, and up to 16.5 months after, the storm. Impact of Hurricane Irma on Coral Reef Sediment Redistribution at \n5 \nLooe Key Reef, Florida, USA \nKimberly K. Yates1, Zachery Fehr2, Selena Johnson1, David Zawada1 \n1U.S. Geological Survey, St. Petersburg, FL, 33701, United States \n2Cherokee Nation System Solutions, Tulsa, OK, 74166, United States, Contractor to the U.S. Geological Survey \nCorrespondence to: Kimberly K. Yates (kyates@usgs.gov) \n10 Impact of Hurricane Irma on Coral Reef Sediment Redistribution at \n5 \nLooe Key Reef, Florida, USA \nKimberly K. Yates1, Zachery Fehr2, Selena Johnson1, David Zawada1 \n1U.S. Geological Survey, St. Petersburg, FL, 33701, United States \n2Cherokee Nation System Solutions, Tulsa, OK, 74166, United States, Contractor to the U.S. Geological Survey \nCorrespondence to: Kimberly K. Yates (kyates@usgs.gov) \n10 rly K. Yates1, Zachery Fehr2, Selena Johnson1, David Zawada1 Abstract. Understanding event-driven sediment transport in coral reef environments is essential to assessing impacts to reef \nspecies, habitats, restoration, and mitigation, yet there remains a global knowledge gap due to limited quantitative studies. Hurricane Irma made landfall in the Lower Florida Keys with sustained 209 km h-1 winds and greater than 8 m waves on 10 \nSeptember 2017, directly impacting the Florida Reef Tract (FRT), and providing an opportunity to perform a unique \ncomprehensive, quantitative assessment of its impact on coral reef structure and sediment redistribution. We used lidar and \n15 \nmultibeam derived digital elevation models (DEMs) collected before and after the passing of Hurricane Irma over a 15.98 km2 \narea along the Lower FRT including Looe Key Reef to quantify changes in seafloor elevation, volume, and structure due to \nstorm impacts. Elevation change was calculated at over 4-million point-locations across 10 habitat types within this study area \nfor two time periods using data collected from 1) approximately one year before the passing of Irma and three to six months \nfollowing the storm’s impact, and 2) from three to six months after, and up to 16.5 months after, the storm. Elevation-change \n20 \ndata were then used to generate Triangulated Irregular Network (TIN) models in ArcMap to calculate changes in seafloor \nvolume during each time-period. Our results indicate that Hurricane Irma was primarily a depositional event that increased \nmean seafloor elevation and volume at this study site by 0.34 m and up to 5.4 Mm3, respectively. Sediment was transported \nprimarily west-southwest (WSW) and downslope modifying geomorphic seafloor features including the migration of sand \nwaves and rubble fields, formation of scour marks in shallow seagrass habitat, and burial of seagrass and coral-dominated \n25 \nhabitat. Approximately 16.5 months after Hurricane Irma (during a 13-month period between 2017 and 2019), net erosion was \nobserved across all habitats with mean elevation-change of -0.15 m and net volume change up to -2.46 Mm3. https://doi.org/10.5194/egusphere-2023-3000\nPreprint. Discussion started: 22 December 2023\nPub ic domain CC\n1.0. l\n. 0 https://doi.org/10.5194/egusphere-2023-3000\nPreprint. Discussion started: 22 December 2023\nPub ic domain CC\n1.0. l\n. 0 1 Introduction Coral coverage \nhas been reported at less than 7% along the Florida Keys Reef Tract and less than 3% along the northern FRT in recent years \n50 \n(Jackson et al., 2014; Walton et al, 2018; Knowlton, 2020); and many of Florida’s reefs are in a net erosional state (Yates et \nal., 2017; Morris et al, 2022). Additionally, seagrass has been decreasing in coverage since early Thalassia testudinum die-\noffs in 1987 and more contemporary die-offs in 2015 following storm events and water quality variations (Hall et al., 2016). al., 2009; Gorstein et al., 2016, Towle et al., 2020). Benthic communities of the Florida Reef Tract (FRT) have been degrading \n40 \nfor the past several decades. Coral coverage has declined across the Caribbean and Florida reefs by more than 50% since the \n1970’s due to coral disease and bleaching (Porter et al., 2001; Patterson et al., 2002; Williams and Miller, 2012; Joyner et al., \n2015; Walker et al., 2018), pollution and overfishing (Littler et al., 1986; Lapointe & Clark, 1992; and Hughes 1994), and \nmass-mortality of macroalgal grazers (e.g., Lessios et al. 1983). Progression of climate change has increased thermal stress, al., 2009; Gorstein et al., 2016, Towle et al., 2020). Benthic communities of the Florida Reef Tract (FRT) have been degrading \n40 \nfor the past several decades. Coral coverage has declined across the Caribbean and Florida reefs by more than 50% since the \n1970’s due to coral disease and bleaching (Porter et al., 2001; Patterson et al., 2002; Williams and Miller, 2012; Joyner et al., \n2015; Walker et al., 2018), pollution and overfishing (Littler et al., 1986; Lapointe & Clark, 1992; and Hughes 1994), and \nmass-mortality of macroalgal grazers (e.g., Lessios et al. 1983). Progression of climate change has increased thermal stress, coral bleaching and disease, ocean acidification, and corallivory (predation of corals) (Wilkinson 1996; Mumby et al., 2006; \n45 \nBrandt and McManus, 2009; Soto et al., 2011; Kuffner et al., 2015; Randall and van Woesik, 2015; Muehllehner et al., 2016; \nHughes et al., 2017; Rice et al., 2019). These multiple stressors and increased storm occurrences have caused a shift from \nstony-coral-dominated reefs to macroalgae and octocoral dominated reefs (Bohsnack 1983; Hughs, 1994; Knowlton, 1992; \nMiller et al., 2002; Norstrom et al., 2009; Bruno et al., 2009; Ruzicka et al., 2013 and Jackson et al., 2014). Impact of Hurricane Irma on Coral Reef Sediment Redistribution at \n5 \nLooe Key Reef, Florida, USA \nKimberly K. Yates1, Zachery Fehr2, Selena Johnson1, David Zawada1 \n1U.S. Geological Survey, St. Petersburg, FL, 33701, United States \n2Cherokee Nation System Solutions, Tulsa, OK, 74166, United States, Contractor to the U.S. Geological Survey \nCorrespondence to: Kimberly K. Yates (kyates@usgs.gov) \n10 Elevation-change \n20 \ndata were then used to generate Triangulated Irregular Network (TIN) models in ArcMap to calculate changes in seafloor \nvolume during each time-period. Our results indicate that Hurricane Irma was primarily a depositional event that increased \nmean seafloor elevation and volume at this study site by 0.34 m and up to 5.4 Mm3, respectively. Sediment was transported \nprimarily west-southwest (WSW) and downslope modifying geomorphic seafloor features including the migration of sand following the storm’s impact, and 2) from three to six months after, and up to 16.5 months after, the storm. Elevation-change \n20 \ndata were then used to generate Triangulated Irregular Network (TIN) models in ArcMap to calculate changes in seafloor \nvolume during each time-period. Our results indicate that Hurricane Irma was primarily a depositional event that increased \nmean seafloor elevation and volume at this study site by 0.34 m and up to 5.4 Mm3, respectively. Sediment was transported \nprimarily west-southwest (WSW) and downslope modifying geomorphic seafloor features including the migration of sand waves and rubble fields, formation of scour marks in shallow seagrass habitat, and burial of seagrass and coral-dominated \n25 \nhabitat. Approximately 16.5 months after Hurricane Irma (during a 13-month period between 2017 and 2019), net erosion was \nobserved across all habitats with mean elevation-change of -0.15 m and net volume change up to -2.46 Mm3. Rates of elevation \nchange during this post-storm period were one to two orders of magnitude greater than decadal and multi-decadal rates of \nchange in the same location, and changes showed erosion of approximately 50% of sediment deposited during the storm event as seafloor sediment distribution began to re-equilibrate to non-storm sea state conditions. Our results suggest higher resolution \n30 \nelevation-change data collected over seasonal and annual time periods could enhance characterization and understanding of \nshort-term and long-term rates and processes of seafloor change and help guide post-storm recovery and restoration of benthic \nhabitats in topographically complex coral reef systems. as seafloor sediment distribution began to re-equilibrate to non-storm sea state conditions. Our results suggest higher resolution \n30 \nelevation-change data collected over seasonal and annual time periods could enhance characterization and understanding of \nshort-term and long-term rates and processes of seafloor change and help guide post-storm recovery and restoration of benthic \nhabitats in topographically complex coral reef systems. 1 1 Introduction Coral reefs provide a variety of services to coastal communities including protection from coastal hazards such as storms, \n35 \nwaves, and erosion (Ferrario et al., 2014; Storlazzi et al., 2021); socioeconomic benefits such as fisheries, recreation, and \ntourism (Moberg and Folk, 1999; Hall et al. 2020); and they support numerous habitats and diverse marine species (Knowlton, \n2020). Socioeconomic benefits of Florida reefs have an estimated value of over 8 billion dollars a year, supporting 39,000 \nSouth Florida jobs and 70,400 total jobs, with at least 2.9 billion dollars contributing directly to the local economy (Krediet et Coral reefs provide a variety of services to coastal communities including protection from coastal hazards such as storms, \n35 \nwaves, and erosion (Ferrario et al., 2014; Storlazzi et al., 2021); socioeconomic benefits such as fisheries, recreation, and \ntourism (Moberg and Folk, 1999; Hall et al. 2020); and they support numerous habitats and diverse marine species (Knowlton, \n2020). Socioeconomic benefits of Florida reefs have an estimated value of over 8 billion dollars a year, supporting 39,000 \nSouth Florida jobs and 70,400 total jobs, with at least 2.9 billion dollars contributing directly to the local economy (Krediet et \nal., 2009; Gorstein et al., 2016, Towle et al., 2020). Benthic communities of the Florida Reef Tract (FRT) have been degrading \n40 \nfor the past several decades. Coral coverage has declined across the Caribbean and Florida reefs by more than 50% since the \n1970’s due to coral disease and bleaching (Porter et al., 2001; Patterson et al., 2002; Williams and Miller, 2012; Joyner et al., \n2015; Walker et al., 2018), pollution and overfishing (Littler et al., 1986; Lapointe & Clark, 1992; and Hughes 1994), and \nmass-mortality of macroalgal grazers (e.g., Lessios et al. 1983). Progression of climate change has increased thermal stress, \ncoral bleaching and disease, ocean acidification, and corallivory (predation of corals) (Wilkinson 1996; Mumby et al., 2006; \n45 \nBrandt and McManus, 2009; Soto et al., 2011; Kuffner et al., 2015; Randall and van Woesik, 2015; Muehllehner et al., 2016; \nHughes et al., 2017; Rice et al., 2019). These multiple stressors and increased storm occurrences have caused a shift from \nstony-coral-dominated reefs to macroalgae and octocoral dominated reefs (Bohsnack 1983; Hughs, 1994; Knowlton, 1992; \nMiller et al., 2002; Norstrom et al., 2009; Bruno et al., 2009; Ruzicka et al., 2013 and Jackson et al., 2014). 1 Introduction Coral coverage has been reported at less than 7% along the Florida Keys Reef Tract and less than 3% along the northern FRT in recent years \n50 \n(Jackson et al., 2014; Walton et al, 2018; Knowlton, 2020); and many of Florida’s reefs are in a net erosional state (Yates et \nal., 2017; Morris et al, 2022). Additionally, seagrass has been decreasing in coverage since early Thalassia testudinum die-\noffs in 1987 and more contemporary die-offs in 2015 following storm events and water quality variations (Hall et al., 2016). Multi-decadal seafloor elevation-change analyses along the FRT indicate that degradation of coral reefs and surrounding \n55 \nseafloor habitats has led to substantial erosion and loss of elevation from the 1930’s to 2002 and increased water depths to \nlevels not expected until near the year 2100 (Yates et al., 2017). Projected socioeconomic impacts due to continued FRT coral \nreef degradation and loss of seafloor elevation estimate increases of flooding risk from storms and coastal inundation to more \nthan 7,300 people and $823.6 million (2010 U.S. dollars, USD) in direct and indirect damage to housing, buildings, and Multi-decadal seafloor elevation-change analyses along the FRT indicate that degradation of coral reefs and surrounding \n55 \nseafloor habitats has led to substantial erosion and loss of elevation from the 1930’s to 2002 and increased water depths to \nlevels not expected until near the year 2100 (Yates et al., 2017). Projected socioeconomic impacts due to continued FRT coral \nreef degradation and loss of seafloor elevation estimate increases of flooding risk from storms and coastal inundation to more \nthan 7,300 people and $823.6 million (2010 U.S. dollars, USD) in direct and indirect damage to housing, buildings, and businesses, annually (Storlazzi et al., 2021). Storm frequency and strength are projected to increase as sea-surface temperatures \n60 \nand atmospheric energy increase due to climate change and global warming (Elsner et al., 2008, Bhatia et al., 2019; Knutson \net al., 2020). While advances have been made in understanding long-term change in seafloor elevation and structure and its \npotential socioeconomic consequences, understanding the effects of event-driven changes to seafloor geomorphology due to \nstorms remains a major knowledge gap. businesses, annually (Storlazzi et al., 2021). Storm frequency and strength are projected to increase as sea-surface temperatures \n60 \nand atmospheric energy increase due to climate change and global warming (Elsner et al., 2008, Bhatia et al., 2019; Knutson \net al., 2020). https://doi.org/10.5194/egusphere-2023-3000\nPreprint. Discussion started: 22 December 2023\nPub ic domain CC\n1.0. l\n. 0 https://doi.org/10.5194/egusphere-2023-3000\nPreprint. Discussion started: 22 December 2023\nPub ic domain CC\n1.0. l\n. 0 Major tropical storms persistently impact the state of Florida with historical hurricane impacts estimated to have caused more \nthan $450 billion dollars of damage across the state from the early 1900’s to 2007 (Malmstadt et al., 2009). The Middle to \nLower Florida Keys (from Islamorada to Key West) has been impacted by 15 major hurricane landfall events (Category 3 \nthrough 5) and numerous tropical storm and Category 1 and 2 hurricanes from the early 1900’s to 2022 (NOAA, 2022a). Hurricane Irma made landfall at Cudjoe Key in the Lower Florida Keys after passing directly over Looe Key Reef on 10 \n70 \nSeptember 2017 as a category 4 hurricane with maximum wind speeds of 213 km h-1 (115 kts) (Cangialosi et al., 2021) and \nsignificant wave heights of approximately 14 m a few kilometers offshore of the Florida Keys (Xian et al., 2018, Fig. 1a, b). Satellite imagery showed extensive sediment plumes throughout South Florida and the FRT caused by sediment resuspension \nand transport during the storm (Fig. 1c, d). The storm damaged up to 75% of buildings near its landfall point and caused Hurricane Irma made landfall at Cudjoe Key in the Lower Florida Keys after passing directly over Looe Key Reef on 10 \n70 \nSeptember 2017 as a category 4 hurricane with maximum wind speeds of 213 km h-1 (115 kts) (Cangialosi et al., 2021) and \nsignificant wave heights of approximately 14 m a few kilometers offshore of the Florida Keys (Xian et al., 2018, Fig. 1a, b). Satellite imagery showed extensive sediment plumes throughout South Florida and the FRT caused by sediment resuspension \nand transport during the storm (Fig. 1c, d). The storm damaged up to 75% of buildings near its landfall point and caused \napproximately 50 billion USD of wind and water damage across the state of Florida (Xian et al., 2018; Cangialosi et al., 2021; \n75 \nNOAA, 2022b). Prior to Hurricane Irma, the most recent, direct impact to Looe Key Reef from a tropical storm was in 2008 \nduring Tropical Storm Fay (NOAA, 2022a). Diver\nbased surveys of coral reefs at 57 locations along the FRT by the National Oceanic and Atmospheric Administration showed \n80 \nhighest levels of damage in the Middle to Lower Keys including dislodged and fractured corals, clogged and damaged sponges, \nheavy sedimentation, burial of corals, displaced rubble and sand, reef erosion, fractured substrate, and marine debris; 14% of \nsites showed severe impact, 33% showed moderate impact, and 53% showed minimal impact (Viehman et al., 2018). Looe \nKey Reef, located near the hurricane landfall location, showed more than 26% prevalence of hurricane-impacted corals (Florida based surveys of coral reefs at 57 locations along the FRT by the National Oceanic and Atmospheric Administration showed \n80 \nhighest levels of damage in the Middle to Lower Keys including dislodged and fractured corals, clogged and damaged sponges, \nheavy sedimentation, burial of corals, displaced rubble and sand, reef erosion, fractured substrate, and marine debris; 14% of \nsites showed severe impact, 33% showed moderate impact, and 53% showed minimal impact (Viehman et al., 2018). Looe \nKey Reef, located near the hurricane landfall location, showed more than 26% prevalence of hurricane-impacted corals (Florida Resilience Program, 2017). Similar surveys along the northern FRT from Key Biscayne north showed from approximately 5% \n85 \nto 17% of 62 sites with impacts to corals including dislodged and buried colonies, and at least one site with slabs of hardbottom \n2 to 5 m in size fractured and displaced several meters (Walker, 2018). Analyses of long-term monitoring-transect data at 40 \nsites throughout the Florida Keys National Marine Sanctuary (FKNMS) showed instantaneous losses in seagrass and \ncalcareous green macroalgae density after the storm passed, particularly in the Lower Florida Keys near where Hurricane Irma Resilience Program, 2017). Similar surveys along the northern FRT from Key Biscayne north showed from approximately 5% \n85 \nto 17% of 62 sites with impacts to corals including dislodged and buried colonies, and at least one site with slabs of hardbottom \n2 to 5 m in size fractured and displaced several meters (Walker, 2018). Analyses of long-term monitoring-transect data at 40 \nsites throughout the Florida Keys National Marine Sanctuary (FKNMS) showed instantaneous losses in seagrass and \ncalcareous green macroalgae density after the storm passed, particularly in the Lower Florida Keys near where Hurricane Irma made landfall (Wilson et al., 2020). 1 Introduction While advances have been made in understanding long-term change in seafloor elevation and structure and its \npotential socioeconomic consequences, understanding the effects of event-driven changes to seafloor geomorphology due to \nstorms remains a major knowledge gap. 65 2 2 Hurricane Irma made landfall at Cudjoe Key in the Lower Florida Keys after passing directly over Looe Key Reef on 10 \n70 \nSeptember 2017 as a category 4 hurricane with maximum wind speeds of 213 km h-1 (115 kts) (Cangialosi et al., 2021) and \nsignificant wave heights of approximately 14 m a few kilometers offshore of the Florida Keys (Xian et al., 2018, Fig. 1a, b). Satellite imagery showed extensive sediment plumes throughout South Florida and the FRT caused by sediment resuspension \nand transport during the storm (Fig. 1c, d). The storm damaged up to 75% of buildings near its landfall point and caused \napproximately 50 billion USD of wind and water damage across the state of Florida (Xian et al., 2018; Cangialosi et al., 2021; \n75 approximately 50 billion USD of wind and water damage across the state of Florida (Xian et al., 2018; Cangialosi et al., 2021; \n75 \nNOAA, 2022b). Prior to Hurricane Irma, the most recent, direct impact to Looe Key Reef from a tropical storm was in 2008 \nduring Tropical Storm Fay (NOAA, 2022a). Numerous rapid assessments of seafloor habitats were conducted along the FRT in the weeks following Hurricane Irma. Diver-\nbased surveys of coral reefs at 57 locations along the FRT by the National Oceanic and Atmospheric Administration showed \n80 \nhighest levels of damage in the Middle to Lower Keys including dislodged and fractured corals, clogged and damaged sponges, \nheavy sedimentation, burial of corals, displaced rubble and sand, reef erosion, fractured substrate, and marine debris; 14% of \nsites showed severe impact, 33% showed moderate impact, and 53% showed minimal impact (Viehman et al., 2018). Looe \nKey Reef, located near the hurricane landfall location, showed more than 26% prevalence of hurricane-impacted corals (Florida \nResilience Program, 2017). Similar surveys along the northern FRT from Key Biscayne north showed from approximately 5% \n85 \nto 17% of 62 sites with impacts to corals including dislodged and buried colonies, and at least one site with slabs of hardbottom \n2 to 5 m in size fractured and displaced several meters (Walker, 2018). Analyses of long-term monitoring-transect data at 40 \nsites throughout the Florida Keys National Marine Sanctuary (FKNMS) showed instantaneous losses in seagrass and \ncalcareous green macroalgae density after the storm passed, particularly in the Lower Florida Keys near where Hurricane Irma \nmade landfall (Wilson et al., 2020). Additionally, several locations showed moderate burial of seagrass with up to 5 to 10 cm \n90 \nof sand, while other locations showed heavy erosion or moderate seagrass canopy thinning (Wilson et al., 2020). Reef Visual \nCensus (RVC) surveys including structure from motion (SfM) habitat photogrammetry at sites in the Lower Florida Keys from \nFebruary 2017 to December 2018 showed a 30% decrease in macroalgae at the Looe Key Sanctuary Preservation Area (SPA) \nand a 30% increase at the Looe Key Special Use Area (SPU) post Irma; while both Looe Key locations showed a 10% decrease \nin live coral cover and a 20% increase in octocoral cover (Simmons et al., 2022). Comparison of restored (outplant) coral \n95 \nsurvival rates at two fore reef and two patch reef sites near Tavernier Key in the Upper Florida Keys showed approximately \n85% outplant survival at all locations prior to the passage of Hurricane Irma; however, no outplants survived at the fore reef \nsites and only 51% of outplants survived at the patch reef sites post-Irma, the difference likely due to protection of the patch \nreefs from dissipation of wave energy by the reef crest (Lohr et al., 2020). Examination of Diadema antillarum sea urchins (a Numerous rapid assessments of seafloor habitats were conducted along the FRT in the weeks following Hurricane Irma. Diver-\nbased surveys of coral reefs at 57 locations along the FRT by the National Oceanic and Atmospheric Administration showed \n80 \nhighest levels of damage in the Middle to Lower Keys including dislodged and fractured corals, clogged and damaged sponges, \nheavy sedimentation, burial of corals, displaced rubble and sand, reef erosion, fractured substrate, and marine debris; 14% of \nsites showed severe impact, 33% showed moderate impact, and 53% showed minimal impact (Viehman et al., 2018). Looe \nKey Reef, located near the hurricane landfall location, showed more than 26% prevalence of hurricane-impacted corals (Florida Numerous rapid assessments of seafloor habitats were conducted along the FRT in the weeks following Hurricane Irma. Additionally, several locations showed moderate burial of seagrass with up to 5 to 10 cm \n90 \nof sand, while other locations showed heavy erosion or moderate seagrass canopy thinning (Wilson et al., 2020). Reef Visual \nCensus (RVC) surveys including structure from motion (SfM) habitat photogrammetry at sites in the Lower Florida Keys from \nFebruary 2017 to December 2018 showed a 30% decrease in macroalgae at the Looe Key Sanctuary Preservation Area (SPA) \nand a 30% increase at the Looe Key Special Use Area (SPU) post Irma; while both Looe Key locations showed a 10% decrease in live coral cover and a 20% increase in octocoral cover (Simmons et al., 2022). Comparison of restored (outplant) coral \n95 \nsurvival rates at two fore reef and two patch reef sites near Tavernier Key in the Upper Florida Keys showed approximately \n85% outplant survival at all locations prior to the passage of Hurricane Irma; however, no outplants survived at the fore reef \nsites and only 51% of outplants survived at the patch reef sites post-Irma, the difference likely due to protection of the patch \nreefs from dissipation of wave energy by the reef crest (Lohr et al., 2020). Examination of Diadema antillarum sea urchins (a 3 https://doi.org/10.5194/egusphere-2023-3000\nPreprint. Discussion started: 22 December 2023\nPub ic domain CC\n1.0. l\n. 0 https://doi.org/10.5194/egusphere-2023-3000\nPreprint. Discussion started: 22 December 2023\nPub ic domain CC\n1.0. l\n. 0 100 re 1. Location of the Florida Keys Reef Tract, Hurricane Irma trackline and impact. (a) NOAA, National Weather Service WSR-\nradar image (decibels, DBZ) from south Florida on 10 September 2017 at 5:22 am Eastern Daylight Time (EDT) showing approach of\nicane Irma (inset black line = hurricane trackline). (b) Significant wave height (m) from the U.S. Geological Survey (USGS) Coupled\nan, Atmosphere, Wave, Sediment Transport (COAWST) model on 10 September 2017 at 5:00 am EDT (Warner et al., 2010, image\nit: Patricia Dalyander, USGS). (c) Satellite imagery from 30 August 2017, 11 days prior to landfall of Hurricane Irma in the Florida\ns (NASA, 2023, EOSDIS Worldview Imagery). (d) Satellite imagery from 13 September 2017, 3 days after Hurricane Irma landfall in\nFlorida Keys showing extensive resuspended sediment plume (NASA, 2023, EOSDIS Worldview Imagery). Red boxes show the location\nooe Key Reef relative to other reefs along the reef tract Figure 1. Location of the Florida Keys Reef Tract, Hurricane Irma trackline and impact. https://doi.org/10.5194/egusphere-2023-3000\nPreprint. Discussion started: 22 December 2023\nPub ic domain CC\n1.0. l\n. 0 https://doi.org/10.5194/egusphere-2023-3000\nPreprint. Discussion started: 22 December 2023\nPub ic domain CC\n1.0. l\n. 0 key reef grazer) density, size structure, and coral reef community structure before and 2.5 months after Irma at 10 locations in \n110 \nthe Middle and Upper Florida Keys showed a significant decrease in D. antillarum density with increased sedimentation, \nsuggesting sediment transport caused mortality through abrasion and burial (Kobelt et al. 2019). key reef grazer) density, size structure, and coral reef community structure before and 2.5 months after Irma at 10 locations in \n110 \nthe Middle and Upper Florida Keys showed a significant decrease in D. antillarum density with increased sedimentation, \nsuggesting sediment transport caused mortality through abrasion and burial (Kobelt et al. 2019). While observational data from several locations indicate seafloor sediments were transporte While observational data from several locations indicate seafloor sediments were transported and likely caused damage to \nbenthic habitats, the direct impact of Hurricane Irma or other tropical storms on seafloor elevation and geomorphologic \n115 \nstructures has not previously been quantified along the FRT. In this study, we used high-resolution light-detection-and-ranging \n(lidar) and multibeam bathymetry data collected before and after the passage of Hurricane Irma to quantify seafloor elevation \nand volume change of benthic habitats and geomorphological structures resulting from the storm’s impact and post-storm re-\nequilibration of seafloor sediments at more than 4-million point-locations at the Looe Key Reef system in the Lower FRT. benthic habitats, the direct impact of Hurricane Irma or other tropical storms on seafloor elevation and geomorphologic \n115 \nstructures has not previously been quantified along the FRT. In this study, we used high-resolution light-detection-and-ranging \n(lidar) and multibeam bathymetry data collected before and after the passage of Hurricane Irma to quantify seafloor elevation \nand volume change of benthic habitats and geomorphological structures resulting from the storm’s impact and post-storm re-\nequilibration of seafloor sediments at more than 4-million point-locations at the Looe Key Reef system in the Lower FRT. 2.1 Looe Key Reef Study Site The FRT is the only living coral barrier reef in the continental United States, and it spans more than 580 km along the east \ncoast of Florida from St. Lucie Inlet to the Dry Tortugas, with total reef area of approximately 1,179 km2 (Finkl and Andrews, \n2008; Jackson et al., 2014; Florida Department of Environmental Protection, 2022). Water depth along the FRT is up to approximately 20 m with discontinuous spur and groove formations and patch reefs separated by tidal passes, and it is \n125 \ncharacterized by both coral-dominated and non-coral dominated seafloor habitat as characterized and mapped by the Florida \nFish and Wildlife Conservation Commission-Fish and Wildlife Research Institute (FWC, 2015). Much of the FRT is protected \nby the FKNMS, Biscayne National Park, and Dry Tortugas National Park, and includes several sanctuary preservation areas \n(SPAs) and special use areas (SPUs) within FKNMS, including the Looe Key SPA and SPU, that together protect over 6000 approximately 20 m with discontinuous spur and groove formations and patch reefs separated by tidal passes, and it is \n125 \ncharacterized by both coral-dominated and non-coral dominated seafloor habitat as characterized and mapped by the Florida \nFish and Wildlife Conservation Commission-Fish and Wildlife Research Institute (FWC, 2015). Much of the FRT is protected \nby the FKNMS, Biscayne National Park, and Dry Tortugas National Park, and includes several sanctuary preservation areas \n(SPAs) and special use areas (SPUs) within FKNMS, including the Looe Key SPA and SPU, that together protect over 6000 marine species (Keller and Donahue, 2006). Looe Key Reef is a barrier bank reef located approximately 10 km offshore in the \n130 \nLower Florida Keys, south of Cudjoe Key, and it is characterized by a prominent, shallow reef crest with two extensive coral \nrubble fields, a fore reef with a spur-and-groove formation, a forereef terrace and deep reef zone, and a back reef area with \nseagrass communities, patch reefs, and individual coral heads (Fig. 2a-d). Seagrass beds and sandflats with intermittent patch \nreefs extend shoreward from Looe Key Reef proper to Hawk Channel, approximately 2 km to the north. Looe Key SPA, marine species (Keller and Donahue, 2006). (a) NOAA, National Weather Service WSR-\n88D radar image (decibels, DBZ) from south Florida on 10 September 2017 at 5:22 am Eastern Daylight Time (EDT) showing approach of \nHurricane Irma (inset black line = hurricane trackline). (b) Significant wave height (m) from the U.S. Geological Survey (USGS) Coupled \nOcean, Atmosphere, Wave, Sediment Transport (COAWST) model on 10 September 2017 at 5:00 am EDT (Warner et al., 2010, image \ncredit: Patricia Dalyander, USGS). (c) Satellite imagery from 30 August 2017, 11 days prior to landfall of Hurricane Irma in the Florida \nKeys (NASA, 2023, EOSDIS Worldview Imagery). (d) Satellite imagery from 13 September 2017, 3 days after Hurricane Irma landfall in \nthe Florida Keys showing extensive resuspended sediment plume (NASA, 2023, EOSDIS Worldview Imagery). Red boxes show the location \nof Looe Key Reef relative to other reefs along the reef tract. 105 4 2.1 Looe Key Reef Study Site Looe Key Reef is a barrier bank reef located approximately 10 km offshore in the \n130 \nLower Florida Keys, south of Cudjoe Key, and it is characterized by a prominent, shallow reef crest with two extensive coral \nrubble fields, a fore reef with a spur-and-groove formation, a forereef terrace and deep reef zone, and a back reef area with \nseagrass communities, patch reefs, and individual coral heads (Fig. 2a-d). Seagrass beds and sandflats with intermittent patch \nreefs extend shoreward from Looe Key Reef proper to Hawk Channel, approximately 2 km to the north. Looe Key SPA, located at approximately 24° 32' N, 81° 24' W, is just over 18 km2 and surrounds LKR proper which is less than 1.7 km2. Looe \n135 \nKey Reef contains a coral nursery and several restoration sites for coral outplants; it is one of seven FKNMS iconic reefs, and \nthe focus of a major collaborative habitat restoration effort known as Mission: Iconic reefs (NOAA Fisheries, 2022). located at approximately 24° 32' N, 81° 24' W, is just over 18 km2 and surrounds LKR proper which is less than 1.7 km2. Looe \n135 \nKey Reef contains a coral nursery and several restoration sites for coral outplants; it is one of seven FKNMS iconic reefs, and \nthe focus of a major collaborative habitat restoration effort known as Mission: Iconic reefs (NOAA Fisheries, 2022). 5\nThe northeastern eyewall of Hurricane Irma passed directly over LKR with the storm’s center passing approximately 9 km \nwest of LKR. However, the storm was approximately 684 km in diameter and covered the entire FRT and much of South \n140 \nFlorida. The National Weather Service’s technical summary of the storm reported tropical storm force winds more than 640 The northeastern eyewall of Hurricane Irma passed directly over LKR with the storm’s center passing approximately 9 km 5\nwest of LKR. However, the storm was approximately 684 km in diameter and covered the entire FRT and much of South \n140 \nFlorida. The National Weather Service’s technical summary of the storm reported tropical storm force winds more than 640 5\nwest of LKR. However, the storm was approximately 684 km in diameter and covered the entire FRT and much of South \n140 \nFlorida. The National Weather Service’s technical summary of the storm reported tropical storm force winds more than 640 5 https://doi.org/10.5194/egusphere-2023-3000\nPreprint. Discussion started: 22 December 2023\nPub ic domain CC\n1.0. l\n. 0 https://doi.org/10.5194/egusphere-2023-3000\nPreprint. Discussion started: 22 December 2023\nPub ic domain CC\n1.0. l\n. 0 km away from the storm’s center, and hurricane force winds more than 125 km from the storm’s center (NWS, 2022b). Gale \nforce winds (sustained winds above 63 km h-1) were detected by the evening of 9 September 2017 at the National Oceanic and \nAtmospheric Administration - National Ocean Service (NOAA-NOS), Tides and Currents Station at Vaca Key (number \n8723970), 35 km to the northeast of LKR; maximum sustained winds of 213 km h-1 were reported as the storm made landfall, 155 and latent gale force winds were detected after the storm passed on the evening of 10 September 2017 (NOAA-NOS, 2023). 155 \nThe average wind direction for this period was 67.01 degrees indicating winds moved from ENE toward WSW. Wind speeds \nfell sharply below gale force after the storm, shifting north eastward. Wind conditions were relatively quiescent from July \n2016 through January 2019 (except during Hurricane Irma) with wind speeds occasionally ranging up to approximately 56 km \nh-1 during winter storms (NOAA-NOS, 2023). and latent gale force winds were detected after the storm passed on the evening of 10 September 2017 (NOAA-NOS, 2023). 155 \nThe average wind direction for this period was 67.01 degrees indicating winds moved from ENE toward WSW. Wind speeds \nfell sharply below gale force after the storm, shifting north eastward. Wind conditions were relatively quiescent from July \n2016 through January 2019 (except during Hurricane Irma) with wind speeds occasionally ranging up to approximately 56 km \nh-1 during winter storms (NOAA-NOS, 2023). https://doi.org/10.5194/egusphere-2023-3000\nPreprint. Discussion started: 22 December 2023\nPub ic domain CC\n1.0. l\n. 0 gure 2. Looe Key Reef location, bathymetry, and seafloor habitats. (a) Location of the Florida Keys along the southern coast of Florida, \nckline of Hurricane Irma and the location of its landfall (red box). (b) Proximity of Hurricane Irma’s trackline to the Looe Key Reef study \ne (purple box), location of landfall at Cudjoe Key, and location of Vaca Key where the nearest NOAA-NOS (2023) Tides and Currents \ntion was located. (c) 2016 lidar bathymetric map of the Looe Key Reef study site showing location of Florida Keys National Marine \nnctuary Special Preservation Area (SPA), Special Use Area (SPU), and geomorphic features of focused investigation for this study. (d) \nbitat distribution at the Looe Key Study site from Florida Fish and Wildlife Conservation Commission-Fish and Wildlife Research \ntitute (2015). Figure 2. Looe Key Reef location, bathymetry, and seafloor habitats. (a) Location of the Florida Keys along the southern coast of Florida, \ntrackline of Hurricane Irma and the location of its landfall (red box). (b) Proximity of Hurricane Irma’s trackline to the Looe Key Reef study \nsite (purple box), location of landfall at Cudjoe Key, and location of Vaca Key where the nearest NOAA-NOS (2023) Tides and Currents \n145 \nstation was located. (c) 2016 lidar bathymetric map of the Looe Key Reef study site showing location of Florida Keys National Marine \nSanctuary Special Preservation Area (SPA), Special Use Area (SPU), and geomorphic features of focused investigation for this study. (d) \nHabitat distribution at the Looe Key Study site from Florida Fish and Wildlife Conservation Commission-Fish and Wildlife Research \nInstitute (2015). 150 150 6 The 2016 lidar DEM refers to data that were collected on 23 July 2016 (13.5 months before the passage of Hurricane Irma) by \nthe NOAA Office for Coastal Management, National Geodetic Survey, Topobathy Lidar Dem Block 1 dataset (Office for \nCoastal Management, 2017). The 2017 multibeam DEM refers to multibeam bathymetry data collected by the U.S. Geological \nSurvey in December 2017, and February–March 2018 at Looe Key Reef (between three and six months after the passage of \nHurricane Irma), specifically to examine impacts from the storm (Fredericks et al., 2019). The 2019 lidar DEM refers to data \n170 \ncollected January 8–31, 2019 by NOAA NGS Topobathy Lidar DEM Hurricane Irma: Miami to Marquesas Keys, FL dataset \n(National Geodetic Survey, 2022). The Florida Fish and Wildlife Conservation Commission (FWC) Unified Florida Reef Tract \n(UFRT) Map version 2.0, Level 2 habitat categories (FWC, 2015) were used to delineate geographic boundaries for 10 habitat 2.2 Elevation and Habitat Data \n160 Three Digital Elevation Models (DEMs) derived from lidar or multibeam bathymetric surveys were used for seafloor elevation- \nand volume-change analyses and are referenced in this study as 2016 lidar, 2017 multibeam, and 2019 lidar (Table 1). Table 1: Elevation datasets used in this analysis; collection dates are specific to the geographic extent of this study. Digital Elevation Model \nSource \nCollection Dates \nHorizontal Resolution/ \nVertical RMSE \n(meters/meters) \n2016 NOAA NGS Topobathy \nLidar DEM:Florida Keys \nOuter Reef Block 01 \nOffice for \nCoastal \nManagement, \n2017 \n23 July 2016 \n \n1.0/0.15 \nMultibeam bathymetry data \ncollected in December 2017, \nFebruary and March 2018 at \nLooe Key, the Florida Keys \nFredericks et al, \n2019 \nLeg 1: 12 December 2017 – 16 December 2017 \nLeg 2: 2 February 2018 – 9 February 2018 \nLeg 3: 9 March 2018 –11 March 2018 \n \n1.0/0.14 \n2018-2019 NOAA NGS \nTopobathy Lidar Hurricane \nIrma: Miami to Marquesas \nKeys, FL \nNational \nGeodetic Survey, \n2022 \n8 January 2019– 31 January 2019 \n \n1.0/0.11 \nRMSE = root mean square error. 165 165 The 2016 lidar DEM refers to data that were collected on 23 July 2016 (13.5 months before the passage of Hurricane Irma) by \nthe NOAA Office for Coastal Management, National Geodetic Survey, Topobathy Lidar Dem Block 1 dataset (Office for \nCoastal Management, 2017). The 2017 multibeam DEM refers to multibeam bathymetry data collected by the U.S. Geological \nSurvey in December 2017, and February–March 2018 at Looe Key Reef (between three and six months after the passage of \nHurricane Irma), specifically to examine impacts from the storm (Fredericks et al., 2019). The 2019 lidar DEM refers to data \n170 \ncollected January 8–31, 2019 by NOAA NGS Topobathy Lidar DEM Hurricane Irma: Miami to Marquesas Keys, FL dataset \n(National Geodetic Survey, 2022). The Florida Fish and Wildlife Conservation Commission (FWC) Unified Florida Reef Tract \n(UFRT) Map version 2.0, Level 2 habitat categories (FWC, 2015) were used to delineate geographic boundaries for 10 habitat The 2016 lidar DEM refers to data that were collected on 23 July 2016 (13.5 months before the passage of Hurricane Irma) by \nthe NOAA Office for Coastal Management, National Geodetic Survey, Topobathy Lidar Dem Block 1 dataset (Office for \nCoastal Management, 2017). The 2017 multibeam DEM refers to multibeam bathymetry data collected by the U.S. https://doi.org/10.5194/egusphere-2023-3000\nPreprint. Discussion started: 22 December 2023\nPub ic domain CC\n1.0. l\n. 0 https://doi.org/10.5194/egusphere-2023-3000\nPreprint. Discussion started: 22 December 2023\nPub ic domain CC\n1.0. l\n. 0 types within the LKR study site (Fig. 2d). Habitat labelled as ‘not classified’ was indistinguishable during mapping due to \nturbidity, cloud cover, water depth, or other interferences with obtaining an optical signature of the seafloor (Zitello et al., \n175 \n2009). turbidity, cloud cover, water depth, or other interferences with obtaining an optical signature of the seafloor (Zitello et al., \n175 \n2009). turbidity, cloud cover, water depth, or other interferences with obtaining an optical signature of the seafloor (Zitello et al., \n175 \n2009). 2.2 Elevation and Habitat Data \n160 Geological \nSurvey in December 2017, and February–March 2018 at Looe Key Reef (between three and six months after the passage of Hurricane Irma), specifically to examine impacts from the storm (Fredericks et al., 2019). The 2019 lidar DEM refers to data \n170 \ncollected January 8–31, 2019 by NOAA NGS Topobathy Lidar DEM Hurricane Irma: Miami to Marquesas Keys, FL dataset \n(National Geodetic Survey, 2022). The Florida Fish and Wildlife Conservation Commission (FWC) Unified Florida Reef Tract \n(UFRT) Map version 2.0, Level 2 habitat categories (FWC, 2015) were used to delineate geographic boundaries for 10 habitat Hurricane Irma), specifically to examine impacts from the storm (Fredericks et al., 2019). The 2019 lidar DEM refers to data \n170 \ncollected January 8–31, 2019 by NOAA NGS Topobathy Lidar DEM Hurricane Irma: Miami to Marquesas Keys, FL dataset \n(National Geodetic Survey, 2022). The Florida Fish and Wildlife Conservation Commission (FWC) Unified Florida Reef Tract \n(UFRT) Map version 2.0, Level 2 habitat categories (FWC, 2015) were used to delineate geographic boundaries for 10 habitat 7 7 2.3 Elevation- and Volume-Change Analyses A 2-m XY grid was created in Global Mapper and clipped to the same footprint. Elevation values were then extracted \nfrom each of the three DEMs at the center points of co-aligned 2-m grid boxes. Elevation change between time periods was \ncalculated for each of 4,007,961 paired elevation values (e.g., 2017 elevation – 2016 elevation, and 2019 elevation – 2017 extent of each DEM was then clipped to the areal extent of the common overlapping footprint prior to elevation change analysis \n185 \nusing the ‘Clip’ tool in ArcMap. The following steps were performed in Global Mapper 22.1 due to file size limitations in \nArcMap. A 2-m XY grid was created in Global Mapper and clipped to the same footprint. Elevation values were then extracted \nfrom each of the three DEMs at the center points of co-aligned 2-m grid boxes. Elevation change between time periods was \ncalculated for each of 4,007,961 paired elevation values (e.g., 2017 elevation – 2016 elevation, and 2019 elevation – 2017 elevation). Elevation-change (XYZ) point maps were generated as shapefiles for each time-period of change for the total study \n190 \nsite; positive values indicate an increase in elevation and negative values indicate a decrease in elevation. Data are available \nfrom Fehr et al. (2021). Vertical uncertainty of elevation change analyses were estimated using methods of Yates et al. 2017 \nand the reported vertical accuracy of the lidar and multibeam data sets (typically reported as the 95% root-mean-square error, \nRMSE, Table 1) to calculate a composite RMSE (RMSETotal) for each elevation change analysis (Fig. 3b). The RMSE of lidar and multibeam data sets used for elevation-change analyses in our study ranged from 0.11 to 0.15 m (Table 1). These values \n195 \nare consistent with RMSEs determined in performance evaluations of lidar sensors that ranged from 0.08 to 0.52 m (Fernandez-\nDiaz et al., 2014; Legleiter et al., 2016; Kinzel et al., 2013; Tonina et al., 2019; Yoshida et al., 2022). Composite RMSE values \nfor elevation-change analyses based on comparison of lidar to multibeam DEMs ranged from 0.19 to 0.21m in our study. These \nvalues are consistent with RMSEs determined in performance evaluations of lidar sensors against multibeam echosounders and multibeam data sets used for elevation-change analyses in our study ranged from 0.11 to 0.15 m (Table 1). 2.3 Elevation- and Volume-Change Analyses The RMSE of lidar \nand multibeam data sets used for elevation-change analyses in our study ranged from 0.11 to 0.15 m (Table 1). These values \n195 \nare consistent with RMSEs determined in performance evaluations of lidar sensors that ranged from 0.08 to 0.52 m (Fernandez-\nDiaz et al., 2014; Legleiter et al., 2016; Kinzel et al., 2013; Tonina et al., 2019; Yoshida et al., 2022). Composite RMSE values \nfor elevation-change analyses based on comparison of lidar to multibeam DEMs ranged from 0.19 to 0.21m in our study. These \nvalues are consistent with RMSEs determined in performance evaluations of lidar sensors against multibeam echosounders \nthat ranged from 0.02 to 0.23 m (Awadallah et al., 2023). The FWC UFRT habitat map was clipped to the intersect footprint \n200 \nfor each elevation-change analysis using ArcMap 10.7. Each total-study-site elevation-change data set was then clipped to \nindividual habitat polygons to create individual elevation-change shapefiles for each habitat type. of the three 1-m resolution digital elevation models (DEMs) in ArcMap 10.7 and were used to create a common footprint \n180 \npolygon shapefile for the total LKR study site encompassing the overlapping area among the three datasets. The original (full \nareal extent, or unclipped) 2016–2017 elevation-change data set was 19.71 km2 and included 4,934,364 data points. The \noverlapping areal extent for the 2016, 2017, and 2019 DEMs was 15.98 km2 and excluded areas where water depths were too \nshallow for boat access to collect multibeam data in 2017 and areas of coarse interpolation within the 2017 DEM. The areal of the three 1-m resolution digital elevation models (DEMs) in ArcMap 10.7 and were used to create a common footprint \n180 \npolygon shapefile for the total LKR study site encompassing the overlapping area among the three datasets. The original (full \nareal extent, or unclipped) 2016–2017 elevation-change data set was 19.71 km2 and included 4,934,364 data points. The \noverlapping areal extent for the 2016, 2017, and 2019 DEMs was 15.98 km2 and excluded areas where water depths were too \nshallow for boat access to collect multibeam data in 2017 and areas of coarse interpolation within the 2017 DEM. The areal 180 extent of each DEM was then clipped to the areal extent of the common overlapping footprint prior to elevation change analysis \n185 \nusing the ‘Clip’ tool in ArcMap. The following steps were performed in Global Mapper 22.1 due to file size limitations in \nArcMap. 2.3 Elevation- and Volume-Change Analyses Seafloor elevation- and volume-change analyses were conducted using the methods of Yates et al. techniques of Murphy et al. (2022) (Fig. 3a). Briefly, individual geographic footprint areas (poly Seafloor elevation- and volume-change analyses were conducted using the methods of Yates et al. (2017) and 2-m grid spacing \ntechniques of Murphy et al. (2022) (Fig. 3a). Briefly, individual geographic footprint areas (polygons) were created for each Seafloor elevation- and volume-change analyses were conducted using the methods of Yates et al. (2017) and 2-m grid spacing \ntechniques of Murphy et al. (2022) (Fig. 3a). Briefly, individual geographic footprint areas (polygons) were created for each \nof the three 1-m resolution digital elevation models (DEMs) in ArcMap 10.7 and were used to create a common footprint \n180 \npolygon shapefile for the total LKR study site encompassing the overlapping area among the three datasets. The original (full \nareal extent, or unclipped) 2016–2017 elevation-change data set was 19.71 km2 and included 4,934,364 data points. The \noverlapping areal extent for the 2016, 2017, and 2019 DEMs was 15.98 km2 and excluded areas where water depths were too \nshallow for boat access to collect multibeam data in 2017 and areas of coarse interpolation within the 2017 DEM. The areal \nextent of each DEM was then clipped to the areal extent of the common overlapping footprint prior to elevation change analysis \n185 \nusing the ‘Clip’ tool in ArcMap. The following steps were performed in Global Mapper 22.1 due to file size limitations in \nArcMap. A 2-m XY grid was created in Global Mapper and clipped to the same footprint. Elevation values were then extracted \nfrom each of the three DEMs at the center points of co-aligned 2-m grid boxes. Elevation change between time periods was \ncalculated for each of 4,007,961 paired elevation values (e.g., 2017 elevation – 2016 elevation, and 2019 elevation – 2017 \nelevation). Elevation-change (XYZ) point maps were generated as shapefiles for each time-period of change for the total study \n190 \nsite; positive values indicate an increase in elevation and negative values indicate a decrease in elevation. Data are available \nfrom Fehr et al. (2021). Vertical uncertainty of elevation change analyses were estimated using methods of Yates et al. 2017 \nand the reported vertical accuracy of the lidar and multibeam data sets (typically reported as the 95% root-mean-square error, \nRMSE, Table 1) to calculate a composite RMSE (RMSETotal) for each elevation change analysis (Fig. 3b). 2.3 Elevation- and Volume-Change Analyses These values \n195 \nare consistent with RMSEs determined in performance evaluations of lidar sensors that ranged from 0.08 to 0.52 m (Fernandez-\nDiaz et al., 2014; Legleiter et al., 2016; Kinzel et al., 2013; Tonina et al., 2019; Yoshida et al., 2022). Composite RMSE values \nfor elevation-change analyses based on comparison of lidar to multibeam DEMs ranged from 0.19 to 0.21m in our study. These \nvalues are consistent with RMSEs determined in performance evaluations of lidar sensors against multibeam echosounders that ranged from 0.02 to 0.23 m (Awadallah et al., 2023). The FWC UFRT habitat map was clipped to the intersect footprint \n200 \nfor each elevation-change analysis using ArcMap 10.7. Each total-study-site elevation-change data set was then clipped to \nindividual habitat polygons to create individual elevation-change shapefiles for each habitat type. that ranged from 0.02 to 0.23 m (Awadallah et al., 2023). The FWC UFRT habitat map was clipped to the intersect footprint \n200 \nfor each elevation-change analysis using ArcMap 10.7. Each total-study-site elevation-change data set was then clipped to \nindividual habitat polygons to create individual elevation-change shapefiles for each habitat type. Elevation-change data from each time-period were then used to generate TIN (Triangulated Irregular Network) surface mode Elevation-change data from each time-period were then used to generate TIN (Triangulated Irregular Network) surface models \nin ArcMap for calculation of volume change. TIN models were clipped to the original overall study site intersect footprint to \n205 \nremove interpolation across areas where no data were collected. Lower bound (conservative) volume-change was calculated Elevation-change data from each time-period were then used to generate TIN (Triangulated Irregular Network) surface models in ArcMap for calculation of volume change. TIN models were clipped to the original overall study site intersect footprint to \n205 \nremove interpolation across areas where no data were collected. Lower bound (conservative) volume-change was calculated in ArcMap for calculation of volume change. TIN models were clipped to the original overall study site intersect footprint to \n205 \nremove interpolation across areas where no data were collected. Lower bound (conservative) volume-change was calculated 8 https://doi.org/10.5194/egusphere-2023-3000\nPreprint. Discussion started: 22 December 2023\nPub ic domain CC\n1.0. l\n. 0 𝑅𝑀𝑆𝐸𝑡𝑜𝑡𝑎𝑙= √(𝑅𝑀𝑆𝐸𝑆𝑜𝑢𝑟𝑐𝑒 𝐷𝑎𝑡𝑎𝑠𝑒𝑡 1\n2\n+ 𝑅𝑀𝑆𝐸𝑆𝑜𝑢𝑟𝑐𝑒 𝐷𝑎𝑡𝑎𝑠𝑒𝑡 2\n2\n) 𝑅𝑀𝑆𝐸𝑡𝑜𝑡𝑎𝑙= √(𝑅𝑀𝑆𝐸𝑆𝑜𝑢𝑟𝑐𝑒 𝐷𝑎𝑡𝑎𝑠𝑒𝑡 1\n2\n+ 𝑅𝑀𝑆𝐸𝑆𝑜𝑢𝑟𝑐𝑒 𝐷𝑎𝑡𝑎𝑠𝑒𝑡 2\n2\n) \nation and volume change methods. 2.3 Elevation- and Volume-Change Analyses (a) Flowchart outlining generalized geoprocessing steps in ArcMap and Global \nh 3), and in the Seafloor Elevation Change Analysis Tool (SECAT, step 4) for seafloor elevation and volume change \ns et al. (2017), Murphy et al. (2022), and Zieg and Zawada (2021). (b) Composite RMSE (RMSETotal) for each \nis (2016 to 2017 and 2017 to 2019) calculated using reported RMSE for lidar and multibeam source data and methods \nBlack boxes indicate source data files. Blue boxes indicate steps using geoprocessing tools from ArcMap or Global \ndicate data analysis conducted using SECAT. 𝑅𝑀𝑆𝐸𝑡𝑜𝑡𝑎𝑙= √(𝑅𝑀𝑆𝐸𝑆𝑜𝑢𝑟𝑐𝑒 𝐷𝑎𝑡𝑎𝑠𝑒𝑡 1\n2\n+ 𝑅𝑀𝑆𝐸𝑆𝑜𝑢𝑟𝑐𝑒 𝐷𝑎𝑡𝑎𝑠𝑒𝑡 2\n2\n) \nand volume change methods. (a) Flowchart outlining generalized geoprocessin\nnd in the Seafloor Elevation Change Analysis Tool (SECAT, step 4) for seafloor\nl. (2017), Murphy et al. (2022), and Zieg and Zawada (2021). (b) Composite\n6 to 2017 and 2017 to 2019) calculated using reported RMSE for lidar and multib\nboxes indicate source data files. Blue boxes indicate steps using geoprocessing \ndata analysis conducted using SECAT. 𝑅𝑀𝑆𝐸𝑡𝑜𝑡𝑎𝑙= √(𝑅𝑀𝑆𝐸𝑆𝑜𝑢𝑟𝑐𝑒 𝐷𝑎𝑡𝑎𝑠𝑒𝑡 1\n2\n+ 𝑅𝑀𝑆𝐸𝑆𝑜𝑢𝑟𝑐𝑒 𝐷𝑎𝑡𝑎𝑠𝑒𝑡 2\n2\n) 𝑅𝑀𝑆𝐸𝑡𝑜𝑡𝑎𝑙= √(𝑅𝑀𝑆𝐸𝑆𝑜𝑢\n2 𝑅𝑀𝑆𝐸𝑡𝑜𝑡𝑎𝑙= √(𝑅𝑀𝑆𝐸𝑆𝑜𝑢𝑟𝑐𝑒 𝐷𝑎𝑡𝑎𝑠𝑒𝑡 1\n2\n+ 𝑅𝑀𝑆𝐸𝑆𝑜𝑢𝑟𝑐𝑒 𝐷𝑎𝑡𝑎𝑠𝑒𝑡 2\n2\n) Figure 3. Seafloor elevation and volume change methods. (a) Flowchart outlining generalized geop\n0 Figure 3. Seafloor elevation and volume change methods. (a) Flowchart outlining generalized geoprocessing steps in ArcMap and Global \nMapper (steps 1 through 3), and in the Seafloor Elevation Change Analysis Tool (SECAT, step 4) for seafloor elevation and volume change \n210 \nanalyses based on Yates et al. (2017), Murphy et al. (2022), and Zieg and Zawada (2021). (b) Composite RMSE (RMSETotal) for each \nelevation change analysis (2016 to 2017 and 2017 to 2019) calculated using reported RMSE for lidar and multibeam source data and methods \nof Yates et al. (2017). Black boxes indicate source data files. Blue boxes indicate steps using geoprocessing tools from ArcMap or Global \nMapper. Green boxes indicate data analysis conducted using SECAT. Figure 3. Seafloor elevation and volume change methods. (a) Flowchart outlining generalized geoprocessing steps in ArcMap and Global \nMapper (steps 1 through 3), and in the Seafloor Elevation Change Analysis Tool (SECAT, step 4) for seafloor elevation and volume change \n210 \nanalyses based on Yates et al. (2017), Murphy et al. (2022), and Zieg and Zawada (2021). 2.4 Geomorphic Feature Analyses Sub-areas or geomorphic features of high-magnitude elevation change (greater than approximately ±0.5 m) were delineated \non each total-study-site elevation-change point map by manually drawing polygons in ArcMap 10.7 and creating elevation-\nchange shapefiles for each sub-area. Each sub-area was clipped to individual habitat polygons to create individual shapefiles \n230 \nfor each habitat type within a given sub-area. Elevation- volume-change statistics were computed for each geomorphic feature \nof interest, and each habitat within sub-areas of interest using SECAT and methods described in section 2.3. Sub-areas or geomorphic features of high-magnitude elevation change (greater than approximately ±0.5 m) were delineated \non each total-study-site elevation-change point map by manually drawing polygons in ArcMap 10.7 and creating elevation-\nchange shapefiles for each sub-area. Each sub-area was clipped to individual habitat polygons to create individual shapefiles \n230 \nfor each habitat type within a given sub area Elevation volume change statistics were computed for each geomorphic feature Sub-areas or geomorphic features of high-magnitude elevation change (greater than approximately ±0.5 m) were delineated \non each total-study-site elevation-change point map by manually drawing polygons in ArcMap 10.7 and creating elevation- on each total study site elevation change point map by manually drawing polygons in ArcMap 10.7 and creating elevation\nchange shapefiles for each sub-area. Each sub-area was clipped to individual habitat polygons to create individual shapefiles \n230 \nfor each habitat type within a given sub-area. Elevation- volume-change statistics were computed for each geomorphic feature \nof interest, and each habitat within sub-areas of interest using SECAT and methods described in section 2.3. We examined elevation and elevation-change along four 200 to 300 m transects across exam We examined elevation and elevation-change along four 200 to 300 m transects across examples of high-elevation change \ngeomorphic features. Elevation profiles for 2016, 2017, and 2019 were created for each feature of interest by extracting \n235 \nelevation values from each DEM along transect lines across the areas of greatest elevation change for each feature using \nArcMap. Points were selected using the Select Feature by Line tool in ArcMap, and the selected features were then exported \nas a new shapefile. Positions and types of geomorphic features of interest were verified through in-situ observation by SCUBA \ndivers using methods of Fehr and Yates (2020) at 30 diver reconnaissance sites throughout the total study site. https://doi.org/10.5194/egusphere-2023-3000\nPreprint. Discussion started: 22 December 2023\nPub ic domain CC\n1.0. l\n. 0 https://doi.org/10.5194/egusphere-2023-3000\nPreprint. Discussion started: 22 December 2023\nPub ic domain CC\n1.0. l\n. 0 based on areal volume above and below surface plane heights corresponding to plus and minus the RMSETotal of the elevation \nchange analysis (RMSETotal = 21 cm for 2016 to 2017, and 19 cm for 2017 to 2019 change analyses). Upper bound volume \nchanges were calculated based on area volume above and below a plane height of 0 m. The attribute values stored within the \nelevation-change and TIN surface shapefiles were then used to compute elevation and volume change statistics for the total LKR study site and each habitat type using the Seafloor Elevation Change Analysis Tool (SECAT) custom Python script of \n220 \nZieg and Zawada (2021). Pearson correlation and linear regression analyses were performed using Microsoft Excel Version \n2302 (build 16.0.16130.20690) to assess relationships between mean habitat water depth, elevation change, and area-\nnormalized volume change for each habitat type including: 1) 2016 mean water depth (estimated from mean elevation) and \nmean elevation change; 2) 2016 mean water depth and area-normalized volume change; 3) 2017 mean water depth and mean LKR study site and each habitat type using the Seafloor Elevation Change Analysis Tool (SECAT) custom Python script of \n220 \nZieg and Zawada (2021). Pearson correlation and linear regression analyses were performed using Microsoft Excel Version \n2302 (build 16.0.16130.20690) to assess relationships between mean habitat water depth, elevation change, and area-\nnormalized volume change for each habitat type including: 1) 2016 mean water depth (estimated from mean elevation) and \nmean elevation change; 2) 2016 mean water depth and area-normalized volume change; 3) 2017 mean water depth and mean elevation change; 4) 2017 mean water depth and area-normalized volume change; and 5) 2017 to 2019 mean elevation change \n225 \nand 2016 to 2017 mean elevation. 2.3 Elevation- and Volume-Change Analyses (b) Composite RMSE (RMSETotal) for each \nelevation change analysis (2016 to 2017 and 2017 to 2019) calculated using reported RMSE for lidar and multibeam source data and methods \nof Yates et al. (2017). Black boxes indicate source data files. Blue boxes indicate steps using geoprocessing tools from ArcMap or Global \nMapper. Green boxes indicate data analysis conducted using SECAT. 215 9 3.1 Elevation and Volume Change Analyses Elevation-change results for 4,007,961 point-locations at LKR between 2016–2017 (approximately 13.5 months before and 3 \nto 6 months after Hurricane Irma) and between 2017–2019 (from approximately 3 to 16.5 months after Hurricane Irma) are \nshown in Fig. 4a and b, respectively. Mean elevation-change for the total LKR study site from 2016–2017 was 0.34 m ± 0.21; \nand all ten habitat types (Fig. 4c) showed increases in mean elevation (accretion) ranging from 0.20 m to 0.54 m (Table 2). 245 \nLargest mean elevation changes were associated with ‘aggregate reef’ (mean 2016 elevation -13.41 m) and ‘not classified’ 10 https://doi.org/10.5194/egusphere-2023-3000\nPreprint. Discussion started: 22 December 2023\nPub ic domain CC\n1.0. l\n. 0 Table 2. Elevation change data by habitat type associated with each period and geomorphic feature subarea. Habitat type \nTotal points \nArea Mean elevation (m) \nMean elevation change (m) (SD) \n(no.) \n(km2) \n2016 \n2017 \n2019 \n2016 to 2017 2017 to 2019 \nOverall Looe Key Study Site \n Total Study Site \n4007961 \n15.98 \n-8.87 \n-8.53 \n-8.69 \n0.34 (0.21) \n-0.15 (0.11) \n Aggregate Reef \n76647 \n0.30 \n-13.41 -12.91 \n-13.16 \n0.51 (0.20) \n-0.25 (0.20) \n Colonized Pavement \n750 \n0.0028 \n-10.65 -10.33 \n-10.44 \n0.32 (0.12) \n-0.11 (0.08) \n Individual or Aggregate Patch Reef \n54414 \n0.22 \n-8.66 \n-8.33 \n-8.51 \n0.34 (0.15) \n-0.19 (0.10) \n Not Classified \n6932 \n0.026 \n-15.84 -15.30 \n-15.55 \n0.54 (0.25) \n-0.25 (0.17) \n Pavement \n645001 \n2.57 \n-10.00 \n-9.62 \n-9.79 \n0.37 (0.16) \n-0.16 (0.11) \n Reef Rubble \n80987 \n0.32 \n-6.19 \n-5.99 \n-6.17 \n0.20 (0.36) \n-0.18 (0.12) \n Seagrass Continuous \n402458 \n1.60 \n-7.69 \n-7.42 \n-7.54 \n0.27 (0.18) \n-0.12 (0.09) \n Seagrass Discontinuous \n1067504 \n4.26 \n-7.24 \n-6.96 \n-7.10 \n0.28 (0.21) \n-0.14 (0.10) \n Spur and Groove \n184875 \n0.74 \n-9.82 \n-9.45 \n-9.65 \n0.37 (0.25) \n-0.19 (0.19) \n Unconsolidated Sediment \n1488416 \n5.94 \n-9.63 \n-9.26 \n-9.42 \n0.37 (0.21) \n-0.16 (0.09) \nSand Wave \n Total Accretion Area \n15336 \n0.060 \n-6.32 \n-5.53 \n-5.68 \n0.79 (0.45) \n-0.15 (0.12) \n Seagrass Discontinuous \n7345 \n0.029 \n-5.98 \n-5.08 \n-5.23 \n0.90 (0.49) \n-0.15 (0.13) \n Unconsolidated Sediment \n7991 \n0.031 \n-6.63 \n-5.95 \n-6.09 \n0.68 (0.37) \n-0.14 (0.10) \n Total Erosion Area \n11265 \n0.043 \n-5.40 \n-5.75 \n-5.90 \n-0.36 (0.28) \n-0.15 (0.06) \n Seagrass Discontinuous \n580 \n0.002 \n-5.72 \n-5.87 \n-6.02 \n-0.15 (0.15) \n-0.15 (0.08) \n Unconsolidated Sediment \n10685 \n0.041 \n-5.38 \n-5.74 \n-5.90 \n-0.37 (0.29) \n-0.15 (0.05) \nScour Marks \n Scour Mark 1 \n202 \n0.00071 \n-7.03 \n-7.51 \n-7.41 \n-0.49 (0.26) \n0.10 (0.12) \n Seagrass Discontinuous \n197 \n0.00071 \n-7.02 \n-7.51 \n-7.41 \n-0.49 (0.26) \n0.11 (0.12) \n Unconsolidated Sediment \n5 <0.00001 \n-7.34 \n-7.47 \n-7.52 \n-0.12 (0.03) \n-0.05 (0.02) \n Scour Mark 2 \n388 \n0.0014 \n-5.41 \n-5.91 \n-5.71 \n-0.50 (0.27) \n0.20 (0.20) \n Seagrass Continuous \n338 \n0.00124 \n-5.41 \n-5.94 \n-5.70 \n-0.53 (0.27) \n0.24 (0.18) \n Unconsolidated Sediment \n50 \n0.00016 \n-5.42 \n-5.67 \n-5.72 \n-0.26 (0.16) \n-0.05 (0.13) \n Scour Mark 3 \n518 \n0.00188 \n-5.64 \n-6.14 \n-6.02 \n-0.50 (0.29) \n0.12 (0.19) \n Seagrass Continuous \n518 \n0.00188 \n-5.64 \n-6.14 \n-6.02 \n-0.50 (0.29) \n0.12 (0.19) \n Scour Mark 4 \n417 \n0.00152 \n-5.20 \n-5.74 \n-5.63 \n-0.54 (0.28) \n0.12 (0.21) \n Seagrass Continuous \n411 \n0.00151 \n-5.19 \n-5.74 \n-5.62 \n-0.55 (0.27) \n0.12 (0.21) \n Unconsolidated Sediment \n6 \n0.00001 \n-5.69 \n-5.74 \n-5.84 \n-0.06 (0.05) \n-0.10 (0.05) \nReef Rubble Field \n Total Accretion Area \n7216 \n0.028 \n-4.22 \n-3.32 \n-3.57 \n0.89 (0.45) \n-0.24 (0.30) \n Reef Rubble \n3102 \n0.012 \n-3.71 \n-2.84 \n-3.05 \n0.87 (0.44) \n-0.21 (0.36) \n Seagrass Discontinuous \n3489 \n0.014 \n-4.66 \n-3.67 \n-3.97 \n0.99 (0.42) \n-0.30 (0.24) \n Unconsolidated Sediment \n628 \n0.00237 \n-4.25 \n-3.82 \n-3.91 \n0.43 (0.26) \n-0.10 (0.12) \n Total Erosion Area \n6043 \n0.023 \n-3.00 \n-3.64 \n-3.74 \n-0.63 (0.48) \n-0.10 (0.19) \n Reef Rubble \n3409 \n0.013 \n-2.61 \n-3.39 \n-3.44 \n-0.77 (0.50) \n-0.06 (0.20) \n Seagrass Discontinuous \n1941 \n0.00708 \n-3.51 \n-4.05 \n-4.22 \n-0.54 (0.43) \n-0.17 (0.15) \n Unconsolidated Sediment \n694 \n0.00248 \n-3.50 \n-3.70 \n-3.82 \n-0.20 (0.15) \n-0.12 (0.13) \nSand Lobe \n Total Area \n67389 \n0.266 \n-12.41 -11.90 \n-12.10 \n0.51 (0.29) -0.20 (0.09) \n Unconsolidated Sediment \n67389 \n0.266 \n-12.41 -11.90 \n-12.10 \n0.51 (0.29) -0.20 (0.09) \n *58 data points fell on borders between habitats and were counted twice during habitat analysis. https://doi.org/10.5194/egusphere-2023-3000\nPreprint. Discussion started: 22 December 2023\nPub ic domain CC\n1.0. l\n. 0 https://doi.org/10.5194/egusphere-2023-3000\nPreprint. Discussion started: 22 December 2023\nPub ic domain CC\n1.0. l\n. 0 Figure 4. Elevation-change results for 4,007,961 point-locations at Looe Key Reef. Elevation change between (a) 2016 \nmonths before and 3 to 6 months after Hurricane Irma) and (b) between 2017 to 2019 (from approximately 3 to 16.5 months af\nrma), and (c) corresponding seafloor habitats (FWC, 2015). The Hurricane Irma best track data in the panel b’s inset is fro\nNHC Irma Storm Track resource page (NHC, 2018, see also Figure 2b). Boundaries for the Looe Key Sanctuary Protection Ar\npecial Protection Unit (SPU) are shown as pink polygons. Geomorphic features of interest are indicated with black polygons\nreas indicate locations where water depth was too shallow for collection of multibeam bathymetric data. Figure 4. Elevation-change results for 4,007,961 point-locations at Looe Key Reef. Elevation change between (a) 2016 to 2017 (13.5 \nmonths before and 3 to 6 months after Hurricane Irma) and (b) between 2017 to 2019 (from approximately 3 to 16.5 months after Hurricane \nIrma), and (c) corresponding seafloor habitats (FWC, 2015). The Hurricane Irma best track data in the panel b’s inset is from the NOAA \n250 \nNHC Irma Storm Track resource page (NHC, 2018, see also Figure 2b). Boundaries for the Looe Key Sanctuary Protection Area (SPA) and \nSpecial Protection Unit (SPU) are shown as pink polygons. Geomorphic features of interest are indicated with black polygons. Gaps in map \nareas indicate locations where water depth was too shallow for collection of multibeam bathymetric data. 255 11 https://doi.org/10.5194/egusphere-2023-3000\nPreprint. Discussion started: 22 December 2023\nPub ic domain CC\n1.0. l\n. 0 https://doi.org/10.5194/egusphere-2023-3000\nPreprint. Discussion started: 22 December 2023\nPub ic domain CC\n1.0. l\n. 0 habitat types showed increases in net volume (accretion) with upper bound ranges from 0.001 to 2.19 Mm3 (Table 3). Largest \nnet volume changes were associated with habitats covering the largest areal extent of the study area including ‘pavement,’ \n‘discontinuous seagrass,’ and ‘unconsolidated sediment.’ Pearson correlation analysis also indicated a very strong positive \ncorrelation (r(8) = 0.99, p = 0.000) and linear relationship (r2 = 0.92, Fig. 5b)) between 2016 mean habitat water depth and habitat types showed increases in net volume (accretion) with upper bound ranges from 0.001 to 2.19 Mm3 (Table 3). Largest \nnet volume changes were associated with habitats covering the largest areal extent of the study area including ‘pavement,’ \n‘discontinuous seagrass,’ and ‘unconsolidated sediment.’ Pearson correlation analysis also indicated a very strong positive \ncorrelation (r(8) = 0.99, p = 0.000) and linear relationship (r2 = 0.92, Fig. 5b)) between 2016 mean habitat water depth and \narea-normalized volume change; area-normalized volume gains increased significantly with increasing water depth. Largest \n270 \narea-normalized volume changes of 0.51 mM3 and 0.54 mM3 were observed for ‘aggregate reef’ and ‘not classified’ habitats, \nrespectively; and smallest changes of 0.20 to 0.27 mM3 were observed for ‘reef rubble’ and ‘continuous seagrass’ habitats \n(Table 3), consistent with mean elevation changes for those habitats. Mean elevation-change values of the 2016–2017 elevation \nchange data set that was clipped to an area of 15.98 km2 and used for this analysis were within ±0.01 m, and area-normalized \nvolumes were within ±0.016 Mm3 km-2, of values calculated in the original 19.71 km2 published data set (unclipped) for the \n275 \noverall study site and all habitats (Yates et al., 2019). area-normalized volume change; area-normalized volume gains increased significantly with increasing water depth. Largest \n270 \narea-normalized volume changes of 0.51 mM3 and 0.54 mM3 were observed for ‘aggregate reef’ and ‘not classified’ habitats, \nrespectively; and smallest changes of 0.20 to 0.27 mM3 were observed for ‘reef rubble’ and ‘continuous seagrass’ habitats \n(Table 3), consistent with mean elevation changes for those habitats. SD = standard deviation. Elevation change data by habitat type associated with each period and geomorphic feature subarea. elevation -6.19 m) and ‘seagrass continuous’ (mean 2016 elevation -7.69 m) habitats (Table 2). Only 4% of all data points \n260 \nshowed losses in elevation (erosion) ranging from -0.01 m to -0.44 m, while 96% of all data points showed gains in elevation \nranging from 0.31 m to 0.55 m across all habitats. Pearson correlation analysis showed a very strong positive correlation (r(8) \n= 0.96, p = 0.000) and linear relationship (r2 = 0.92, Fig. 5a) between 2016 mean habitat water depth (estimated from mean \nelevation) and mean elevation change; mean elevation gains increased significantly with increasing water depth (i.e., \ndecreasing seafloor elevation). Net volume change was up to 5.36 mM3 over the total 15.98 km2 Looe Key study site; and all \n265 260 12 5c) between estimated to -0.25 m (Fig. 4b, Table 2). Largest mean elevation changes were associated with ‘aggregate reef’ and ‘not classified’ habitat \n280 \ntypes, and smallest changes were associated with ‘colonized pavement’ and ‘continuous seagrass’ habitats (Table 2). Only 5% \nof all data points showed gains in elevation with mean accretion ranging from 0.04 m to 0.19 m, while 95% of all data points \nshowed losses in elevation with mean erosion ranging from -0.13 m to -0.27 m across all habitat types. Pearson correlation \nanalysis indicated a moderate correlation (r(8) = -0.67, p = 0.035) and linear relationship (r2 = 0.45, Fig. 5c) between estimated 2017 mean habitat water depth and mean elevation change; mean elevation loss generally increased with increasing water \n285 \ndepth. Net volume change was up to -2.46 mM3 over the total 15.98 km2 Looe Key study site and area-normalized volume \nchange was -0.15 Mm3km-2. Losses in net volume up to -0.931 Mm3 (erosion) were observed across all habitat types (Table \n4). 2017 mean habitat water depth and mean elevation change; mean elevation loss generally increased with increasing water \n285 \ndepth. Net volume change was up to -2.46 mM3 over the total 15.98 km2 Looe Key study site and area-normalized volume \nchange was -0.15 Mm3km-2. Losses in net volume up to -0.931 Mm3 (erosion) were observed across all habitat types (Table \n4). Largest net volume changes were associated with habitats covering the largest areal extent of the study area including \n290 \n‘pavement,’ ‘discontinuous seagrass,’ and ‘unconsolidated sediment.’ Pearson correlation analysis indicated a moderate \ncorrelation (r(8) = -0.67, p = 0.035) and linear relationship (r2 = 0.45, Fig. 5d) between 2017 mean habitat water depth and \narea-normalized volume change; area-normalized volume losses generally increased with increasing water depth. Largest area-\nnormalized volume changes were observed for ‘aggregate reef’ and ‘not classified’ habitats, -0.254 and -0.247 Mm3 km-2, Largest net volume changes were associated with habitats covering the largest areal extent of the study area including \n290 \n‘pavement,’ ‘discontinuous seagrass,’ and ‘unconsolidated sediment.’ Pearson correlation analysis indicated a moderate \ncorrelation (r(8) = -0.67, p = 0.035) and linear relationship (r2 = 0.45, Fig. 5d) between 2017 mean habitat water depth and \narea-normalized volume change; area-normalized volume losses generally increased with increasing water depth. Mean elevation-change values of the 2016–2017 elevation \nchange data set that was clipped to an area of 15.98 km2 and used for this analysis were within ±0.01 m, and area-normalized area-normalized volume change; area-normalized volume gains increased significantly with increasing water depth. Largest \n270 \narea-normalized volume changes of 0.51 mM3 and 0.54 mM3 were observed for ‘aggregate reef’ and ‘not classified’ habitats, \nrespectively; and smallest changes of 0.20 to 0.27 mM3 were observed for ‘reef rubble’ and ‘continuous seagrass’ habitats \n(Table 3), consistent with mean elevation changes for those habitats. Mean elevation-change values of the 2016–2017 elevation \nchange data set that was clipped to an area of 15.98 km2 and used for this analysis were within ±0.01 m, and area-normalized \nvolumes were within ±0.016 Mm3 km-2, of values calculated in the original 19.71 km2 published data set (unclipped) for the \n275 \noverall study site and all habitats (Yates et al 2019) Mean elevation-change during a 13-month time-period between December 2017 to June 2019 (up to approximately 16.5 \nmonths after Hurricane Irma) was -0.15 ± 0.11 m, and all habitat types showed losses in mean elevation ranging from -0.11 m )\n,\nyp\ng g\nto -0.25 m (Fig. 4b, Table 2). Largest mean elevation changes were associated with ‘aggregate reef’ and ‘not classified’ habitat \n280 \ntypes, and smallest changes were associated with ‘colonized pavement’ and ‘continuous seagrass’ habitats (Table 2). Only 5% \nof all data points showed gains in elevation with mean accretion ranging from 0.04 m to 0.19 m, while 95% of all data points \nshowed losses in elevation with mean erosion ranging from -0.13 m to -0.27 m across all habitat types. Pearson correlation \nanalysis indicated a moderate correlation (r(8) = -0.67, p = 0.035) and linear relationship (r2 = 0.45, Fig. 5c) between estimated to -0.25 m (Fig. 4b, Table 2). Largest mean elevation changes were associated with ‘aggregate reef’ and ‘not classified’ habitat \n280 \ntypes, and smallest changes were associated with ‘colonized pavement’ and ‘continuous seagrass’ habitats (Table 2). Only 5% \nof all data points showed gains in elevation with mean accretion ranging from 0.04 m to 0.19 m, while 95% of all data points \nshowed losses in elevation with mean erosion ranging from -0.13 m to -0.27 m across all habitat types. Pearson correlation \nanalysis indicated a moderate correlation (r(8) = -0.67, p = 0.035) and linear relationship (r2 = 0.45, Fig. Largest area-\nnormalized volume changes were observed for ‘aggregate reef’ and ‘not classified’ habitats, -0.254 and -0.247 Mm3 km-2, Largest net volume changes were associated with habitats covering the largest areal extent of the study area including \n290 \n‘pavement,’ ‘discontinuous seagrass,’ and ‘unconsolidated sediment.’ Pearson correlation analysis indicated a moderate \ncorrelation (r(8) = -0.67, p = 0.035) and linear relationship (r2 = 0.45, Fig. 5d) between 2017 mean habitat water depth and \narea-normalized volume change; area-normalized volume losses generally increased with increasing water depth. Largest area-\nnormalized volume changes were observed for ‘aggregate reef’ and ‘not classified’ habitats, -0.254 and -0.247 Mm3 km-2, respectively; smallest changes were observed for ‘colonized pavement’ and ‘continuous seagrass’ habitats, -0.112 to -0.118 \n295 \nMm3 km-2 respectively (Table 4), consistent with mean elevation changes for those habitats. Pearson correlation analysis \nindicated a strong negative correlation (r(8) = -0.74, p = 0.014) and linear relationship (r2 = 0.55, Fig. 5e) between 2017 to \n2019 mean habitat elevation change and 2016 to 2017 mean habitat elevation change; mean elevation losses during 2017 to \n2019 were significantly greater in habitats with larger mean elevation gains during 2016 to 2017. Mean elevation change (loss) respectively; smallest changes were observed for ‘colonized pavement’ and ‘continuous seagrass’ habitats, -0.112 to -0.118 \n295 \nMm3 km-2 respectively (Table 4), consistent with mean elevation changes for those habitats. Pearson correlation analysis \nindicated a strong negative correlation (r(8) = -0.74, p = 0.014) and linear relationship (r2 = 0.55, Fig. 5e) between 2017 to \n2019 mean habitat elevation change and 2016 to 2017 mean habitat elevation change; mean elevation losses during 2017 to \n2019 were significantly greater in habitats with larger mean elevation gains during 2016 to 2017. Mean elevation change (loss) 13 during 2017 to 2019 was 35 to 55% of the mean elevation change (gain) during 2016 to 2017 for all habitats except for reef \n00 \nrubble which was 92% and had the shallowest mean depth (6.0 m) of all habitats. https://doi.org/10.5194/egusphere-2023-3000\nPreprint. Discussion started: 22 December 2023\nPub ic domain CC\n1.0. l\n. 0 https://doi.org/10.5194/egusphere-2023-3000\nPreprint. Discussion started: 22 December 2023\nPub ic domain CC\n1.0. l\n. 0 https://doi.org/10.5194/egusphere-2023-3000\nPreprint. Discussion started: 22 December 2023\nPub ic domain CC\n1.0. l\n. 0 Table 3. Compiled volume change data by habitat type for all study areas during the 2016 to 2017 study period (storm period). Habitat Type \nHabitat Area \n(km2) \nGross erosion \n(Mm3) \nGross accretion \n(Mm3) \nNet volume change \n(Mm3 study-area-1) \nArea-normalized volume \nchange (Mm3 km-2) \nLower \nUpper \nLower Upper \n Lower \nUpper \nLower \nUpper \nOverall Looe Key Study Site \n Total Study Site \n15.98 \n0.053 \n0.134 \n2.456 \n5.490 \n2.403 \n5.356 \n0.150 \n0.335 \n Aggregate Reef \n0.30 \n0 \n0 \n0.090 \n0.154 \n0.090 \n0.154 \n0.296 \n0.505 \n Colonized Pavement \n0.0028 \n0 \n0 \n0 \n0.001 \n0 \n0.001 \n0.124 \n0.323 \n Individual or Aggregate Patch Reef \n0.22 \n0 \n0 \n0.029 \n0.073 \n0.029 \n0.072 \n0.136 \n0.336 \n Not Classified \n0.026 \n0 \n0 \n0.009 \n0.014 \n0.009 \n0.014 \n0.337 \n0.540 \n Pavement \n2.57 \n0 \n0.003 \n0.445 \n0.962 \n0.445 \n0.959 \n0.173 \n0.373 \n Reef Rubble \n0.32 \n0.013 \n0.021 \n0.033 \n0.085 \n0.020 \n0.064 \n0.062 \n0.199 \n Seagrass Continuous \n1.60 \n0.008 \n0.021 \n0.152 \n0.449 \n0.144 \n0.428 \n0.090 \n0.267 \n Seagrass Discontinuous \n4.26 \n0.015 \n0.045 \n0.477 \n1.250 \n0.462 \n1.205 \n0.109 \n0.283 \n Spur and Groove \n0.74 \n0.003 \n0.007 \n0.136 \n0.278 \n0.133 \n0.271 \n0.181 \n0.367 \n Unconsolidated Sediment \n5.94 \n0.013 \n0.037 \n1.083 \n2.226 \n1.070 \n2.189 \n0.180 \n0.369 \nSand Wave \n Total Accretion Area \n0.060 \n0 \n0 \n0.036 \n0.048 \n0.036 \n0.048 \n0.598 \n0.800 \n Seagrass Discontinuous \n0.029 \n0 \n0 \n0.020 \n0.026 \n0.020 \n0.026 \n0.709 \n0.914 \n Unconsolidated Sediment \n0.031 \n0 \n0 \n0.015 \n0.021 \n0.015 \n0.021 \n0.494 \n0.694 \n Total Erosion Area \n0.043 \n0.009 \n0.016 \n0 \n0 \n-0.009 \n-0.016 \n-0.198 \n-0.370 \n Seagrass Discontinuous \n0.002 \n<0.001 \n<0.001 \n0 \n0 \n<-0.001 \n<-0.001 \n-0.045 \n-0.162 \n Unconsolidated Sediment \n0.041 \n0.008 \n0.016 \n0 \n0 \n-0.008 \n-0.016 \n-0.205 \n-0.380 \nScour Marks \n Scour Mark 1 \n0.00071 \n0.0002 \n0.0004 \n0 \n0 \n-0.0002 \n-0.0004 \n-0.3083 \n-0.5114 \n Seagrass Discontinuous \n0.00071 \n0.0002 \n0.0004 \n0 \n0 \n-0.0002 \n-0.0004 \n-0.3118 \n-0.5154 \n Unconsolidated Sediment \n<0.00001 \n0 \n0 \n0 \n0 \n0 <-0.0001 \n-0.0001 \n-0.1479 \n Scour Mark 2 \n0.0014 \n0.0005 \n0.0007 \n0 \n0 \n-0.0005 \n-0.0007 \n-0.3255 \n-0.5271 \n Seagrass Continuous \n0.00124 \n0.0004 \n0.0007 \n0 \n0 \n-0.0004 \n-0.0007 \n-0.3558 \n-0.5595 \n Unconsolidated Sediment \n0.00016 \n0 <-0.0001 \n0 \n0 \n0 <-0.0001 \n-0.0943 \n-0.2790 \n Scour Mark 3 \n0.00188 \n0.0006 \n0.0010 \n0 \n0 \n-0.0006 \n-0.0010 \n-0.3247 \n-0.5232 \n Seagrass Continuous \n0.00188 \n0.0006 \n0.0010 \n0 \n0 \n-0.0006 \n-0.0010 \n-0.3247 \n-0.5232 \n Scour Mark 4 \n0.00152 \n0.0006 \n0.0009 \n0 \n0 \n-0.0006 \n-0.0009 \n-0.3631 \n-0.5661 \n Seagrass Continuous \n0.00151 \n0.0006 \n0.0009 \n0 \n0 \n-0.0006 \n-0.0009 \n-0.3658 \n-0.5697 \n Unconsolidated Sediment \n0.00001 \n0 <-0.0001 \n0 \n0 \n0 <-0.0001 \n0.0000 \n-0.0748 \nReef Rubble Field \n Total Accretion Area \n0.028 \n0 \n0 \n0.020 \n0.025 \n0.020 \n0.025 \n0.707 \n0.914 \n Reef Rubble \n0.012 \n0 \n0 \n0.008 \n0.011 \n0.008 \n0.011 \n0.690 \n0.897 \n Seagrass Discontinuous \n0.014 \n0 \n0 \n0.011 \n0.014 \n0.011 \n0.014 \n0.802 \n1.011 \n Unconsolidated Sediment \n0.002 \n0 \n0 \n0.001 \n0.001 \n0.001 \n0.001 \n0.252 \n0.446 \n Total Erosion Area \n0.023 \n0.011 \n0.015 \n0 \n0 \n-0.011 \n-0.015 \n-0.464 \n-0.661 \n Reef Rubble \n0.013 \n0.008 \n0.010 \n0 \n0 \n-0.008 \n-0.010 \n-0.584 \n-0.788 \n Seagrass Discontinuous \n0.007 \n0.003 \n0.004 \n0 \n0 \n-0.003 \n-0.004 \n-0.382 \n-0.577 \n Unconsolidated Sediment \n0.002 \n0.000 \n0.001 \n0 \n0 \n0.000 \n-0.001 \n-0.064 \n-0.221 \nSand Lobe \n Total Area \n0.27 \n0 \n0.002 \n0.089 \n0.139 \n0.089 \n0.137 \n0.332 \n0.513 \n Unconsolidated Sediment \n0.27 \n0 \n0.002 \n0.089 \n0.139 \n0.089 \n0.137 \n0.332 \n0.513 \n‘Upper’ and ‘lower’ headings refer to the upper and lower bounds of volume change based on total RMSE root mean square error). https://doi.org/10.5194/egusphere-2023-3000\nPreprint. Discussion started: 22 December 2023\nPub ic domain CC\n1.0. l\n. 0 during 2017 to 2019 was 35 to 55% of the mean elevation change (gain) during 2016 to 2017 for all habitats except for reef \n300 \nrubble which was 92% and had the shallowest mean depth (6.0 m) of all habitats. during 2017 to 2019 was 35 to 55% of the mean elevation change (gain) during 2016 to 2017 for all habitats except for reef \n300 \nrubble which was 92% and had the shallowest mean depth (6.0 m) of all habitats. during 2017 to 2019 was 35 to 55% of the mean elevation change (gain) during 2016 to 2017 for all habitats except for reef \n300 \nrubble which was 92% and had the shallowest mean depth (6.0 m) of all habitats. (a) \n \n(b) \n \n(c) \n \n(d) \n \n (e) \n \nFigure 5. Linear relationships between elevation change, volume change, and water depth. Linear relationships and coeffici\ndetermination between (a) mean elevation change, (b) mean area-normalized volume change, and estimated 2016 mean water de\nseafloor habitats of the Looe Key study site between 2016 to 2017. Linear relationships and coefficients of determination between (c\n5 \nelevation change, (d) mean area-normalized volume change, and estimated 2017 mean water depth for seafloor habitats of the Loo\nstudy site between 2017 to 2019 (a, b). Linear relationship between 2017 to 2019 mean elevation change and 2016 to 2017 mean ele\nchange (e). (a) (b) (a) (d) (c) (d) (c) (e) (e) Figure 5. Linear relationships between elevation change, volume change, and water depth. Linear relationships and coefficients of \ndetermination between (a) mean elevation change, (b) mean area-normalized volume change, and estimated 2016 mean water depth for \nseafloor habitats of the Looe Key study site between 2016 to 2017. Linear relationships and coefficients of determination between (c) mean \nelevation change, (d) mean area-normalized volume change, and estimated 2017 mean water depth for seafloor habitats of the Looe Key \nstudy site between 2017 to 2019 (a, b). Linear relationship between 2017 to 2019 mean elevation change and 2016 to 2017 mean elevation \nchange (e). 14 https://doi.org/10.5194/egusphere-2023-3000\nPreprint. Discussion started: 22 December 2023\nPub ic domain CC\n1.0. l\n. 0 https://doi.org/10.5194/egusphere-2023-3000\nPreprint. Discussion started: 22 December 2023\nPub ic domain CC\n1.0. l\n. 0 Table 4. Compiled volume change data by habitat type for all study areas during the 2017 to 2019 study period (post-storm re-\nequilibration period). Table 4. Compiled volume change data by habitat type for all study areas during the 2017 to 2019 study period (post-storm re-\nequilibration period). 4. Compiled volume change data by habitat type for all study areas during the 2017 to 2019 study \nbration period). hange data by habitat type for all study areas during the 2017 to 2019 study period (post-storm re- 320 Lower bounds use tota\nRMSE as a plane height in calculating volume. Table 3. Compiled volume change data by habitat type for all study areas during the 2016 to 2017 study period (storm period). 310 Table 3. Compiled volume change data by habitat type for all study areas during the 2016 to 2017 stud e change data by habitat type for all study areas during the 2016 to 2017 study period (storm period). 315 15 Habitat Type \nHabitat Area \n(km2) \nGross erosion \n(Mm3) \nGross accretion \n(Mm3) \nNet volume change \n(Mm3 study-area-1) \nArea-normalized volume \nchange (Mm3 km-2) \nLower \nUpper \nLower Upper \n Lower \nUpper \nLower \nUpper \nOverall Looe Key Study Site \n Total Study Site \n15.98 \n0.316 \n2.502 \n0.005 \n0.041 \n-0.311 \n-2.461 \n-0.019 \n-0.154 \n Aggregate Reef \n0.30 \n0.028 \n0.078 \n0 <0.001 \n-0.028 \n-0.077 \n-0.093 \n-0.254 \n Colonized Pavement \n0.0028 \n<0.001 \n<0.001 \n0 \n0 \n<-0.001 \n<-0.001 \n-0.004 \n-0.112 \n Individual or Aggregate Patch Reef \n0.22 \n0.006 \n0.040 \n0 \n0 \n-0.006 \n-0.040 \n-0.028 \n-0.186 \n Not Classified \n0.026 \n0.002 \n0.007 \n0 <0.001 \n-0.002 \n-0.006 \n-0.083 \n-0.247 \n Pavement \n2.57 \n0.059 \n0.424 \n0 \n0.004 \n-0.059 \n-0.420 \n-0.023 \n-0.163 \n Reef Rubble \n0.32 \n0.010 \n0.061 \n0.001 \n0.002 \n-0.009 \n-0.059 \n-0.029 \n-0.182 \n Seagrass Continuous \n1.60 \n0.012 \n0.197 \n0.001 \n0.008 \n-0.011 \n-0.189 \n-0.007 \n-0.118 \n Seagrass Discontinuous \n4.26 \n0.064 \n0.612 \n0.001 \n0.015 \n-0.063 \n-0.597 \n-0.015 \n-0.140 \n Spur and Groove \n0.74 \n0.032 \n0.145 \n0.001 \n0.003 \n-0.031 \n-0.141 \n-0.042 \n-0.192 \n Unconsolidated Sediment \n5.94 \n0.102 \n0.938 \n0.000 \n0.007 \n-0.102 \n-0.931 \n-0.017 \n-0.157 \nSand Wave \n Total Accretion Area \n0.060 \n0.0015 \n0.0093 \n0 0.0005 \n-0.0015 \n-0.0088 \n-0.0245 \n-0.1479 \n Seagrass Discontinuous \n0.029 \n0.0010 \n0.0048 \n0 0.0003 \n-0.0010 \n-0.0044 \n-0.0336 \n-0.1544 \n Unconsolidated Sediment \n0.031 \n0.0005 \n0.0045 \n0 0.0001 \n-0.0005 \n-0.0044 \n-0.0159 \n-0.1419 \n Total Erosion Area \n0.043 \n0.0003 \n0.0066 \n0 \n0 \n-0.0003 \n-0.0066 \n-0.0074 \n-0.1529 \n Seagrass Discontinuous \n0.002 \n<0.0001 \n0.0003 \n0 \n0 \n<-0.0001 \n-0.0003 \n-0.0158 \n-0.1521 \n Unconsolidated Sediment \n0.041 \n0.0003 \n0.0063 \n0 \n0 \n-0.0003 \n-0.0063 \n-0.0070 \n-0.1529 \nScour Marks \n Scour Mark 1 \n0.00071 \n0.0000 \n0.0000 \n<0.0001 0.0001 \n<0.0001 \n0.0001 \n0.0171 \n0.1201 \n Seagrass Discontinuous \n0.00071 \n0.0000 \n0.0000 \n<0.0001 0.0001 \n<0.0001 \n0.0001 \n0.0173 \n0.1219 \n Unconsolidated Sediment \n<0.00001 \n0.0000 \n<0.0001 \n0.0000 0.0000 \n0.0000 <-0.0001 \n0.0000 \n-0.0447 \n Scour Mark 2 \n0.0014 \n0 \n0 \n0.0001 0.0003 \n0.0001 \n0.0003 \n0.0880 \n0.2226 \n Seagrass Continuous \n0.00124 \n0 \n0 \n0.0001 0.0003 \n0.0001 \n0.0003 \n0.0996 \n0.2550 \n Unconsolidated Sediment \n0.00016 \n<0.0001 \n<0.0001 \n0 \n0 \n<-0.0001 <-0.0001 \n-0.0009 \n-0.0254 \n Scour Mark 3 \n0.00188 \n0 \n0 \n0.0001 0.0003 \n0.0001 \n0.0003 \n0.0524 \n0.1380 \n Seagrass Continuous \n0.00188 \n0 \n0 \n0.0001 0.0003 \n0.0001 \n0.0003 \n0.0524 \n0.1380 \n Scour Mark 4 \n0.00152 \n0 \n0 \n0.0001 0.0002 \n0.0001 \n0.0002 \n0.0615 \n0.1334 \n Seagrass Continuous \n0.00151 \n0 \n0 \n0.0001 0.0002 \n0.0001 \n0.0002 \n0.0620 \n0.1351 \n Unconsolidated Sediment \n0.00001 \n0.0000 \n<0.0001 \n0.0000 0.0000 \n0.0000 <-0.0001 \n0.0000 \n-0.1029 \nReef Rubble Field \n Total Accretion Area \n0.028 \n0.0040 \n0.0080 \n0.0010 0.0010 \n-0.0040 \n-0.0070 \n-0.1310 \n-0.2480 \n Reef Rubble \n0.012 \n0.0020 \n0.0040 \n0 0.0010 \n-0.0010 \n-0.0030 \n-0.1260 \n-0.2180 \n Seagrass Discontinuous \n0.014 \n0.0020 \n0.0040 \n0 \n0 \n-0.0020 \n-0.0040 \n-0.1560 \n-0.3020 \n Unconsolidated Sediment \n0.002 \n<0.0001 \n<0.0001 \n0 \n0 \n<-0.0001 <-0.0001 \n-0.0070 \n-0.0910 \n Total Erosion Area \n0.023 \n0.0005 \n0.0031 \n0.0003 0.0008 \n-0.0002 \n-0.0023 \n-0.0084 \n-0.1026 \n Reef Rubble \n0.013 \n0.0001 \n0.0015 \n0.0003 0.0007 \n0.0002 \n-0.0008 \n0.0113 \n-0.0612 \n Seagrass Discontinuous \n0.007 \n0.0003 \n0.0013 \n0 0.0001 \n-0.0003 \n-0.0012 \n-0.0429 \n-0.1741 \n Unconsolidated Sediment \n0.002 \n<0.0001 \n0.0003 \n0 \n0 \n<-0.0001 \n-0.0003 \n-0.0152 \n-0.1188 \nSand Lobe \n Total Area \n0.27 \n0.010 \n0.055 \n0 <0.001 \n-0.010 \n-0.054 \n-0.038 \n-0.204 \n Unconsolidated Sediment \n0.27 \n0.010 \n0.055 \n0 <0.001 \n-0.010 \n-0.054 \n-0.038 \n-0.204 \n‘Upper’ and ‘lower’ headings refer to the upper and lower bounds of volume change based on total RMSE (root mean square error). https://doi.org/10.5194/egusphere-2023-3000\nPreprint. Discussion started: 22 December 2023\nPub ic domain CC\n1.0. l\n. 0 https://doi.org/10.5194/egusphere-2023-3000\nPreprint. Discussion started: 22 December 2023\nPub ic domain CC\n1.0. l\n. 0 Several scour features developed in ‘discontinuous seagrass’ and ‘unconsolidated sediment’ habitats of the Looe Key back \nreef, indicated by areas of erosion that appear as pits. Rubble fields within and near the Looe Key SPA area were displaced, \nas indicated by adjacent areas of accretion and erosion. Substantial deposition of sediments occurred along a sand lobe at the \nbase of the Looe Key Reef ‘spur and groove’ habitat. 330 330 3.2.1 Sand Wave Total net volume change for the accretion area of the feature was 0.048 Mm3 and area-normalized \nvolume change was 0.800 Mm3 km-2 (Table 3). Mean elevation-change and area-normalized volume change was greatest within the discontinuous seagrass habitat (0.90 m and 0.914 Mm3 km-2, respectively), approximately 2.7 times greater than \n345 \nmean elevation change and area-normalized volume change for the overall Looe Key study site. It accounted for 55% of total \nnet volume gain, indicating burial of seagrass habitat during migration of the sand wave. Net volume change of the erosion \narea was approximately -0.016 Mm3 and area-normalized volume change -0.37 Mm3 km-2 with 98% of net volume change \nassociated with erosion of unconsolidated sediment habitat (Table 3). within the discontinuous seagrass habitat (0.90 m and 0.914 Mm3 km-2, respectively), approximately 2.7 times greater than \n345 \nmean elevation change and area-normalized volume change for the overall Looe Key study site. It accounted for 55% of total \nnet volume gain, indicating burial of seagrass habitat during migration of the sand wave. Net volume change of the erosion \narea was approximately -0.016 Mm3 and area-normalized volume change -0.37 Mm3 km-2 with 98% of net volume change \nassociated with erosion of unconsolidated sediment habitat (Table 3). 350 \nBetween 2017 and 2019, the sand wave (accretion area) showed mean elevation and net volume change of approximately -\n0.15 m and approximately -0.009 Mm3, respectively (Table 2, 4 and Fig. 6b). Similar mean elevation change values were \nobserved for discontinuous seagrass and unconsolidated sediment habitats associated with the feature, and net volume change \nfor each habitat was approximately 50% of the total net volume change (Table 4). Area-normalized volume change was similar 350 \nBetween 2017 and 2019, the sand wave (accretion area) showed mean elevation and net volume change of approximately -\n0.15 m and approximately -0.009 Mm3, respectively (Table 2, 4 and Fig. 6b). Similar mean elevation change values were \nobserved for discontinuous seagrass and unconsolidated sediment habitats associated with the feature, and net volume change \nfor each habitat was approximately 50% of the total net volume change (Table 4). Area-normalized volume change was similar 350 \nBetween 2017 and 2019, the sand wave (accretion area) showed mean elevation and net volume change of approximately -\n0.15 m and approximately -0.009 Mm3, respectively (Table 2, 4 and Fig. 6b). 3.2 Geomorphic Feature Analyses Large-scale geomorphic features that were 10s to 100s of m2 in areal extent and showed extensive erosion and/or accretion \nwith elevation-changes greater than 0.5 m were observed between 2016 and 2017 (Fig. 6 and 7). Examples of these features \n325 \nincluded migration of a sand wave in the back reef area of Looe Key reef indicated by adjacent areas of erosion and accretion. 16 3.2.1 Sand Wave An accretion of 0.060 km2 included \napproximately 50% discontinuous seagrass and 50% unconsolidated sediment habitat. of approximately 5.6 m. Transect elevation profiles showed the location of this feature in 2016, westward migration of \n335 \napproximately 78 m (crest to crest) in 2017, and minor erosion in 2019 (Fig. 7a). An accretion of 0.060 km2 included \napproximately 50% discontinuous seagrass and 50% unconsolidated sediment habitat. of approximately 5.6 m. Transect elevation profiles showed the location of this feature in 2016, westward migration of \n335 \napproximately 78 m (crest to crest) in 2017, and minor erosion in 2019 (Fig. 7a). An accretion of 0.060 km2 included \napproximately 50% discontinuous seagrass and 50% unconsolidated sediment habitat. Between 2016 and 2017, mean elevation change of the accretion area (2017 location of the sand wave) was 0.79 m (Table 2) \nwith a maximum elevation gain at the crest of 1.84m. An adjacent area of erosion was approximately 630 x 122 m in length \n340 \nand width (0.043 km2) and included approximately 5% discontinuous seagrass and 95% unconsolidated sediment. Mean \nelevation-change of the erosion area was -0.36 m (Table 2) with a maximum elevation loss of -1.23 m near the 2016 location \nof the sand wave crest. Total net volume change for the accretion area of the feature was 0.048 Mm3 and area-normalized \nvolume change was 0.800 Mm3 km-2 (Table 3). Mean elevation-change and area-normalized volume change was greatest g\nwith a maximum elevation gain at the crest of 1.84m. An adjacent area of erosion was approximately 630 x 122 m in length \n340 \nand width (0.043 km2) and included approximately 5% discontinuous seagrass and 95% unconsolidated sediment. Mean \nelevation-change of the erosion area was -0.36 m (Table 2) with a maximum elevation loss of -1.23 m near the 2016 location \nof the sand wave crest. Total net volume change for the accretion area of the feature was 0.048 Mm3 and area-normalized \nvolume change was 0.800 Mm3 km-2 (Table 3). Mean elevation-change and area-normalized volume change was greatest with a maximum elevation gain at the crest of 1.84m. An adjacent area of erosion was approximately 630 x 122 m in length \n340 \nand width (0.043 km2) and included approximately 5% discontinuous seagrass and 95% unconsolidated sediment. Mean \nelevation-change of the erosion area was -0.36 m (Table 2) with a maximum elevation loss of -1.23 m near the 2016 location \nof the sand wave crest. 3.2.1 Sand Wave Migration of a sand wave was observed in the back reef area of Looe Key Reef between 2016 and 2017, with minor erosion \nof this feature occurring between 2017 and 2019 (Fig. 6a, b, and c). The sand wave was approximately 733 m long and 104 m \nwide at its widest point in 2017, 2 m in height from the crest to base on the deepest (western) edge, with average water depth \nof approximately 5.6 m. Transect elevation profiles showed the location of this feature in 2016, westward migration of \n335 \napproximately 78 m (crest to crest) in 2017, and minor erosion in 2019 (Fig. 7a). An accretion of 0.060 km2 included \napproximately 50% discontinuous seagrass and 50% unconsolidated sediment habitat. Migration of a sand wave was observed in the back reef area of Looe Key Reef between 2016 and 2017, with minor erosion \nof this feature occurring between 2017 and 2019 (Fig. 6a, b, and c). The sand wave was approximately 733 m long and 104 m \nwide at its widest point in 2017, 2 m in height from the crest to base on the deepest (western) edge, with average water depth \nof approximately 5.6 m. Transect elevation profiles showed the location of this feature in 2016, westward migration of \n335 \ni\nl\n78\n(\n) i\n2017\nd\ni\ni\ni\n2019 (Fi\n7 ) A\ni\nf 0 060 k\n2 i\nl d d Migration of a sand wave was observed in the back reef area of Looe Key Reef between 2016 and 2017, with minor erosion \nof this feature occurring between 2017 and 2019 (Fig. 6a, b, and c). The sand wave was approximately 733 m long and 104 m \nwide at its widest point in 2017, 2 m in height from the crest to base on the deepest (western) edge, with average water depth of this feature occurring between 2017 and 2019 (Fig. 6a, b, and c). The sand wave was approximately 733 m long and 104 m \nwide at its widest point in 2017, 2 m in height from the crest to base on the deepest (western) edge, with average water depth \nof approximately 5.6 m. Transect elevation profiles showed the location of this feature in 2016, westward migration of \n335 \napproximately 78 m (crest to crest) in 2017, and minor erosion in 2019 (Fig. 7a). 3.2.1 Sand Wave Similar mean elevation change values were \nobserved for discontinuous seagrass and unconsolidated sediment habitats associated with the feature, and net volume change \nfor each habitat was approximately 50% of the total net volume change (Table 4). Area-normalized volume change was similar for the total area of the sand wave and the sub-areas within it, including discontinuous seagrass and unconsolidated sediment \n355 \nhabitats, ranging from approximately -0.148 to -0.154 Mm3/km2. The adjacent erosion area (original 2016 location of the sand \nwave) also showed a mean elevation change of -0.15 m with similar values for the associated discontinuous seagrass and \nunconsolidated sediment habitats. Net volume change of the erosion area was approximately -0.007 Mm3 with approximately for the total area of the sand wave and the sub-areas within it, including discontinuous seagrass and unconsolidated sediment \n355 \nhabitats, ranging from approximately -0.148 to -0.154 Mm3/km2. The adjacent erosion area (original 2016 location of the sand \nwave) also showed a mean elevation change of -0.15 m with similar values for the associated discontinuous seagrass and \nunconsolidated sediment habitats. Net volume change of the erosion area was approximately -0.007 Mm3 with approximately 17 https://doi.org/10.5194/egusphere-2023-3000\nPreprint. Discussion started: 22 December 2023\nPub ic domain CC\n1.0. l\n. 0 https://doi.org/10.5194/egusphere-2023-3000\nPreprint. Discussion started: 22 December 2023\nPub ic domain CC\n1.0. l\n. 0 95% of this loss associated with unconsolidated sediment (Table 4). Area-normalized volume change was also consistent \nacross the total erosion feature area, discontinuous seagrass, and unconsolidated sediment habitats at -0.15 Mm3 km-2. 360 95% of this loss associated with unconsolidated sediment (Table 4). Area-normalized volume change was also consistent \nacross the total erosion feature area, discontinuous seagrass, and unconsolidated sediment habitats at -0.15 Mm3 km-2. 360 360 Figure 6. Elevation change data and transect positions for each geomorphic feature subarea. Geomorphic features included a s\nwave (a, b, c), scour marks (d, e, f), western rubble field (g, h, i), and sand lobe subareas (j, k, l). These feature locations and correspond\nhabitat are also shown in Fig. 4. Elevation-change from 2016 to 2017 (a, d, g, j), 2017 to 2019 (b, e, h, k), and corresponding reconnaissa\n5 \nimagery (c, f, i, l) Transect positions are indicated by black lines and lowercase letters in the elevation change panels (see also Fig. 7). Sc\nmarks in panels d and e are labelled SM1 through 4. Photo credit: Mitch Lemon, Cherokee Nations System Solutions for U.S. Geolog\nSurvey. Figure 6. Elevation change data and transect positions for each geomorphic feature subarea. Geomorphic features included a sand \nwave (a, b, c), scour marks (d, e, f), western rubble field (g, h, i), and sand lobe subareas (j, k, l). These feature locations and corresponding \nhabitat are also shown in Fig. 4. Elevation-change from 2016 to 2017 (a, d, g, j), 2017 to 2019 (b, e, h, k), and corresponding reconnaissance \n365 \nimagery (c, f, i, l) Transect positions are indicated by black lines and lowercase letters in the elevation change panels (see also Fig. 7). Scour \nmarks in panels d and e are labelled SM1 through 4. Photo credit: Mitch Lemon, Cherokee Nations System Solutions for U.S. Geological \nSurvey. 18 Figure 7. Elevation transects across geomorphic features in 2016, 2017, and 2019. Geomorphic features included a sand wave (a, e\nscour marks (b, f), western reef rubble field (c, g), and a sand lobe (d, h). Lowercase letters indicate direction of transects as shown in Figur\n6. Vertical red lines indicate areas of erosion and vertical blue lines indicate areas of accretion between (a-d) 2016 and 2017 (before an\nafter Hurricane Irma) and between (e-f) 2017 and 2019. SM = scour mark. Figure 7. Elevation transects across geomorphic features in 2016, 2017, and 2019. Geomorphic features included a sand wave (a, e), \n370 \nscour marks (b, f), western reef rubble field (c, g), and a sand lobe (d, h). Lowercase letters indicate direction of transects as shown in Figure \n6. Vertical red lines indicate areas of erosion and vertical blue lines indicate areas of accretion between (a-d) 2016 and 2017 (before and \nafter Hurricane Irma) and between (e-f) 2017 and 2019. SM = scour mark. 3.2.2 Scour Marks \n375 Development of scour marks was observed in seagrass and unconsolidated sediment habitats in the back reef area of Looe Key \nReef between 2016 and 2017 (Fig. 6d, e, and f). These features ranged from approximately 30 to 60 m in length and width \nwith average depths of approximately 5.7 to 7.5 m in 2017. Visual validation of select scour features indicated they developed 19 mean elevation change of 0.10 m and a net volume change of less than 0.001 Mm3 (Tables 2 and 4). Area-normalized volume \n390 \nchange was approximately 0.12 Mm3 km-2. Scour mark 2 was 1,400 m2 with 88% of the area consisting of continuous seagrass \nand 12% unconsolidated sediment. Between 2016 and 2017, mean elevation change was -0.50 m (Table 2) with maximum \nobserved change of -1.28 m. Net volume change was less than -0.001 Mm3 and area-normalized volume change was \napproximately -0.53 Mm3 km-2 (Table 3). Ninety-four percent of net volume change was associated with continuous seagrass mean elevation change of 0.10 m and a net volume change of less than 0.001 Mm3 (Tables 2 and 4). Area-normalized volume \n390 \nchange was approximately 0.12 Mm3 km-2. Scour mark 2 was 1,400 m2 with 88% of the area consisting of continuous seagrass \nand 12% unconsolidated sediment. Between 2016 and 2017, mean elevation change was -0.50 m (Table 2) with maximum \nobserved change of -1.28 m. Net volume change was less than -0.001 Mm3 and area-normalized volume change was \napproximately -0.53 Mm3 km-2 (Table 3). Ninety-four percent of net volume change was associated with continuous seagrass habitat, which also had the highest area-normalized volume change of -0.56 Mm3 km-2. Between 2017 and 2019, this feature \n395 \nshowed accretion with mean elevation change of 0.20 m and net volume change of 0.0003 Mm3 (Tables 2 and 4). Continuous \nseagrass showed an increase in mean elevation (0.24 m) and net volume (0.0003 Mm3) while unconsolidated sediment showed \na decrease in mean elevation (-0.05 m) and net volume (less than -0.0001 Mm3). Area-normalized volume change across the \nentire scour mark was approximately 0.22 Mm3 km-2. Scour mark 3 was 1,882 m2 with 100% of the area consisting of habitat, which also had the highest area-normalized volume change of -0.56 Mm3 km-2. Between 2017 and 2019, this feature \n395 \nshowed accretion with mean elevation change of 0.20 m and net volume change of 0.0003 Mm3 (Tables 2 and 4). Continuous \nseagrass showed an increase in mean elevation (0.24 m) and net volume (0.0003 Mm3) while unconsolidated sediment showed \na decrease in mean elevation (-0.05 m) and net volume (less than -0.0001 Mm3). Area-normalized volume change across the \nentire scour mark was approximately 0.22 Mm3 km-2. Scour mark 3 was 1,882 m2 with 100% of the area consisting of continuous seagrass. https://doi.org/10.5194/egusphere-2023-3000\nPreprint. Discussion started: 22 December 2023\nPub ic domain CC\n1.0. l\n. 0 between 2016 and 2017 at the edges of seagrass beds where small (approximately 0.5 m) ledges marked the transition between \nthe slightly higher elevation of seagrass beds and lower elevation of adjacent unconsolidated sediment (Fig. 6f). Transect \n380 \nanalyses showed considerable erosion of the western boundaries of seagrass beds, development of pit-like features up to \napproximately 20 m in diameter and 1 m deep, transport of sediment westward, and burial of seagrass between scour features \n(Fig. 7b). Scour marks showed some infilling between 2017 and 2019. Validation imagery showed exposed rhizomatous \ngrowth at the western edges of seagrass beds (Fig. 8). 380 385 \nElevation- and volume-change analyses were performed on four examples of these features (Fig. 6d and e). Scour mark 1 was \n714 m2 with 99% of the area consisting of discontinuous seagrass. Between 2016 and 2017, mean elevation change was -0.49 \nm (Table 2) with a maximum observed change of -1.09 m. Net volume change was less than -0.001 Mm3, and area-normalized \nvolume change was approximately -0.51 Mm3 km-2 (Table 3). Between 2017 and 2019, this feature showed accretion with 385 \nElevation- and volume-change analyses were performed on four examples of these features (Fig. 6d and e). Scour mark 1 was \n714 m2 with 99% of the area consisting of discontinuous seagrass. Between 2016 and 2017, mean elevation change was -0.49 \nm (Table 2) with a maximum observed change of -1.09 m. Net volume change was less than -0.001 Mm3, and area-normalized \nvolume change was approximately -0.51 Mm3 km-2 (Table 3). Between 2017 and 2019, this feature showed accretion with Elevation- and volume-change analyses were performed on four examples of these features (Fig. 6d and e). Scour mark 1 was \n714 m2 with 99% of the area consisting of discontinuous seagrass. Between 2016 and 2017, mean elevation change was -0.49 \nm (Table 2) with a maximum observed change of -1.09 m. Net volume change was less than -0.001 Mm3, and area-normalized \nvolume change was approximately -0.51 Mm3 km-2 (Table 3). Between 2017 and 2019, this feature showed accretion with \nmean elevation change of 0.10 m and a net volume change of less than 0.001 Mm3 (Tables 2 and 4). Area-normalized volume \n390 \nchange was approximately 0.12 Mm3 km-2. Scour mark 2 was 1,400 m2 with 88% of the area consisting of continuous seagrass \nand 12% unconsolidated sediment. Between 2016 and 2017, mean elevation change was -0.50 m (Table 2) with maximum \nobserved change of -1.28 m. Net volume change was less than -0.001 Mm3 and area-normalized volume change was \napproximately -0.53 Mm3 km-2 (Table 3). Ninety-four percent of net volume change was associated with continuous seagrass \nhabitat, which also had the highest area-normalized volume change of -0.56 Mm3 km-2. Between 2017 and 2019, this feature \n395 \nshowed accretion with mean elevation change of 0.20 m and net volume change of 0.0003 Mm3 (Tables 2 and 4). Continuous \nseagrass showed an increase in mean elevation (0.24 m) and net volume (0.0003 Mm3) while unconsolidated sediment showed \na decrease in mean elevation (-0.05 m) and net volume (less than -0.0001 Mm3). Area-normalized volume change across the \nentire scour mark was approximately 0.22 Mm3 km-2. Scour mark 3 was 1,882 m2 with 100% of the area consisting of \ncontinuous seagrass. Between 2016 and 2017, mean elevation change was -0.50 m with a maximum observed change of -1.25 \n400 \nm (Table 2). Net volume change was -0.001 Mm3 and area-normalized volume change was approximately -0.52 Mm3 km-2 \n(Table 3). Between 2017 to 2019, this feature showed accretion with mean elevation change of 0.12 m and net volume change \nof 0.0003 Mm3 (Tables 2 and 4). Area-normalized volume change was approximately 0.14 Mm3 km-2. Scour mark 4 was 1,520 \nm2 with 99% of area consisting of continuous seagrass and 1% unconsolidated sediment. Between 2016 and 2017, mean \nelevation change was -0.54 m with a maximum observed change of -1.29 m (Table 2). Net volume change was -0.0009 Mm3 \n405 \nand area-normalized volume change was approximately -0.57 Mm3 km-2 (Table 3). Ninety-nine percent of net volume change \nwas associated with continuous seagrass habitat which also had the highest area-normalized volume change of -0.57 Mm3 km-\n2. Between 2017 to 2019, this feature showed accretion with mean elevation change of 0.12 m and net volume change of -\n0.0002 Mm3 (Tables 2 and 4). Area-normalized volume change was approximately 0.13 Mm3 km-2. More than 99% of net \nvolume change was associated with continuous seagrass habitat which also had the highest area-normalized volume change of \n410 \napproximately 0.14 Mm3 km-2. High erosion was noted between 2016 and 201\non the sand flat (western) side of the habitat transition and minimal accretion was noted on the seagrass bed (eastern) side of the habit Figure 8. Cardinal orientation imagery (a, b, d, and e represent north, west, east, and south, respectively) and elevation change (c) \nat a scour mark location used to validate benthic features observed in elevation change data. East and west arrows show the boundaries \n415 \nbetween seagrass beds and sand flats in the elevation change data (c) and imagery (b and d). High erosion was noted between 2016 and 2017 \non the sand flat (western) side of the habitat transition and minimal accretion was noted on the seagrass bed (eastern) side of the habitat \ntransition. Photo credit: Mitch Lemon, Cherokee Nations System Solutions for U.S. Geological Survey. 415 Between 2016 and 2017, mean elevation change was -0.50 m with a maximum observed change of -1.25 \n400 \nm (Table 2). Net volume change was -0.001 Mm3 and area-normalized volume change was approximately -0.52 Mm3 km-2 \n(Table 3). Between 2017 to 2019, this feature showed accretion with mean elevation change of 0.12 m and net volume change \nof 0.0003 Mm3 (Tables 2 and 4). Area-normalized volume change was approximately 0.14 Mm3 km-2. Scour mark 4 was 1,520 \nm2 with 99% of area consisting of continuous seagrass and 1% unconsolidated sediment. Between 2016 and 2017, mean elevation change was -0.54 m with a maximum observed change of -1.29 m (Table 2). Net volume change was -0.0009 Mm3 \n405 \nand area-normalized volume change was approximately -0.57 Mm3 km-2 (Table 3). Ninety-nine percent of net volume change \nwas associated with continuous seagrass habitat which also had the highest area-normalized volume change of -0.57 Mm3 km-\n2. Between 2017 to 2019, this feature showed accretion with mean elevation change of 0.12 m and net volume change of -\n0.0002 Mm3 (Tables 2 and 4). Area-normalized volume change was approximately 0.13 Mm3 km-2. More than 99% of net volume change was associated with continuous seagrass habitat which also had the highest area-normalized volume change of \n410 \napproximately 0.14 Mm3 km-2. 20 https://doi.org/10.5194/egusphere-2023-3000\nPreprint. Discussion started: 22 December 2023\nPub ic domain CC\n1.0. l\n. 0 Figure 8. Cardinal orientation imagery (a, b, d, and e represent north, west, east, and south, respectively) and elevation change (c) \nat a scour mark location used to validate benthic features observed in elevation change data. East and west arrows show the boundaries \n415 \nbetween seagrass beds and sand flats in the elevation change data (c) and imagery (b and d). High erosion was noted between 2016 and 2017 \non the sand flat (western) side of the habitat transition and minimal accretion was noted on the seagrass bed (eastern) side of the habitat \ntransition. Photo credit: Mitch Lemon, Cherokee Nations System Solutions for U.S. Geological Survey. Figure 8. Cardinal orientation imagery (a, b, d, and e represent north, west, east, and south, respectively) and elevation change (\nat a scour mark location used to validate benthic features observed in elevation change data. East and west arrows show the boundarie\n415 \nbetween seagrass beds and sand flats in the elevation change data (c) and imagery (b and d). The adjacent erosion \n440 \narea (original 2016 location of the rubble field) showed a mean elevation change of -0.10 m (Table 2) with a maximum \nelevation loss of -0.52 m. Total net volume change was approximately -0.002 Mm3 and area-normalized volume change was \n-0.103 Mm3 km-2 with 53% of net volume change associated with discontinuous seagrass (Table 4). Highest mean elevation \nand area-normalized volume changes were also associated with discontinuous seagrass. Mean elevation and volume losses \ngenerally decreased with increasing mean habitat depth in the erosion area (Tables 2 and 4). 445 loss in mean elevation and area-normalized volume change and accounted for 59% of net volume change. The adjacent erosion \n440 \narea (original 2016 location of the rubble field) showed a mean elevation change of -0.10 m (Table 2) with a maximum \nelevation loss of -0.52 m. Total net volume change was approximately -0.002 Mm3 and area-normalized volume change was \n-0.103 Mm3 km-2 with 53% of net volume change associated with discontinuous seagrass (Table 4). Highest mean elevation \nand area-normalized volume changes were also associated with discontinuous seagrass. Mean elevation and volume losses \ngenerally decreased with increasing mean habitat depth in the erosion area (Tables 2 and 4). 445 3.2.3 Rubble Fields \n420 Migration of reef rubble fields was observed in areas north and northeast of Looe Key Reef between 2016 and 2017. The \nlargest of these features was approximately 418 m long and x 122 m wide at its widest point in 2017, 3 m in height from the \ncrest to base on the deepest (western) edge, with average water depth of approximately 3.3 m (Fig 6g, h, and i). Transect \nelevation profiles showed the location of this feature in 2016, westward migration of approximately 80 m (crest to crest) in Migration of reef rubble fields was observed in areas north and northeast of Looe Key Reef between 2016 and 2017. The \nlargest of these features was approximately 418 m long and x 122 m wide at its widest point in 2017, 3 m in height from the \ncrest to base on the deepest (western) edge, with average water depth of approximately 3.3 m (Fig 6g, h, and i). Transect \nelevation profiles showed the location of this feature in 2016, westward migration of approximately 80 m (crest to crest) in 2017, and minor eastward migration of 8 m (crest to crest) in 2019 (Fig. 7c). The accretion area of this feature covered an area \n425 \nof about 0.03 km2 including approximately 43% reef rubble, 49% discontinuous seagrass, and 9% unconsolidated sediment. Between 2016 and 2017, mean elevation change of the accretion area (2017 location of the rubble field) was 0.89 m (Table 2) \nwith a maximum elevation gain of 2.21 m. Total net volume change was 0.025 Mm3 and area-normalized volume change was 21 https://doi.org/10.5194/egusphere-2023-3000\nPreprint. Discussion started: 22 December 2023\nPub ic domain CC\n1.0. l\n. 0 https://doi.org/10.5194/egusphere-2023-3000\nPreprint. Discussion started: 22 December 2023\nPub ic domain CC\n1.0. l\n. 0 0.914 Mm3 km-2 with discontinuous seagrass accounting for 54% of net volume change indicating burial of seagrass during \nmigration of the rubble field (Table 3). Highest mean elevation and area-normalized volume changes were also associated with \n430 \ndiscontinuous seagrass habitat. An area of erosion (0.023 km2) was observed in 2017 at the original 2016 location of the rubble \nfield that was approximately 428 m long and x 78 m wide including 58% reef rubble, 31% discontinuous seagrass, and 11% \nunconsolidated sediment. Mean elevation-change of the erosion area between 2016 and 2017 was -0.63 m (Table 2) with a \nmaximum elevation loss of -2.11 m. Total net volume change was approximately -0.015 Mm3 and area-normalized volume \nchange was -0.661 Mm3 km-2 with 69% of net volume change associated with reef rubble (Table 3). Highest mean elevation \n435 \nand area-normalized volume changes were also associated with reef rubble. 0.914 Mm3 km-2 with discontinuous seagrass accounting for 54% of net volume change indicating burial of seagrass during \nmigration of the rubble field (Table 3). Highest mean elevation and area-normalized volume changes were also associated with \n430 \ndiscontinuous seagrass habitat. An area of erosion (0.023 km2) was observed in 2017 at the original 2016 location of the rubble \nfield that was approximately 428 m long and x 78 m wide including 58% reef rubble, 31% discontinuous seagrass, and 11% \nunconsolidated sediment. Mean elevation-change of the erosion area between 2016 and 2017 was -0.63 m (Table 2) with a \nmaximum elevation loss of -2.11 m. Total net volume change was approximately -0.015 Mm3 and area-normalized volume change was -0.661 Mm3 km-2 with 69% of net volume change associated with reef rubble (Table 3). Highest mean elevation \n435 \nand area-normalized volume changes were also associated with reef rubble. Between 2017 and 2019, the rubble field (accretion area) showed mean elevation change of -0.24 m, net volume change of -\n0.007 Mm3, and area-normalized volume change of -0.248 Mm3 km-2 (Tables 2 and 4). Discontinuous seagrass showed greatest 0.007 Mm3, and area-normalized volume change of -0.248 Mm3 km-2 (Tables 2 and 4). Discontinuous seagrass showed greatest \nloss in mean elevation and area-normalized volume change and accounted for 59% of net volume change. 3.2.4 Sand Lobe Substantial accretion was observed along a sand lobe located near the base of the fore-reef slope of Looe Key Reef between \n2016 and 2017 (Fig. 6j, k, and l). This feature was approximately 1,383 m long and 344 m wide (approximately 0.27 km2) at \nthe widest point with an average water depth of approximately 11.9 m in 2017 and included only unconsolidated sediment \nhabitat. Between 2016 and 2017, mean elevation change was 0.51 m (Table 2) with maximum gains in elevation up to 1.5 m \n450 \nalong the southern (seaward) downslope section of this feature and maximum elevation losses of -0.58 m along the northern \nlandward section, nearest to the base of the of the fore-reef slope (Fig. 7d). Total net volume change was 0.14 Mm3 and area-\nnormalized volume change was 0.51 Mm3 km-2 (Table 3). Between 2017and 2019, mean elevation change was -0.20 m with \nmaximum elevation losses up to -1.12 m (Table 2, Fig. 7d). Only 852 of 67,389 elevation points analysed for this feature \nshowed gains in elevation after 2017, averaging 0.05 m. Transect elevation profiles showed relatively consistent losses in \n455 \nelevation (erosion) across the sand lobe north to south (landward to seaward) during this time-period. Total net volume change \nwas -0.05 Mm3 and area-normalized volume change was -0.20 Mm3 km-2 (Table 4). Substantial accretion was observed along a sand lobe located near the base of the fore-reef slope of Looe Key Reef between \n2016 and 2017 (Fig. 6j, k, and l). This feature was approximately 1,383 m long and 344 m wide (approximately 0.27 km2) at \nthe widest point with an average water depth of approximately 11.9 m in 2017 and included only unconsolidated sediment (\ng\nj, ,\n)\npp\ny ,\ng\n( pp\ny\n)\nthe widest point with an average water depth of approximately 11.9 m in 2017 and included only unconsolidated sediment \nhabitat. Between 2016 and 2017, mean elevation change was 0.51 m (Table 2) with maximum gains in elevation up to 1.5 m \n450 \nalong the southern (seaward) downslope section of this feature and maximum elevation losses of -0.58 m along the northern \nlandward section, nearest to the base of the of the fore-reef slope (Fig. 7d). Total net volume change was 0.14 Mm3 and area-\nnormalized volume change was 0.51 Mm3 km-2 (Table 3). https://doi.org/10.5194/egusphere-2023-3000\nPreprint. Discussion started: 22 December 2023\nPub ic domain CC\n1.0. l\n. 0 https://doi.org/10.5194/egusphere-2023-3000\nPreprint. Discussion started: 22 December 2023\nPub ic domain CC\n1.0. l\n. 0 3.2.4 Sand Lobe Between 2017and 2019, mean elevation change was -0.20 m with \nmaximum elevation losses up to -1.12 m (Table 2, Fig. 7d). Only 852 of 67,389 elevation points analysed for this feature \nshowed gains in elevation after 2017, averaging 0.05 m. Transect elevation profiles showed relatively consistent losses in \n455 \nelevation (erosion) across the sand lobe north to south (landward to seaward) during this time-period. Total net volume change \nwas -0.05 Mm3 and area-normalized volume change was -0.20 Mm3 km-2 (Table 4). habitat. Between 2016 and 2017, mean elevation change was 0.51 m (Table 2) with maximum gains in elevation up to 1.5 m \n450 \nalong the southern (seaward) downslope section of this feature and maximum elevation losses of -0.58 m along the northern \nlandward section, nearest to the base of the of the fore-reef slope (Fig. 7d). Total net volume change was 0.14 Mm3 and area-\nnormalized volume change was 0.51 Mm3 km-2 (Table 3). Between 2017and 2019, mean elevation change was -0.20 m with \nmaximum elevation losses up to -1.12 m (Table 2, Fig. 7d). Only 852 of 67,389 elevation points analysed for this feature showed gains in elevation after 2017, averaging 0.05 m. Transect elevation profiles showed relatively consistent losses in\n455 \nelevation (erosion) across the sand lobe north to south (landward to seaward) during this time-period. Total net volume change\nwas -0.05 Mm3 and area-normalized volume change was -0.20 Mm3 km-2 (Table 4). 22 4 Discussion Our 2016 to 2017 elevation change results showed general movement of sediment \nand migration of major geomorphic features from ENE to WSW in shallow areas (ranging from approximately 2 to 5.5 m \nwater depth in 2016) of the reef proper and back reef area, consistent with the direction of sustained, high magnitude winds \nduring the passing of Hurricane Irma (Fig. 4; Fig. 6a, d, and g). For example, large sand waves and rubble fields (approximately \n475 \n0.02 to 0.06 km2 in area) migrated westward approximately 80 m (Fig. 6a and g) causing burial of seagrass habitat. Scour \nmarks developed due to erosion of the western edges of seagrass beds and westward transport of sediment, causing burial of \nadjacent seagrass beds between scour marks (Fig. 6d). Numerical modelling of the impact of hurricane-induced wave-current \ninteractions on the transport of material along the FRT during Hurricane Irma showed that wave radiation stress primarily \naffected particle transport trajectories during the passage of the hurricane (Dobbelaere et al., 2022). Additionally, wave energy \n480 \ndissipation occurred through depth-induced wave breaking and bottom dissipation at the shelf break and over the coral reefs. Furthermore, after the passage of the hurricane, suspended particles were transported northeastward by the Florida Current \n(Fig. 1d) and were advected (via Stokes drift) from the outer shelf to inshore for approximately 2 days (Dobbelaere et al., storm and, therefore, included any persistent change that occurred during quiescent sea state conditions before and after the \n465 \npassing of Irma. However, observations during several rapid reef assessments after the storm indicated broad-scale sediment \ndeposition as a direct result of Hurricane Irma (Viehman et al., 2018; Walker, 2018; Wilson et al., 2020; Kobelt et al. 2019), \nwhich corroborates our findings of increased mean elevation and sediment accretion resulting from this storm event. Furthermore, wind conditions were relatively quiescent from the 2016 lidar acquisition date up to the passing of Hurricane storm and, therefore, included any persistent change that occurred during quiescent sea state conditions before and after the \n465 \npassing of Irma. However, observations during several rapid reef assessments after the storm indicated broad-scale sediment \ndeposition as a direct result of Hurricane Irma (Viehman et al., 2018; Walker, 2018; Wilson et al., 2020; Kobelt et al. 2019), \nwhich corroborates our findings of increased mean elevation and sediment accretion resulting from this storm event. 4 Discussion Furthermore, wind conditions were relatively quiescent from the 2016 lidar acquisition date up to the passing of Hurricane Irma and after the storm, and historical aerial imagery of LKR from 2014 and 18 March 2017 (3 years and 6 months prior to \n470 \nHurricane Irma, respectively, Fig. 9) show that patterns of major sedimentary features were mostly static (Finkl and Vollmer, \n2017) in the few years prior to the storm. Our 2016 to 2017 elevation change results showed general movement of sediment \nand migration of major geomorphic features from ENE to WSW in shallow areas (ranging from approximately 2 to 5.5 m \nwater depth in 2016) of the reef proper and back reef area, consistent with the direction of sustained, high magnitude winds Irma and after the storm, and historical aerial imagery of LKR from 2014 and 18 March 2017 (3 years and 6 months prior to \n470 \nHurricane Irma, respectively, Fig. 9) show that patterns of major sedimentary features were mostly static (Finkl and Vollmer, \n2017) in the few years prior to the storm. Our 2016 to 2017 elevation change results showed general movement of sediment \nand migration of major geomorphic features from ENE to WSW in shallow areas (ranging from approximately 2 to 5.5 m \nwater depth in 2016) of the reef proper and back reef area, consistent with the direction of sustained, high magnitude winds Irma and after the storm, and historical aerial imagery of LKR from 2014 and 18 March 2017 (3 years and 6 months prior to \n470 \nHurricane Irma, respectively, Fig. 9) show that patterns of major sedimentary features were mostly static (Finkl and Vollmer, \n2017) in the few years prior to the storm. Our 2016 to 2017 elevation change results showed general movement of sediment \nand migration of major geomorphic features from ENE to WSW in shallow areas (ranging from approximately 2 to 5.5 m \nwater depth in 2016) of the reef proper and back reef area, consistent with the direction of sustained, high magnitude winds during the passing of Hurricane Irma (Fig. 4; Fig. 6a, d, and g). For example, large sand waves and rubble fields (approximately \n475 \n0.02 to 0.06 km2 in area) migrated westward approximately 80 m (Fig. 6a and g) causing burial of seagrass habitat. 4 Discussion There are few comprehensive assessments of the effects of major hurricanes on seafloor elevation and geomorphology on coral \nreefs; and no quantitative studies of reef-scale seafloor elevation change resulting from tropical storm impacts have previously \n460 \nbeen conducted in the Florida Keys. Our results showed Hurricane Irma was primarily a depositional event that increased mean \nseafloor elevation and volume over a 15.98 km2 section of Looe Key Reef by 0.34 m (annualized elevation-change rate of up \nto 247 mm yr-1) and up to 5.4 Mm3, respectively, with area-normalized volume change of approximately 0.34 Mm3km-2. Our \nobservations were based on elevation measurements collected 13.5 months before the storm and three to six months after the There are few comprehensive assessments of the effects of major hurricanes on seafloor elevation and geomorphology on coral \nreefs; and no quantitative studies of reef-scale seafloor elevation change resulting from tropical storm impacts have previously \n460 \nbeen conducted in the Florida Keys. Our results showed Hurricane Irma was primarily a depositional event that increased mean \nseafloor elevation and volume over a 15.98 km2 section of Looe Key Reef by 0.34 m (annualized elevation-change rate of up \nto 247 mm yr-1) and up to 5.4 Mm3, respectively, with area-normalized volume change of approximately 0.34 Mm3km-2. Our \nobservations were based on elevation measurements collected 13.5 months before the storm and three to six months after the \nstorm and, therefore, included any persistent change that occurred during quiescent sea state conditions before and after the \n465 \npassing of Irma. However, observations during several rapid reef assessments after the storm indicated broad-scale sediment \ndeposition as a direct result of Hurricane Irma (Viehman et al., 2018; Walker, 2018; Wilson et al., 2020; Kobelt et al. 2019), \nwhich corroborates our findings of increased mean elevation and sediment accretion resulting from this storm event. Furthermore, wind conditions were relatively quiescent from the 2016 lidar acquisition date up to the passing of Hurricane \nIrma and after the storm, and historical aerial imagery of LKR from 2014 and 18 March 2017 (3 years and 6 months prior to \n470 \nHurricane Irma, respectively, Fig. 9) show that patterns of major sedimentary features were mostly static (Finkl and Vollmer, \n2017) in the few years prior to the storm. 4 Discussion Scour \nmarks developed due to erosion of the western edges of seagrass beds and westward transport of sediment, causing burial of \nadjacent seagrass beds between scour marks (Fig. 6d). Numerical modelling of the impact of hurricane-induced wave-current \ninteractions on the transport of material along the FRT during Hurricane Irma showed that wave radiation stress primarily during the passing of Hurricane Irma (Fig. 4; Fig. 6a, d, and g). For example, large sand waves and rubble fields (approximately \n475 \n0.02 to 0.06 km2 in area) migrated westward approximately 80 m (Fig. 6a and g) causing burial of seagrass habitat. Scour \nmarks developed due to erosion of the western edges of seagrass beds and westward transport of sediment, causing burial of \nadjacent seagrass beds between scour marks (Fig. 6d). Numerical modelling of the impact of hurricane-induced wave-current \ninteractions on the transport of material along the FRT during Hurricane Irma showed that wave radiation stress primarily affected particle transport trajectories during the passage of the hurricane (Dobbelaere et al., 2022). Additionally, wave energy \n480 \ndissipation occurred through depth-induced wave breaking and bottom dissipation at the shelf break and over the coral reefs. Furthermore, after the passage of the hurricane, suspended particles were transported northeastward by the Florida Current \n(Fig. 1d) and were advected (via Stokes drift) from the outer shelf to inshore for approximately 2 days (Dobbelaere et al., \n2022). affected particle transport trajectories during the passage of the hurricane (Dobbelaere et al., 2022). Additionally, wave energy \n480 \ndissipation occurred through depth-induced wave breaking and bottom dissipation at the shelf break and over the coral reefs. Furthermore, after the passage of the hurricane, suspended particles were transported northeastward by the Florida Current \n(Fig. 1d) and were advected (via Stokes drift) from the outer shelf to inshore for approximately 2 days (Dobbelaere et al., \n2022). 485 \nSimilar geomorphic seafloor changes have been documented for other category 4 hurricanes in the Florida Keys based on \nphotographic air and ground surveys, maps, sediment cores, and bottom markers. 4 Discussion In 1967, Hurricane Donna approached from \nthe southeast and passed over the central islands of the Florida Keys in September 1960 with sustained winds of 226 km h-1 \n(category 4) and with breaking waves and storm currents causing broken coral rubble up to a meter in diameter, shoreward 485 \nSimilar geomorphic seafloor changes have been documented for other category 4 hurricanes in the Florida Keys based on \nphotographic air and ground surveys, maps, sediment cores, and bottom markers. In 1967, Hurricane Donna approached from \nthe southeast and passed over the central islands of the Florida Keys in September 1960 with sustained winds of 226 km h-1 \n(category 4) and with breaking waves and storm currents causing broken coral rubble up to a meter in diameter, shoreward \ntransport of gravel to boulder sized rubble and sand approximately 60 to 150 m shoreward, and burial of seagrass with 15 cm \n490 485 \nSimilar geomorphic seafloor changes have been documented for other category 4 hurricanes in the Florida Keys based on \nphotographic air and ground surveys, maps, sediment cores, and bottom markers. In 1967, Hurricane Donna approached from \nthe southeast and passed over the central islands of the Florida Keys in September 1960 with sustained winds of 226 km h-1 \n(category 4) and with breaking waves and storm currents causing broken coral rubble up to a meter in diameter, shoreward \ntransport of gravel to boulder sized rubble and sand approximately 60 to 150 m shoreward, and burial of seagrass with 15 cm \n490 transport of gravel to boulder sized rubble and sand approximately 60 to 150 m shoreward, and burial of seagrass with 15 cm \n490 23 Figure 9. Historical satellite and aerial imagery of Looe Key Reef. Imagery from (a) 17 December 2014, before Hurricane Irma; (b) 18\nMarch 2017, before Hurricane Irma; (c) 30 December 2017, 3 months after Hurricane Irma; (d) 2019, 16.5 months after Hurricane Irma; \n2023. Panel d source: 2019 NOAA National Geodetic Survey via NOAA Digital Coast, downloaded 11 September 2023, \nhttps://www.fisheries.noaa.gov/inport/item/63292. and (e) from 1975 (Lidz et al., 2016). Panels a, b, and c source: Maxar 2023 via © Google Earth Pro 7.3.6.9345, downloaded 11 Septembe Figure 9. Historical satellite and aerial imagery of Looe Key Reef. 4 Discussion Imagery from (a) 17 December 2014, before Hurricane Irma; (b) 18 \nMarch 2017, before Hurricane Irma; (c) 30 December 2017, 3 months after Hurricane Irma; (d) 2019, 16.5 months after Hurricane Irma; \n2023. Panel d source: 2019 NOAA National Geodetic Survey via NOAA Digital Coast, downloaded 11 September 2023, \nhttps://www.fisheries.noaa.gov/inport/item/63292. 495 \nand (e) from 1975 (Lidz et al., 2016). Panels a, b, and c source: Maxar 2023 via © Google Earth Pro 7.3.6.9345, downloaded 11 September of sediment (Ball et al., 1967). Hurricane Betsy approached from the west and passed over the Florida Keys approximately 25 \nkm north of Hurricane Donna’s landfall in September 1965 with sustained winds of up to 226 km h-1. While both storms had \n500 \nsimilar destructive effects to corals on the outer reefs, Hurricane Betsy produced less rubble, showed an overall effect of \nerosion and recycling of sediment in the environment, and caused sediment plumes from the mainland to the edge of the Gulf km north of Hurricane Donna’s landfall in September 1965 with sustained winds of up to 226 km h-1. While both storms had \n500 \nsimilar destructive effects to corals on the outer reefs, Hurricane Betsy produced less rubble, showed an overall effect of \nerosion and recycling of sediment in the environment, and caused sediment plumes from the mainland to the edge of the Gulf 24 Fourqurean and Rutten (2004) showed that seagrass recovery was slowest at sites that were \neroded; losses by mechanical thinning and burial with only a few centimeters of sediment recovered quickly; and seagrass \nburied with 10s of centimeters of sediment hadn’t recovered by three years after the storm. Results from these studies show \n515 \nthe variability in storm impacts due complex interactions among factors such as location, fetch, wind speed, duration, storm \nhistory, and water depth (Fourqurean and Rutten, 2004), and demonstrate the value of comprehensive, quantitative post storm \nassessments of geological and ecological impacts. with maximum sustained winds of only 145 km h-1. However, data from 30 seagrass monitoring transects showed a 3% decline \n510 \nin density of Thalassia testudinum and 19% decline in density of Syringodium filiforme seagrasses, with complete loss of \nseagrass beds at 3 monitoring stations, burial of one station with 50cm of sediment, substantial erosion at two stations \n(Fourqurean and Rutten, 2004). Fourqurean and Rutten (2004) showed that seagrass recovery was slowest at sites that were \neroded; losses by mechanical thinning and burial with only a few centimeters of sediment recovered quickly; and seagrass with maximum sustained winds of only 145 km h-1. However, data from 30 seagrass monitoring transects showed a 3% decline \n510 \nin density of Thalassia testudinum and 19% decline in density of Syringodium filiforme seagrasses, with complete loss of \nseagrass beds at 3 monitoring stations, burial of one station with 50cm of sediment, substantial erosion at two stations \n(Fourqurean and Rutten, 2004). Fourqurean and Rutten (2004) showed that seagrass recovery was slowest at sites that were \neroded; losses by mechanical thinning and burial with only a few centimeters of sediment recovered quickly; and seagrass buried with 10s of centimeters of sediment hadn’t recovered by three years after the storm. Results from these studies show \n515 \nthe variability in storm impacts due complex interactions among factors such as location, fetch, wind speed, duration, storm \nhistory, and water depth (Fourqurean and Rutten, 2004), and demonstrate the value of comprehensive, quantitative post storm \nassessments of geological and ecological impacts. buried with 10s of centimeters of sediment hadn’t recovered by three years after the storm. Results from these studies show \n515 \nthe variability in storm impacts due complex interactions among factors such as location, fetch, wind speed, duration, storm \nhistory, and water depth (Fourqurean and Rutten, 2004), and demonstrate the value of comprehensive, quantitative post storm \nassessments of geological and ecological impacts. 520 \nA previous analysis of seafloor elevation change at LKR during the decade prior to Hurricane Irma (from 2004–2016, during \nwhich only one minor tropical storm impacted this location in 2008) indicated an increase in mean elevation of 0.39 m \n(annualized elevation-change rate of 32.5 mm yr-1), net volume gain of up to 6.4 Mm3 and area-normalized volume change of \n0.39 Mm3 km-2, with accretion observed across all habitat types and some WSW movement of sand waves (Yates et al., 2019). 520 \nA previous analysis of seafloor elevation change at LKR during the decade prior to Hurricane Irma (from 2004–2016, during \nwhich only one minor tropical storm impacted this location in 2008) indicated an increase in mean elevation of 0.39 m \n(annualized elevation-change rate of 32.5 mm yr-1), net volume gain of up to 6.4 Mm3 and area-normalized volume change of \n0.39 Mm3 km-2, with accretion observed across all habitat types and some WSW movement of sand waves (Yates et al., 2019). 520 \nA previous analysis of seafloor elevation change at LKR during the decade prior to Hurricane Irma (from 2004–2016, during \nwhich only one minor tropical storm impacted this location in 2008) indicated an increase in mean elevation of 0.39 m \n(annualized elevation-change rate of 32.5 mm yr-1), net volume gain of up to 6.4 Mm3 and area-normalized volume change of \n0.39 Mm3 km-2, with accretion observed across all habitat types and some WSW movement of sand waves (Yates et al., 2019). Our results showed that sediment deposited during the approximately 16.5 to 19.5-month time-period including impacts from \n525 \nHurricane Irma caused changes in seafloor elevation and volume across all habitat types similar in magnitude to net changes \nobserved over the past decade and at accumulation rates one order of magnitude greater. Previous studies on several coral reefs \naround St. Croix, U.S. https://doi.org/10.5194/egusphere-2023-3000\nPreprint. Discussion started: 22 December 2023\nPub ic domain CC\n1.0. l\n. 0 https://doi.org/10.5194/egusphere-2023-3000\nPreprint. Discussion started: 22 December 2023\nPub ic domain CC\n1.0. l\n. 0 Stream for several days after the Hurricane passed (Perkins and Enos, 1968). Perkins and Enos (1968) noted the difference in \nwind directions for the two storms caused different effects, and that it is difficult to extrapolate quantitative sedimentation rates \nfrom the sedimentary record of one hurricane and frequency of recorded hurricanes. Hurricane Andrew made landfall along \n505 \nthe southeast coast of Florida just south of Miami also with sustained winds of 226 km h-1 with maximum wave heights of less \nthan 2 m. Branching corals were broken, massive coral heads were toppled, seafans and sponges were ripped loose, and shallow \nreefs sustained the most damage (Orr and Ogden, 1992); however, there was little damage to seagrass beds immediately \nseaward of coastal mangroves (Tilmant et al., 1994). Hurricane Georges was a category 2 storm that passed over Key West Stream for several days after the Hurricane passed (Perkins and Enos, 1968). Perkins and Enos (1968) noted the difference in \nwind directions for the two storms caused different effects, and that it is difficult to extrapolate quantitative sedimentation rates \nfrom the sedimentary record of one hurricane and frequency of recorded hurricanes. Hurricane Andrew made landfall along \n505 \nthe southeast coast of Florida just south of Miami also with sustained winds of 226 km h-1 with maximum wave heights of less \nthan 2 m. Branching corals were broken, massive coral heads were toppled, seafans and sponges were ripped loose, and shallow \nreefs sustained the most damage (Orr and Ogden, 1992); however, there was little damage to seagrass beds immediately \nseaward of coastal mangroves (Tilmant et al., 1994). Hurricane Georges was a category 2 storm that passed over Key West \nwith maximum sustained winds of only 145 km h-1. However, data from 30 seagrass monitoring transects showed a 3% decline \n510 \nin density of Thalassia testudinum and 19% decline in density of Syringodium filiforme seagrasses, with complete loss of \nseagrass beds at 3 monitoring stations, burial of one station with 50cm of sediment, substantial erosion at two stations \n(Fourqurean and Rutten, 2004). https://doi.org/10.5194/egusphere-2023-3000\nPreprint. Discussion started: 22 December 2023\nPub ic domain CC\n1.0.\nl\n.\n0 Virgin Islands showed that physical transport of sediment is primarily due to wave-induced oscillatory \nand unidirectional currents, and that storms can increase sediment transport by an order of magnitude higher than during non- Our results showed that sediment deposited during the approximately 16.5 to 19.5-month time-period including impacts from \n525 \nHurricane Irma caused changes in seafloor elevation and volume across all habitat types similar in magnitude to net changes \nobserved over the past decade and at accumulation rates one order of magnitude greater. Previous studies on several coral reefs \naround St. Croix, U.S. Virgin Islands showed that physical transport of sediment is primarily due to wave-induced oscillatory \nand unidirectional currents, and that storms can increase sediment transport by an order of magnitude higher than during non- storm conditions (Hubbard et al., 1981; Hubbard, 1986). Measurements from 15 locations around St. Croix showed sediment \n530 \ntransport rates ranging from 0.009 to 0.3 Mm3 km-2yr-1 during non-storm conditions, and 0.09 to 1.5 Mm3 km-2yr-1 during storm \nconditions (Hubbard et al., 1981; Yates et al., 2017). Sediment trap studies along the southwest coast of Puerto Rico showed \nmedian sediment accumulation rates increased by an order of magnitude (from approximately 6 to 68 mg m-2 d-1) after the \npassage of Hurricane Maria in September of 2017 (a category 4 storm) and a large October 2017 storm that caused resuspension storm conditions (Hubbard et al., 1981; Hubbard, 1986). Measurements from 15 locations around St. Croix showed sediment \n530 \ntransport rates ranging from 0.009 to 0.3 Mm3 km-2yr-1 during non-storm conditions, and 0.09 to 1.5 Mm3 km-2yr-1 during storm \nconditions (Hubbard et al., 1981; Yates et al., 2017). Sediment trap studies along the southwest coast of Puerto Rico showed \nmedian sediment accumulation rates increased by an order of magnitude (from approximately 6 to 68 mg m-2 d-1) after the \npassage of Hurricane Maria in September of 2017 (a category 4 storm) and a large October 2017 storm that caused resuspension 25 https://doi.org/10.5194/egusphere-2023-3000\nPreprint. Discussion started: 22 December 2023\nPub ic domain CC\n1.0. l\n. 0 https://doi.org/10.5194/egusphere-2023-3000\nPreprint. Discussion started: 22 December 2023\nPub ic domain CC\n1.0. l\n. 0 which is considered heavy sedimentation and has been associated with fewer coral species, less live coral, lower coral growth \nrates, reduced coral recruitment and calcification rates, and slower rates of reef accretion (Rogers, 1990). Mean elevation- and area-normalized volume-change from 2016–2017 for habitats examined in our study increased \nsignificantly with water depth suggesting that, in addition to broad-scale sediment deposition across the study site, sediment \n540 \nwas also transported from shallower to deeper habitats (Fig. 5a and b). Notably, greatest increases in elevation (accretion) were \nassociated with habitats in water depths exceeding 11 m including aggregate reef, a sand lobe consisting of unconsolidated \nsediment, and ‘not classified’ habitat located seaward and near the base of the reef’s spur-and-groove formation, suggesting \nsome movement of sediment offshore and downslope (Fig. 4a, Table 2). Additionally, erosion was observed in the shallower, upslope grooves of the spur-and-groove formation, and accretion was observed in the deeper, downslope areas of the grooves \n545 \nfrom 2016 to 2017 further suggesting downslope, offshore movement of sediments (Fig. 10). The sand lobe at the base of the \nspur and groove formation also showed upslope erosion and considerable downslope (seaward) accretion, further suggesting \noffshore transport of sediments (Fig. 7d). Our observations are consistent with previous bathymetric change analyses \nconducted along the northern FRT from 2001 to 2008 (approximately 3 years before Hurricane Ivan and 3 years after Hurricane Katrina) that showed movement of up to 1.8 Mm3 of sediment between these time periods and transport of sediment from the \n550 \ninner shelf to offshore and beyond the shelf edge through gaps in the barrier reef and diabathic (cross-shore) channels during \nhigh-energy events or when the back reef overfills with sand (Finkl, 2004; Finkl and Vollmer, 2017). These observations are \nalso consistent with results of Yates et al. (2017) that show a multi-decadal trend along the FRT of reef sediment transport \ndown the fore-reef-slope and export offshore. Field observations of currents, waves, and reef sediment grain-size analyses Katrina) that showed movement of up to 1.8 Mm3 of sediment between these time periods and transport of sediment from the \n550 \ninner shelf to offshore and beyond the shelf edge through gaps in the barrier reef and diabathic (cross-shore) channels during \nhigh-energy events or when the back reef overfills with sand (Finkl, 2004; Finkl and Vollmer, 2017). These observations are \nalso consistent with results of Yates et al. (2017) that show a multi-decadal trend along the FRT of reef sediment transport \ndown the fore-reef-slope and export offshore. Field observations of currents, waves, and reef sediment grain-size analyses Katrina) that showed movement of up to 1.8 Mm3 of sediment between these time periods and transport of sediment from the \n550 \ninner shelf to offshore and beyond the shelf edge through gaps in the barrier reef and diabathic (cross-shore) channels during \nhigh-energy events or when the back reef overfills with sand (Finkl, 2004; Finkl and Vollmer, 2017). These observations are \nalso consistent with results of Yates et al. (2017) that show a multi-decadal trend along the FRT of reef sediment transport \ndown the fore-reef-slope and export offshore. Field observations of currents, waves, and reef sediment grain-size analyses coupled with integrated ocean-atmosphere-wave-sediment transport modelling during a one-year study at Crocker reef in the \n555 \nUpper Florida Keys showed that sediment mobility was primarily driven by wave stress exceeding critical shear stress; current \nstress alone only exceeded the critical shear stress for sediment mobility 5% of the time usually due to Florida Current eddies \n(Torres-Garcia et al., 2018). Torres-Garcia (2018) showed that nonbreaking wave stress (characteristic of quiescent sea states) \nmobilizes sand approximately 23 to 59% of the time; and fine-grained material is winnowed from the shallow areas of the reef and deposited to the flanks and offshore, particularly to the southwest. Furthermore, the critical stress threshold of gravel-sized \n560 \nmaterial was exceeded only 1 to 13% of the time, particularly during near-field tropical storm conditions (similar to Hurricane \nWilma, a category 3 hurricane) that cause breaking waves, mobilize and transport gravel material, and can cause physical reef \ndegradation (Torres-Garcia et al., 2018). https://doi.org/10.5194/egusphere-2023-3000\nPreprint. Discussion started: 22 December 2023\nPub ic domain CC\n1.0. l\n. 0 570 \n \nFigure 10. Elevation-change along Looe Key Reef spur and groove formation. (a) Upslope to downslope transects along Looe Key\nReef spur and groove formation (green lines); image source: 2019 NOAA National Geodetic Survey via NOAA Digital Coast, downlo\n11 September 2023, https://www.fisheries.noaa.gov/inport/item/63292 with structure from motion overlay image of Hatcher et al. (202\nAreas of erosion (red circles) and accretion (blue circles) along transect 1 between (b) 2016 and 2017, and between (c) 2017 and 2019. 575 \nAreas of erosion (red circles) and accretion (blue circles) along transect 2 between (d) 2016 and 2017, and between (e) 2017 and 2019. Elevation profiles from 2016, 2017, and 2019 for (f) transect 1 and (g) transect 2. Vertical red lines indicate net erosion and vertical blu\nlines indicate net accretion between 2016 and 2017. 570 Figure 10. Elevation-change along Looe Key Reef spur and groove formation. (a) Upslope to downslope transects along Looe Key \nReef spur and groove formation (green lines); image source: 2019 NOAA National Geodetic Survey via NOAA Digital Coast, downloaded \n11 September 2023, https://www.fisheries.noaa.gov/inport/item/63292 with structure from motion overlay image of Hatcher et al. (2022). Areas of erosion (red circles) and accretion (blue circles) along transect 1 between (b) 2016 and 2017, and between (c) 2017 and 2019. 575 \nAreas of erosion (red circles) and accretion (blue circles) along transect 2 between (d) 2016 and 2017, and between (e) 2017 and 2019. Elevation profiles from 2016, 2017, and 2019 for (f) transect 1 and (g) transect 2. Vertical red lines indicate net erosion and vertical blue \nlines indicate net accretion between 2016 and 2017. Figure 10. Elevation-change along Looe Key Reef spur and groove formation. (a) Upslope to downslope transects along Looe Key \nReef spur and groove formation (green lines); image source: 2019 NOAA National Geodetic Survey via NOAA Digital Coast, downloaded \n11 September 2023, https://www.fisheries.noaa.gov/inport/item/63292 with structure from motion overlay image of Hatcher et al. (2022). Areas of erosion (red circles) and accretion (blue circles) along transect 1 between (b) 2016 and 2017, and between (c) 2017 and 2019. 575 \nAreas of erosion (red circles) and accretion (blue circles) along transect 2 between (d) 2016 and 2017, and between (e) 2017 and 2019. Elevation profiles from 2016, 2017, and 2019 for (f) transect 1 and (g) transect 2. Southwest counter currents due to the formation of Florida Current eddies (Lee and \nWilliams, 1999) and WSW movement of sand wave features over a decadal time-period (Yates et al., 2019b) have also been and deposited to the flanks and offshore, particularly to the southwest. Furthermore, the critical stress threshold of gravel-sized \n560 \nmaterial was exceeded only 1 to 13% of the time, particularly during near-field tropical storm conditions (similar to Hurricane \nWilma, a category 3 hurricane) that cause breaking waves, mobilize and transport gravel material, and can cause physical reef \ndegradation (Torres-Garcia et al., 2018). Southwest counter currents due to the formation of Florida Current eddies (Lee and \nWilliams, 1999) and WSW movement of sand wave features over a decadal time-period (Yates et al., 2019b) have also been observed near LKR. Results from these previous studies suggest that some sediment transport observed in our study could be \n565 \ndue to persistent transport of sand during quiescent sea state conditions; however, the large volume of material transported \n(including gravel-sized and larger reef rubble) during the short time-period of our study from 2016 to 2017 was likely due \nprimarily to storm conditions caused by Hurricane Irma. observed near LKR. Results from these previous studies suggest that some sediment transport observed in our study could be \n565 \ndue to persistent transport of sand during quiescent sea state conditions; however, the large volume of material transported \n(including gravel-sized and larger reef rubble) during the short time-period of our study from 2016 to 2017 was likely due \nprimarily to storm conditions caused by Hurricane Irma. observed near LKR. Results from these previous studies suggest that some sediment transport observed in our study could be \n565 \ndue to persistent transport of sand during quiescent sea state conditions; however, the large volume of material transported \n(including gravel-sized and larger reef rubble) during the short time-period of our study from 2016 to 2017 was likely due \nprimarily to storm conditions caused by Hurricane Irma. 26 Vertical red lines indicate net erosion and vertical blue \nlines indicate net accretion between 2016 and 2017. 580 27 https://doi.org/10.5194/egusphere-2023-3000\nPreprint. Discussion started: 22 December 2023\nPub ic domain CC\n1.0. l\n. 0 https://doi.org/10.5194/egusphere-2023-3000\nPreprint. Discussion started: 22 December 2023\nPub ic domain CC\n1.0. l\n. 0 Approximately 16.5 months after Hurricane Irma (during a 13-month period between 2017 and 2019), net erosion was observed \nacross all habitats with mean elevation-change of -0.15 (annualized elevation change-rate of -139 mm yr-1), net volume change \nup to -2.46 Mm3, and area-normalized volume change of -0.15 Mm3km-2. Newly deposited carbonate sediments typically have \nporosities of 40 to 70% (Choquette and Pray, 1970) at shallow sediment depths of a few hundreds of meters (Schmoker and Halley, 1982). Porosity of carbonate sands on the FRT and in Hawk Channel ranges from 60 to 72% in the upper 22 cm of \n585 \ndeposited sediment (Walter et al. 2007). Schmoker and Halley (1982) showed that there is little or no sediment porosity loss \nat near-surface sediment depths. Application of their exponential function for porosity versus depth of sediment (porosity (%) \n= 41.73e-z/2498, where z = depth below sediment surface) indicates that the decrease in porosity of deposited carbonate sediments \nat 2 m below the sediment-surface is only 0.03%. Carbonate sands have settling velocities ranging from 0.025 to 0.364 m s-1 Halley, 1982). Porosity of carbonate sands on the FRT and in Hawk Channel ranges from 60 to 72% in the upper 22 cm of \n585 \ndeposited sediment (Walter et al. 2007). Schmoker and Halley (1982) showed that there is little or no sediment porosity loss \nat near-surface sediment depths. Application of their exponential function for porosity versus depth of sediment (porosity (%) \n= 41.73e-z/2498, where z = depth below sediment surface) indicates that the decrease in porosity of deposited carbonate sediments \nat 2 m below the sediment-surface is only 0.03%. Carbonate sands have settling velocities ranging from 0.025 to 0.364 m s-1 585 (Riazi et al., 2020). Satellite imagery shows the sediment plume caused by resuspension of sediment during Hurricane Irma \n590 \ncleared within approximately 5 days of the storm’s passing (NASA, 2023). Therefore, it is likely that resuspended sediment \nsettled quickly (within days) when storm conditions subsided; and it is unlikely that the decrease in elevation observed between \n2017 and 2019 was caused by compaction of sediment after the storm. This suggests that approximately 50% of sediment \ndeposited between 2016 and 2017 was eroded by 2019 due to physical transport away from the study site. The sand wave and (Riazi et al., 2020). Satellite imagery shows the sediment plume caused by resuspension of sediment during Hurricane Irma \n590 \ncleared within approximately 5 days of the storm’s passing (NASA, 2023). Therefore, it is likely that resuspended sediment \nsettled quickly (within days) when storm conditions subsided; and it is unlikely that the decrease in elevation observed between \n2017 and 2019 was caused by compaction of sediment after the storm. This suggests that approximately 50% of sediment \ndeposited between 2016 and 2017 was eroded by 2019 due to physical transport away from the study site. The sand wave and reef rubble field showed continued erosion between 2017 to 2019 with some evidence for migration of the crest of the rubble \n595 \nfield back toward its original 2016 position indicated in the elevation profile (Fig. 6 and 7). Shallow areas between the scour \nmarks showed erosion, while the scour mark pits showed infilling (Fig. 6 and 7). Spurs of the spur-and-groove formation \nprimarily showed erosion, while shallow (landward) sections of grooves showed some accretion, likely due to transport of \nsediments from spurs to grooves and downslope from the shallow reef (Fig. 10). Deeper (seaward) areas of grooves and the reef rubble field showed continued erosion between 2017 to 2019 with some evidence for migration of the crest of the rubble \n595 \nfield back toward its original 2016 position indicated in the elevation profile (Fig. 6 and 7). Shallow areas between the scour \nmarks showed erosion, while the scour mark pits showed infilling (Fig. 6 and 7). Spurs of the spur-and-groove formation \nprimarily showed erosion, while shallow (landward) sections of grooves showed some accretion, likely due to transport of \nsediments from spurs to grooves and downslope from the shallow reef (Fig. 10). Deeper (seaward) areas of grooves and the reef rubble field showed continued erosion between 2017 to 2019 with some evidence for migration of the crest of the rubble \n595 \nfield back toward its original 2016 position indicated in the elevation profile (Fig. 6 and 7). Shallow areas between the scour \nmarks showed erosion, while the scour mark pits showed infilling (Fig. 6 and 7). Spurs of the spur-and-groove formation \nprimarily showed erosion, while shallow (landward) sections of grooves showed some accretion, likely due to transport of \nsediments from spurs to grooves and downslope from the shallow reef (Fig. 10). Deeper (seaward) areas of grooves and the sand lobe located at the base of the spur and groove formation showed erosion (Fig. 4 and 6k) suggesting continued downslope, \n600 \noffshore transport of sediments. Historical aerial and satellite imagery from before and after the passing of Hurricane Irma \ncorroborates our elevation-change observations (Fig. 9). Imagery from 2014 and March 2017 shows that major geomorphic \nfeatures of Looe Key proper such as distribution of seagrass beds and the size and position of the sand lobe and rubble fields \nwere relatively static between these time periods leading up to Hurricane Irma (Fig. 9a and b). Imagery from December 2017, sand lobe located at the base of the spur and groove formation showed erosion (Fig. 4 and 6k) suggesting continued downslope, \n600 \noffshore transport of sediments. Historical aerial and satellite imagery from before and after the passing of Hurricane Irma \ncorroborates our elevation-change observations (Fig. 9). Imagery from 2014 and March 2017 shows that major geomorphic \nfeatures of Looe Key proper such as distribution of seagrass beds and the size and position of the sand lobe and rubble fields \nwere relatively static between these time periods leading up to Hurricane Irma (Fig. 9a and b). Imagery from December 2017, 3 months after Hurricane Irma passed, shows broad scale sediment deposition and burial of seagrass beds in the shallow areas \n605 \nof the reef proper, erosion and exposure of deeper, downslope spur-and-groove formation and downslope deposition on the \nsand lobe (Fig. 9c). Imagery from 2019 shows re-exposure of some shallow seagrass beds and deep spur-and-groove formation \nas sediments were eroded (Fig. 9d). Historical areal imagery from 1975 (Fig. 9e, Lidz et al., 2016) shows a distribution of \nseagrass, presence of rubble fields, and patterns of sediment along the sand lobe similar to 2014 and 2017 imagery (before 3 months after Hurricane Irma passed, shows broad scale sediment deposition and burial of seagrass beds in the shallow areas \n605 \nof the reef proper, erosion and exposure of deeper, downslope spur-and-groove formation and downslope deposition on the \nsand lobe (Fig. 9c). Imagery from 2019 shows re-exposure of some shallow seagrass beds and deep spur-and-groove formation \nas sediments were eroded (Fig. 9d). Historical areal imagery from 1975 (Fig. 9e, Lidz et al., 2016) shows a distribution of \nseagrass, presence of rubble fields, and patterns of sediment along the sand lobe similar to 2014 and 2017 imagery (before 3 months after Hurricane Irma passed, shows broad scale sediment deposition and burial of seagrass beds in the shallow areas \n605 \nof the reef proper, erosion and exposure of deeper, downslope spur-and-groove formation and downslope deposition on the \nsand lobe (Fig. 9c). Imagery from 2019 shows re-exposure of some shallow seagrass beds and deep spur-and-groove formation \nas sediments were eroded (Fig. 9d). Historical areal imagery from 1975 (Fig. 9e, Lidz et al., 2016) shows a distribution of \nseagrass, presence of rubble fields, and patterns of sediment along the sand lobe similar to 2014 and 2017 imagery (before Hurricane Irma) indicating these features have persisted over the past several decades despite repeated impact from tropical \n610 \nand seasonal storms. Lidz et al. (2016) suggested the formation of rubble fields in the shallow back reef area is mainly due to \nhistorical passage of hurricanes and winter storms, and our elevation change results suggest that these structures continue to \nmigrate in response to storm conditions. Lidz et al. (2016) also suggested that transport of sediment during hurricanes was Hurricane Irma) indicating these features have persisted over the past several decades despite repeated impact from tropical \n610 \nand seasonal storms. Lidz et al. (2016) suggested the formation of rubble fields in the shallow back reef area is mainly due to \nhistorical passage of hurricanes and winter storms, and our elevation change results suggest that these structures continue to \nmigrate in response to storm conditions. Lidz et al. (2016) also suggested that transport of sediment during hurricanes was Hurricane Irma) indicating these features have persisted over the past several decades despite repeated impact from tropical \n610 \nand seasonal storms. Lidz et al. (2016) suggested the formation of rubble fields in the shallow back reef area is mainly due to \nhistorical passage of hurricanes and winter storms, and our elevation change results suggest that these structures continue to \nmigrate in response to storm conditions. Lidz et al. (2016) also suggested that transport of sediment during hurricanes was 28 https://doi.org/10.5194/egusphere-2023-3000\nPreprint. Discussion started: 22 December 2023\nPub ic domain CC\n1.0. l\n. 0 https://doi.org/10.5194/egusphere-2023-3000\nPreprint. Discussion started: 22 December 2023\nPub ic domain CC\n1.0. l\n. 0 primarily to the north; however, our observations showed primary sediment movement during Hurricane Irma was WSW and \ndownslope from shallow to deep habitats with apparent seaward movement of the sand lobe after the storm passed. 615 615 elevation-change rate of -1.4 mm yr-1 between 1934 and 2004 (Yates et al., 2017). Furthermore, six of nine habitats at LKR \n620 \nshowed elevation loss over those periods, with greatest losses associated with shallow habitats, and mean elevation and \nvolume gains in deep-water habitats including at the base of the spur-and-groove habitat, indicating transport of reef \nsediments down the fore-reef-slope and export offshore (Yates et al., 2017). Our observed rate of mean elevation loss \nbetween 2017 and 2019 (-139 mm yr-1) was two orders of magnitude higher than the multi-decadal rates of Yates et al. (2017). Additionally, elevation loss (erosion) showed a moderate correlation with water depth, and mean elevation losses \n625 \nduring 2017 to 2019 were significantly greater in habitats with larger mean elevation gains during 2016 to 2017, suggesting \nthat sediment distribution was re-equilibrating or stabilizing to quiescent sea-state conditions up to 16.5 months after the \nstorm. The annualized mean rate of elevation-change for LKR from the 2.5-year period between July 2016 to January 2019 examined \nin our study, including sediment accretion from Hurricane Irma and the post-storm erosion and re-equilibration, was \n630 \napproximately 72 mm yr-1, which is almost double the rate of accretion observed in the previous decade of 32.5 mm yr-1 (Yates \net al. 2019b). Numerous field reconnaissance observations immediately after the passing of Hurricane Irma indicated \nbroadscale sediment deposition across the FRT due to the storm (e.g., Viehman et al., 2018; Walker, 2018; Wilson et al., 2020; \nKobelt et al. 2019). Our 2016 to 2019 elevation-change rate is consistent with annualized mean elevation-change rates from The annualized mean rate of elevation-change for LKR from the 2.5-year period between July 2016 to January 2019 examined \nin our study, including sediment accretion from Hurricane Irma and the post-storm erosion and re-equilibration, was \n630 \napproximately 72 mm yr-1, which is almost double the rate of accretion observed in the previous decade of 32.5 mm yr-1 (Yates \net al. 2019b). 5 Conclusion High-resolution lidar and multibeam bathymetric data were used to quantify seafloor elevation and volume change within the \nLooe Key Reef system of the Florida Keys Reef Tract over a 2.5-year period from 2016–2019 and to examine impacts from \ncategory-4 Hurricane Irma and post-storm re-equilibration of seafloor sediments. Analysis of seafloor elevation and volume \nchange over a 16.5-month period from July 2016 to December 2017 showed Hurricane Irma caused broadscale deposition of \n655 \nsediments across all benthic habitats of this reef system and burial of seagrass and coral dominated habitat. Rates of net \nelevation change were one order of magnitude greater during this short-term period that included storm impacts from Hurricane \nIrma than for the previous decade (Yates et al., 2019). Major seafloor geomorphic features such as sand waves and rubble \nfields migrated 10s of meters to the WSW in response to predominant wind conditions during the passing of Hurricane Irma, \nand sediment accretion was significantly greater in deep habitats than shallow habitats, suggesting downslope and offshore \n660 \ntransport of seafloor sediment. High-resolution lidar and multibeam bathymetric data were used to quantify seafloor elevation and volume change within the \nLooe Key Reef system of the Florida Keys Reef Tract over a 2.5-year period from 2016–2019 and to examine impacts from \ncategory-4 Hurricane Irma and post-storm re-equilibration of seafloor sediments. Analysis of seafloor elevation and volume change over a 16.5-month period from July 2016 to December 2017 showed Hurricane Irma caused broadscale deposition of \n655 \nsediments across all benthic habitats of this reef system and burial of seagrass and coral dominated habitat. Rates of net \nelevation change were one order of magnitude greater during this short-term period that included storm impacts from Hurricane \nIrma than for the previous decade (Yates et al., 2019). Major seafloor geomorphic features such as sand waves and rubble \nfields migrated 10s of meters to the WSW in response to predominant wind conditions during the passing of Hurricane Irma, \nand sediment accretion was significantly greater in deep habitats than shallow habitats, suggesting downslope and offshore \n660 \ntransport of seafloor sediment. 655 Loss of mean elevation and volume in all habitats in the period following the storm (from December 2017 to January 2019) \nindicated that 35% to 50% of sediment deposited during the storm had eroded by approximately 16.5 months after the storm \nand that erosion rates were two orders of magnitude greater than historical, multi-decadal rates of erosion. https://doi.org/10.5194/egusphere-2023-3000\nPreprint. Discussion started: 22 December 2023\nPub ic domain CC\n1.0. l\n. 0 Table 5. Annualized mean elevation-change rates (mm yr-1) for event-driven to multi-decadal time periods at the Florida Keys \nReef Tract. Annualized mean elevation change rate (mm yr-1) \nLocation \nEvent-drivena \n2016 to 2017 \nPost-stormb \n2017 to 2019 \nShort-termc \n2016 to 2019 \nDecadal \n2004 to 2016 \nMulti-decadal \n1930s to 2000s \nLooe Key Reef \n247 \n-139 \n72 \n32.5d \n-4.5e \nLower Florida Reef Tract \n(south of Big Pine) \nna \nna \n84f \nna \nna \nFlorida Reef Tract (Miami to \nKey West) \nna \nna \n76f \nna \nna \nUpper Florida Reef Tract \n(Elliott Key to Tavernier Key) \nna \nna \nna \nna \n-1.4e \na = calculated assuming a total time-period of 16.5 months (13.5 month pre- to 3 months post-storm); b = total time period 13.5 months (3 to 16.5 months \npost-storm); c = total time-period approximately 30 months (13.5 months pre- to 16.5 months post-storm); d = using data from Yates et al., 2019; e = using \ndata from Yates et al., 2017; f = using data from Fehr et al., 2021; na = no data available. 0 Table 5. Annualized mean elevation-change rates (mm yr-1) for event-driven to multi-decadal time periods at the Florida Keys \nReef Tract Table 5. Annualized mean elevation-change rates (mm yr-1) for event-driven to multi-decadal time per\nReef Tract. elevation-change rates (mm yr-1) for event-driven to multi-decadal time periods at the Florida Keys a = calculated assuming a total time-period of 16.5 months (13.5 month pre- to 3 months post-storm); b = total time period 13.5 months (3 to 16.5 months \npost-storm); c = total time-period approximately 30 months (13.5 months pre- to 16.5 months post-storm); d = using data from Yates et al., 2019; e = using \ndata from Yates et al., 2017; f = using data from Fehr et al., 2021; na = no data available. 650 Numerous field reconnaissance observations immediately after the passing of Hurricane Irma indicated \nbroadscale sediment deposition across the FRT due to the storm (e.g., Viehman et al., 2018; Walker, 2018; Wilson et al., 2020; \nKobelt et al. 2019). Our 2016 to 2019 elevation-change rate is consistent with annualized mean elevation-change rates from 2016 to 2019 for the Lower FRT from approximately Big Pine Key to Key West of 84 mm yr-1, and for the FRT from Miami \n635 \nto Key West of 76 mm yr-1 (Fehr et al., 2021), further suggesting that sediment distribution may have still been undergoing \npost-storm re-equilibration at our study site and along the broader FRT (Table 5). Collection and analysis of additional elevation-change data sets over shorter time-periods (e.g., seasonal to annual) could Collection and analysis of additional elevation-change data sets over shorter time-periods (e.g., seasonal to annual) could \nimprove characterization of post-storm elevation-change rates and duration of post-storm sediment re-equilibration periods \n640 \nrelative to persistent seasonal, interannual, decadal, and multi-decadal time periods. Our results also suggest that caution should \nbe used in selection of DEMs for use in elevation change and projection modelling to minimize bias that could result from \nselecting elevation surfaces that reflect periods of rapid elevation change due to storm impacts and periods of post-storm re-\nequilibration. improve characterization of post-storm elevation-change rates and duration of post-storm sediment re-equilibration periods \n640 \nrelative to persistent seasonal, interannual, decadal, and multi-decadal time periods. Our results also suggest that caution should \nbe used in selection of DEMs for use in elevation change and projection modelling to minimize bias that could result from \nselecting elevation surfaces that reflect periods of rapid elevation change due to storm impacts and periods of post-storm re-\nequilibration. 645 29 5 Conclusion Sediment erosion \n665 \nafter the storm (2017–2019) was moderately correlated with depth and was significantly greater in habitats that showed greater \naccumulation during the period including Hurricane Irma from 2016–2017, suggesting a period of rapid sediment re-\nequilibration after the storm. Historical satellite and aerial imagery show that major geomorphic features at this location \nincluding rubble fields, sand waves, and a sand lobe at the base of the spur-and-groove formation have persisted over the past several decades despite impacts from storms. However, our elevation-change results indicate these features are highly \n670 \nephemeral, migrating rapidly during storms, re-equilibrating to non-storm sea state conditions between storms, and periodically \nburying seafloor habitat such as seagrass. Such features and the area surrounding them likely represent localized areas of long- \nand short-term seafloor instability that could be less suitable for restoration of slow growing benthic species. Our observed several decades despite impacts from storms. However, our elevation-change results indicate these features are highly \n670 \nephemeral, migrating rapidly during storms, re-equilibrating to non-storm sea state conditions between storms, and periodically \nburying seafloor habitat such as seagrass. Such features and the area surrounding them likely represent localized areas of long- \nand short-term seafloor instability that could be less suitable for restoration of slow growing benthic species. Our observed 30 Data availability Elevation-change and multibeam bathymetric data are publicly available in U.S. Geological Survey Data Releases at \nhttps://doi.org/10.5066/P9CHC95D, \nhttps://doi.org/10.5066/P937LNZF, \nhttps://doi.org/10.5066/P9NXNX61, \nhttps://doi.org/10.5066/P9JTOOMB and https://doi.org/10.5066/P9P2V7L0. Lidar topobathymetric data are publicly available \n690 \nfrom \nthe \nNOAA \nOffice \nfor \nCoastal \nManagement \nat \nhttps://www.fisheries.noaa.gov/inport/item/63018 \nand \nhttps://www.fisheries.noaa.gov/inport/item/48373. Seafloor habitat data are publicly available from the Florida Fish and \nWildlife \nConservation \nCommission, \nFish \nand \nWildlife \nResearch \nInstitute \nat \nhttp://ocean.floridamarine.org/IntegratedReefMap/UnifiedReefTract.htm. p\ng\ng\np\n \n695 \nAuthor contribution \nKY and DZ conceptualized the research, data acquisition approach, and methodology for analysis of seafloor elevation data. KY, ZF, and DZ performed formal analysis of data. DZ developed the SECAT software for statistical analysis of elevation- \nand volume-change data. KY and ZF developed data interpretations and prepared the original manuscript draft with \ncontributions from SJ and DZ. All authors contributed to preparation of the final, published manuscript. 700 \n \nCompeting interests 695 \nAuthor contribution \nKY and DZ conceptualized the research, data acquisition approach, and methodology for analysis of seafloor elevation data. KY, ZF, and DZ performed formal analysis of data. DZ developed the SECAT software for statistical analysis of elevation- \nand volume-change data. KY and ZF developed data interpretations and prepared the original manuscript draft with \ncontributions from SJ and DZ. All authors contributed to preparation of the final, published manuscript. 700 Code availability Python script for the Seafloor Elevation Change Analysis Tool (SECAT), intended to be applied in ArcMap or ArcGIS Pro, is \npublicly \navailable \nas \na \nU. S. Geological \nSurvey \nsoftware \nrelease, \ndoi: \n10.5066/P9D5UUZ0, \nhttps://www.usgs.gov/software/seafloor-elevation-change-analysis-tool. Python script for the Seafloor Elevation Change Analysis Tool (SECAT), intended to be applied in ArcMap or ArcGIS Pro, is \npublicly \navailable \nas \na \nU. S. Geological \nSurvey \nsoftware \nrelease, \ndoi: \n10.5066/P9D5UUZ0, \nhttps://www.usgs.gov/software/seafloor-elevation-change-analysis-tool. https://doi.org/10.5194/egusphere-2023-3000\nPreprint. Discussion started: 22 December 2023\nPub ic domain CC\n1.0. l\n. 0 https://doi.org/10.5194/egusphere-2023-3000\nPreprint. Discussion started: 22 December 2023\nPub ic domain CC\n1.0. l\n. 0 rates of elevation change in the 16-month period after Hurricane Irma were one to two orders of magnitude greater than during \nthe past decade or multi-decadal period (Yates et al., 2017; 2019b) indicating seafloor sediments across all habitats may have \n675 \nstill been re-equilibrating to non-storm sea state conditions up January 2019. Higher resolution elevation-change data collected \nover seasonal and annual time periods could improve characterization and understanding of short-term (event-driven, seasonal, \ninterannual) and long-term (decadal to multi-decadal) rates and processes of seafloor change and help guide benthic habitat \npost-storm recovery and restoration efforts in topographically complex coral reef systems. https://doi.org/10.5194/egusphere-2023-3000\nPreprint. Discussion started: 22 December 2023\nPub ic domain CC\n1.0. l\n. 0 https://doi.org/10.5194/egusphere-2023-3000\nPreprint. Discussion started: 22 December 2023\nPub ic domain CC\n1.0. l\n. 0 Acknowledgement Funding for this study was provided by the U.S. Geological Survey, Coastal and Marine Hazards and Resources Program and \n710 \nby 2018 Hurricane and Wildfire Supplemental Funding provided to the U.S. Geological Survey from the Additional \nSupplemental Appropriations for Disaster Relief Requirements Act of 2018 (P.L. 115-123). We would like to thank J.J. Fredericks, B.J. Reynolds, and A.S. Farmer for collection of the multibeam bathymetry data and J.J. Fredericks for \ndevelopment of the associated multibeam digital elevation model. We also thank J. Zieg for assistance with development of Funding for this study was provided by the U.S. Geological Survey, Coastal and Marine Hazards and Resources Program and \n710 \nby 2018 Hurricane and Wildfire Supplemental Funding provided to the U.S. Geological Survey from the Additional \nSupplemental Appropriations for Disaster Relief Requirements Act of 2018 (P.L. 115-123). We would like to thank J.J. Fredericks, B.J. Reynolds, and A.S. Farmer for collection of the multibeam bathymetry data and J.J. Fredericks for \ndevelopment of the associated multibeam digital elevation model. We also thank J. Zieg for assistance with development of the SECAT software and K. Murphy for assistance with development of methods for sub-sampling large-scale digital elevation \n715 \nmodels. We greatly appreciate reviews of the original draft manuscript and constructive comments from L. Toth and G. Hatcher. Competing interests The authors declare that they have no conflict of interest. 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 òÌÜÛa @ À @ ÐnÛa pb @ Þìy @ òîvy @a @@@@@@@ bèj’  òÌÜÛa @ À @ ÐnÛa pb @ Þìy @ òîvy @a @@@@@@@ bèj’ ﺩ ﺍﳋﺎﻣﺲ ﺍﻟﻌﺪﺩ ﺣﻮﻟﻴﺔ •ﺣ ﻀﻰ ﺍﻟﻠﻐﺔ،)(١ ﻑ ﺍﻟﺒﻴﺎﻥ ﺇﱃ ﻡﺎﻜﺣﻷﺍ «. ﺎﻥ ﻋﻨﻪ ﻋﻠﻴﻪ ﺍﻟﻘﺮﺁﻥ ﲟﻘﺘﻀ ﻢﻬﻨ . ﻲﻫﻭ : ، ﻓﻘﺪ ﺃﺿﺎﻑ ﻮﻝ ﻋﻠﻰ ﺑﻴﺎﻥ ﺎ ﻓﻴﻪ ﺑﻴﺎ ﻭﺭﺩ ﺯﺍﻮﺟ ﲑﺴﻔﺗ ﺔ ﻘﻘﶈﺍﻭ ﲔ ﻨﻣ n ﻞﺤﻨﻟﺍ ٤٤ : ﻚ) .(٢ ﻪ : »ﻪﻧﺇ ﻮﻤﳏ ﻪﻨﻋ  ﺎﻤﻴﻓ ﺐ ﺇﱃ ﻋﺪﻡ ﺟ ﲨﻬﻮﺭ ﺍﳊﻨﺎﺑﻠﺔ Z [ \ n ﳎﺎﻭﺯﺓ ﺫﻟﻚ ﻲ)٣(ﻪﻨﻋ ﻪﻟﻮﻘﺑ ﺻﺢ ﻭﺭﻭﺩﻩ ﺎﺑﻠﺔ ﻣﻦ ﺫﻫﺐ ﲨ ﺑﺄﺩﻟﺔ ﺭﺩﻫﺎ o X Y ﻓﻠﻴﺲ ﺪﺣﻷ ﳋﻄﺎﺏ ﺍﳊﻨﺒﻠﻲ ﺻ ﻗﻔﻪ ﻋﻠﻰ ﻣﺎ ﺕﺎ . ﺇ ﻥ ﻣﻦ ﺍﳊﻨﺎ ﻮﺍ ﻋﻠﻰ ﺫﻟﻚ ﻗﺎﻝ ﺗﻌﺎﱃ: o ﷲ ، ﺍﺬﻟﻭ ﺃﺟﺎﺏ ﺃﺑﻮ ﺍﳋ ﻧﺰﺍﻉ ﰲ ﺗﻮﻗ ﺎﻫﺎﲡﻻ ﻻﻭﺃ : ﻮﻟﺪﺘﺳ ١- ﻗ ﺳﻮﻝ ﺍﷲ ﺪﻗﻭ ﺃ ﺍﺬﻫ ﻻ ،ﺔ)١( ﺩ ﺍﳋﺎﻣﺲ ٨٥١ /٢: ﻭﻧﻘﻞ ﺍﻟﻌﺪﺩ ﻭﺍﻟﻜﻮﻛﺐ ﺍﳌﻨﲑ ٧ ﺓﺩﻮﺴﳌﺍﻭ : ١٧٦ ﲨﺎﻋﺔ ﻣﻦ ﺍﳊﻨﺎﺑﻠﺔ. ﺣﻮﻟﻴﺔ ﺍﺑﻦ ﺍﻟﻠﺤﺎﻡ ٣٧ : ﺿﻲ ﺃﰊ ﻳﻌﻠﻰ ﻭﲨ . •ﺣ ، ﻳﻨﻈﺮ : ﺮﺼﺘﺨﳌﺍ : ﰊ ﺍﳊﺴﲔ ﺑﻦ ﺍﻟﻘﺎ ﻲﻠﺒﻨﳊ :٢/ ٢٨٢ . ٢ ﺣﺪ ﻗﻮﻟﲔ ﻟﻠﺤﻨﺎﺑﻠﺔ ﺮﺃﻱ ﻋﻦ ﺍﻟﻘﺎﺿﻲ ﺃ ﺪ :ﺃﺑﻮ ﺍﳋﻄﺎﺏ ﺍﳊ ﺭ ﻖﺑﺎﺴﻟﺍ ٢٨/٢: )( ١ ﻫﻮ ﺃﺣ ﺍﻟﺮﻫﺬﺍ )٢( ﺪﻴﻬﻤﺘﻟﺍ )٣( ﺭﺪﺼﳌﺍ ﺩ ﺍﳋﺎﻣﺲ ٨٥١ /٢: ﻭﻧﻘﻞ ﺍﻟﻌﺪﺩ ﻭﺍﻟﻜﻮﻛﺐ ﺍﳌﻨﲑ ٧ ﺓﺩﻮﺴﳌﺍﻭ : ١٧٦ ﲨﺎﻋﺔ ﻣﻦ ﺍﳊﻨﺎﺑﻠﺔ. ﺣﻮﻟﻴﺔ ﺍﺑﻦ ﺍﻟﻠﺤﺎﻡ ٣٧ : ﺿﻲ ﺃﰊ ﻳﻌﻠﻰ ﻭﲨ . •ﺣ ، ﻳﻨﻈﺮ : ﺮﺼﺘﺨﳌﺍ : ﰊ ﺍﳊﺴﲔ ﺑﻦ ﺍﻟﻘﺎ ﻲﻠﺒﻨﳊ :٢/ ٢٨٢ . ٢ ﺣﺪ ﻗﻮﻟﲔ ﻟﻠﺤﻨﺎﺑﻠﺔ ﺮﺃﻱ ﻋﻦ ﺍﻟﻘﺎﺿﻲ ﺃ ﺪ :ﺃﺑﻮ ﺍﳋﻄﺎﺏ ﺍﳊ ﺭ ﻖﺑﺎﺴﻟﺍ ٢٨/٢: )( ١ ﻫﻮ ﺃﺣ ﺍﻟﺮﻫﺬﺍ )٢( ﺪﻴﻬﻤﺘﻟﺍ )٣( ﺭﺪﺼﳌﺍ ٨٥١ /٢: ﻭﻧﻘ ﻭﺍﻟﻜﻮﻛﺐ ﺍﳌﻨﲑ ٧ ﺓﺩﻮﺴﳌﺍﻭ : ١٧٦ ﲨﺎﻋﺔ ﻣﻦ ﺍﳊﻨﺎﺑﻠﺔ. ﺍﺑﻦ ﺍﻟﻠﺤﺎﻡ ٣٧ : ﺿﻲ ﺃﰊ ﻳﻌﻠﻰ ﻭﲨ . ، ﻳﻨﻈﺮ : ﺮﺼﺘﺨﳌﺍ : ﰊ ﺍﳊﺴﲔ ﺑﻦ ﺍﻟﻘﺎ ﻲﻠﺒﻨﳊ :٢/ ٢٨٢ . ٢ ﺣﺪ ﻗﻮﻟﲔ ﻟﻠﺤﻨﺎﺑﻠﺔ ﺮﺃﻱ ﻋﻦ ﺍﻟﻘﺎﺿﻲ ﺃ ﺪ :ﺃﺑﻮ ﺍﳋﻄﺎﺏ ﺍﳊ ﺭ ﻖﺑﺎﺴﻟﺍ ٢٨/٢: )( ١ ﻫﻮ ﺃﺣ ﺍﻟﺮﻫﺬﺍ )٢( ﺪﻴﻬﻤﺘﻟﺍ )٣( ﺭﺪﺼﳌﺍ  òÌÜÛa @ À @ ÐnÛa pb @ Þìy @ òîvy @a @@@@@@@ bèj’ ﺩ ﺍﳋﺎﻣﺲ ﻋﻦ ﺭﺳﻮﻝ ﺮﺩ ﻓﻴﻪ ﺑﻴﺎﻥ، ﺔﲨ ،ﻅﺎﻔﻟﻷﺍ ﺰﻧﹶ ﺎ ﺃ ﻣﻭﺩﺪ ﹶ ﻝ ﺯ ﺍﻻﺣﺘﺠﺎﺝ ﰲ ﺍﳊﺪﻭﺩ ﺽ ﻭﺍﻹﻧﺴﺎﻥ، ﰲ ﺗﻔﺼﻴﻞ ﺍﳌﺮﺍﺩ ﺍﻟﻌﺪﺩ ﻓﺈﻥ ﻣﺎ ﻭﺭﺩ ﻭﺃﻣﺎ ﻣﺎ ﱂ ﻳﺮ ﻤﺎ ﻳﺘﻌﻠﻖ ﲨﺮﺘﺑ ﹼ ﻻ ﺪﻮﺍ ﺣﹶﻤ ﻠﻌﻳ ﺪ ﻋﺪﻡ ﺟﻮﺍﺯ ﳓﺘﺞ ﺑﻘﻮﳍﻢ ﺴﻮﺍﺩ ﻭﺍﻟﺒﻴﺎﺽ ﺴﻠﻢ ﻟﻮﺟﻮﻩ: :٢/ ١٦٢ ﺮﻈﻨﻳﻭ ﺎﻤ،ﺪﻌﺑ ﺍﺬﻟﻭ ﻃﺮﻳﻖ ﺁﺧﺮ. ﻟﱰﺍﻉ ﻫﻮ ﻓﻴﻤ ﻥ ﻭﺗﻮﻗﻴﻒ .  òÌÜÛa @ À @ ÐnÛa pb @ Þìy @ òîvy @a @@@@@@@ bèj’ ﻻﹶ ﺃ ﺭﺪﺟﹶ ﺃﹰ ﻭ ﺎﻗﺎ ٩٧ ، ﻓﻬﻲ ﺗﻔﻴﺪ ﻪ : »ﺎﻧﺇ ﻻ ﻨﺎﻫﺎ، ﻣﺜﻞ ﺍﻟﺴ ﻉﻮﺿ ﲑﻏ ﺴﻣ ﰲ ﻋﻠﻮﻡ ﺍﻟﻘﺮﺁﻥ ﺣﻮﻟﻴﺔ ﻣﺎﺳﺔ ﺇﻟﻴﻪ ﻓﻴﻤ ﻃ ﻋﻦ ﺍﻟﻨﻈﺮ ﰲ ﻩ.)١( ﻞﳏﻭ ﱰﻟﺍ ﱂ ﻳﺮﺩ ﻓﻴﻪ ﺑﻴﺎﻥ ﺎﹶ ﻔﹺ ﻧﹰ ﻭ ﺍﺮﹾ ﻔﹸ ﻛ ﺪ ﻢ ﺔﺑﻮﺘﻟﺍ : ٧ .) (٢ ﻲﻠﺒﻨﳊ)٣( ﻪﻟﻮﻘﺑ ﻅﺎﻔﻟﻷﺍ ﺎﻨﻌﻣﻭ . « ﻣﺜﻞ ﻫﺬﺍ ﺍﳌﻮﺿ :١٨٦ ، ﺍﻟﱪﻫﺎﻥ . •ﺣ ﻣ ﻩ، ﻭﺍﳊﺎﺟﺔ ﻋ ﻓﻔﻴﻪ ﻛﻔﺎﻳﺔ ﻞ ﺍﻟﻌﻠﻢ ﺑﻌﺪﻩ ﱂ ﺍﻟﻠﻐﺔ ﻓﻴﻤﺎ ﺍ َ ﻷ ﺪﺷﹶ ﺃ ﺏﺍﺮﻋ ﻢﻴﻜﺣ ﻢﻴﻠﻋ ﻪ ﺴﲑ ﻛﻼﻡ ﺍﷲ. ﺍﳋﻄﺎﺏ ﺍﳊﻨ ﺞ ﺑﻘﻮﳍﻢ ﰲ ﺍ ﻤﻬﻢ ﻓﻼ ﺗﻘﺒﻞ ﺬﻩ ﺍﻵﻳﺔ ﰲ ﺪﻣﺔ ﻛﺘﺎﺏ ﺍﳌﺒﺎﱐ ﻥﺁﺮﻘﻟ :١٢ -٢٠ . . ٢ ﺇﻟﻴﻪ ﰲ ﻋﺼﺮﻩ ﻦﻣ ﻥﺎﻴﺑ ﻓ ﻪﻟ ﻨﺌﺬ ﺓﺮﻜﻓ ﻞﻫﺃ ﻘﺮﺁﻥ ﲟﻘﺘﻀﻰ ﻗﺎﻝ ﺗﻌﺎﱃ:  ﻪﱠ ﺍﻟﻠ ﻭﻪﻮﻟﺳﻰ ﺭ ﻌﺮﺏ ﰲ ﺗﻔﺴ ﺏ ﻋﻨﻪ ﺃﺑﻮ ﺎﻡ، ﻭﺇﳕﺎ ﳛﺘﺞ ﺎﺭﻫﻢ ﻭﺣﻜﻤ  ﺝﺎﺠﺘﺣﻻﺍ ﺀ :١/ ٢٩٠ ، ﻣﻘﺪ  :ﺗﻄﻮﺭ ﺗﻔﺴﲑ ﻘﻟﺍ ﺪ :٢/ ٢٨٢ . ﺭ ﻖﺑﺎﺴﻟﺍ ٢٨/٢: ﺇ ﺔﺟﺎﳊﺍ ﺍﷲ  ﻓﻔﻴﻪ ﺣﻴﻨ ﻭﻓﻬﻢ ﺍﻟﻘ ٢- ﻗ ﻰﹶ ﻠﻋ ﻪﱠ ﻠﻟﺍ ﺑﻜﻼﻡ ﺍﻟﻌ ﺏﺎﺟﺃﻭ ﺎﻜﺣﻷﺍﻭ ﻓﺄﻣﺎ ﺃﺧﺒﺎ ﰒ ﻥﺇ )( ١ ﺀﺎﻴﺣﻹﺍ ﻪﻧﺎﻴﺒﺑ )٢( ﺪﻴﻬﻤﺘﻟﺍ ( ٣) ﺭﺪﺼﳌﺍ ﰲ ﺗﻔﺼﻴﻞ ﺍ :٢٦١ /٢ ﺮﻈﻨﻳﻭ ﰲ ﻋﻠﻮﻡ ﺍﻟﻘﺮﺁﻥ :١٨٦ ، ﺍﻟﱪﻫﺎﻥ . ﺪﻣﺔ ﻛﺘﺎﺏ ﺍﳌﺒﺎﱐ ﻥﺁﺮﻘﻟ :١٢ -٢٠ . . ٢ ﺀ :١/ ٢٩٠ ، ﻣﻘﺪ  :ﺗﻄﻮﺭ ﺗﻔﺴﲑ ﻘﻟﺍ ﺪ :٢/ ٢٨٢ . ﺭ ﻖﺑﺎﺴﻟﺍ ٢٨/٢: )( ١ ﺀﺎﻴﺣﻹﺍ ﻪﻧﺎﻴﺒﺑ )٢( ﺪﻴﻬﻤﺘﻟﺍ ( ٣) ﺭﺪﺼﳌﺍ :٢/٢ ﻥ ١٤٣ ﺩ ﺍﳋﺎﻣﺲ ﺎﺩﻳﺔ، ﻣﻘﺎﺑﻠﺔ ﺑﺎﳌﺪﻳﻨﺔ ﺫﻛﺮ ﻢ ﺃﺷﺪ، ﻷ ﻗﻮﻻ، ﻓﺄﺧﻠﻖ ﺔ ﺝﺎﺠﺘﺣﻻﺍ ﻢﻭﻓﻖ ﻟﺴﺎ ﺲ ﻫﻮ ﺍﳌﻔﺴﺮ ﻥ ﻋﻠﻰ ﻭﻓﻘﻪ ﻨﻬﻢ ﰲ ﻫﺬﻩ ﻳﺪﺧﻞ ﻓﻴﻪ ﻳﺘﺴﺎﻭﻭﺍ ﰲ ﻦ ﺍﻻﺣﺘﺠﺎﺝ ﺔ ﺍﻻﺣﺘﺠﺎﺝ ﺍﻟﻌﺪﺩ ﻣﻦ ﺃﻫﻞ ﺍﻟﺒﺎ ﻮﺍﻝ ﺍﳌ ﺑ ﲔﻘﻓﺎﻨ ﺃﻥ ﻛﻔﺮﻫﻢ ﻗﻮ ﻌﺎ، ﻭﺃﺟﻔﻰ ﺍﻉ ﰲ ﺔﺤﺻ ﺪ ﻭ ﻧﺰﻝ ﻋﻠﻰ ﻣﻪ، ﻟﻴﺲ ﻷﻧﻪ ﺃﻟﻔﺎﻅ ﻥﺁﺮﻘﻟﺍ ﻣﺴﻜﻮﺕ ﻋﻨ ﺿﺖ ﳍﻢ ﻻ ﻻ ﳝﻜﻦ ﺃﻥ ﻪﻴﻠﻋﻭ ﻦﻜﻤﻴﻓ ﳑﺎ ﻳﺮﺩ ﺻﺤﺔ ﲑﺴﻔ .  òÌÜÛa @ À @ ÐnÛa pb @ Þìy @ òîvy @a @@@@@@@ bèj’ ﻜﻔﺮ ﻭﺍﻟﻨﻔﺎﻕ ﺟﻞ ﻭﻋﺰ ﺃﺣﻮ ، ﻓﺄﺧﱪ ﻋﻦ ﻢ ﺃﻏﻠﻆ ﻃﺒﻌ ﻋﻦ ﳏﻞ ﺍﻟﱰﺍ ،ﻼﺻﺃ ﺪﻗﻭ ﺔ ﻋﻨﻪ ﺇﺳﻼﻣ ﺃ ﻐﻮﻱ ﻓﻴﻔﺴﺮ ﻫﻞ ﺍﳊﺎﺿﺮﺓ ﻋﻠﻰ ﻣﻦ ﺗﻌﺮﺿ ﻡ ﻏﲑﻫﻢ، ﻭﻻ ،ﲔﻨﻣﺆﻣ )٢( ﻭ ﳑ ،ﺺ ﻭﻣﻮﺟﺒﻪ ﻟﻌﺮﺏ ﰲ ﺍﻟﺘﻔﺴ ﺣﻮﻟﻴﺔ ﺣﺎﻝ ﺃﻫﻞ ﺍﻟﻜ ﺟ ﺪ ﺃﻥ ﺫﻛﺮ ﺃﻫﻞ ﺍﻟﺒﺎﺩﻳﺔ ﺴﻨﻦ ﻣﻊ ﻛﻮ ﺍﺬﻫ ﻋ ﺝﺭﺎﺧ ﻧﺰﻭﻝ ﺍﻟﻘﺮﺁﻥ ﻦ ﻧﻘﻠﺖ ﺍﻟﻠﻐﺔ ﻪ ﺍﻟﻌﺮﰊ ﺍﻟﻠﻐﻮ ﺍﻟﺒﺎﺩﻳﺔ، ﻭﺃﻫ ﻋ ﺍﻵﻳﺔ ﻫﺬﻩ ﺍﻷﻋﺮﺍﺏ ﺃﻡ ﻦ ﺃﻫﻞ ﺔﻳﺩﺎﺒﻟﺍ ﻣﻘﺘﻀﻰ ﺍﻟﻨﺺ ﺠﺎﺝ ﺑﻜﻼﻡ ﺍﻟ •ﺣ À ﺣ ﺘﺤﺪﺙ ﻋﻦ ﳊﺎﺿﺮﺓ، ﻓﺒﻌﺪ ﻦﻣ ﺏﺍﺮﻋﻷﺍ ﻞ ﺔﻓﺮﻌﻣﻭ ﺴﻟﺍ ،ﻉﺮﺸﻟ)١( ﻫﻭ ﺩﺍﺕ ﺍﻟﻘ ﻥﺁﺮ. ﻗﺎﺋﻤﺔ ﻗﺒﻞ ﻳﺸﺘﺮﻁ ﻓﻴﻤﻦ ﺍﳌﻔﺴﺮ ﻛﻼﻣﻪ ﻫﻢ ﺃﻋﺮﺍ ﺏ ﻚ ﺃﻥ ﺣﻜﻢ ﺀ ﻛﺎﻧﻮﺍ ﻦﻣ ﺇﻧﻜﺎﺭ ﺃﻥ ﻣﻦ ﻣ ﻭﺟﻬﻢ ﻋﻦ ﺯﺍﻮﺟ ﺠﺘﺣﻻﺍ ٢-٢٣٢ . . ٢ - ﻥﺇ ﺍﻵﻳﺔ ﺗﺘﺤ ﳍﻢ ﻣﻦ ﻞﻫﺃ ﺍ ﻥ ﻧﺎﺋﻴﺎ ﻋﻨﻬﺎ ﻦ ﲰﺎﻉ ﺍﻟﺘﱰﻳﻞ ﻤﻮﺍ ﻓﺮﺍﺋﺾ ﺍﻟ ﰲ ﻓﻬﻢ ﻣﻔﺮﺩ ﺎﻬ- ﺇﻥ ﺍﻟﻠﻐﺔ ﺍﻟﻘﺎﻃﻊ، ﻓﻼ ﻭﺇﳕﺎ ﻳﺄﺧﺬ ﻪﺘﺤ. - ﻥﺇ ﺀﻻﺆﻫ ﺎ - ﻻﻭ ﻚﺷ ﻣﻨﻬﻢ، ﺳﻮﺍ ﻛﻤﺎ ﻻ ﳝﻜﻦ ،ﲔﻨﻣﺆﳌ ﻭﺮﳋ ﻳﺔ ﻋﻠﻰ ﻋﺪﻡ ﱯﻃﺮﻘﻟﺍ ١٣٢ /٨: ﺭ ﻖﺑﺎﺴﻟﺍ ٢٣/٨: è ﺎﳍﻭﺃ- ﻣﻊ ﺃﻣﺜﺎﳍ ﻣﻦ ﻛﺎﻥ ﺃﺑﻌﺪ ﻋﻦ ﺃﻻ ﻳﻌﻠﻤﻮ ﻢﺎﺴﻠﺑ ﻬﻴﻧﺎﺛ ﻞﻴﻟﺪﻟﺎﺑ ،ﺮﺷﺎﺒﳌﺍ ﻋﻨﺪ ﺻﺤ ﺎﻬﺜﻟﺎﺛ ﺔﻳﻵﺍ . ﺎﻬﻌﺑﺍﺭ ﻥﻮﻨﻣﺆﳌﺍ ﻚﻟﺫ ﻛ. ﻠﺑ ﺍﳌﺴﺎﻥ ﺬﻩ ﺍﻵﻳﺔ )( ١ ﲑﺴﻔﺗ ( ٢) ﺭﺪﺼﳌﺍ ،ﻉ)١( ﻭ،ﲔ)٢( ﻭ  òÌÜÛa @ À @ ÐnÛa pb @ Þìy @ òîvy @a @@@@@@ bèj’ ﳍﻢ ﺑﻘﻮﻟﻪ :ﻻ ﺍﷲ ﻭﺃﻭﺭﺩ ﻟﺸﻌﺮ ﺃﺻﻼ ﻓﻴﻪ ﻫﻮ ﺑﻴﺎﻥ v w x ، ﻭﻟﻮ ﻛﺎﻥ ﺪ ﺑﺴﻨﺪﻩ ﻋﻦ ﺪ :ﻳﻌﲏ ﻛﺎﻥ ﳍ ﻣﻊ ﻭﺻﻔﻪ ﺗﻔﺴﲑ ﻡﻼﻛ ﺸﻌﺮ ﳚﻌﻞ ﺍ ﺔﻳﺎﻏ ﻓ ﺮﻣﻷﺍ ﻝ: o u v n ﺀﺍﺮﻌﺸﻟﺍ : ١٩٥ ﻭﻯ ﺃﺑﻮ ﻋﺒﻴﺪ ﻗﺎﻝ ﺃﺑﻮ ﻋﺒﻴﺪ ﱂ ﺴﻳﻤ،ﻢﻬ ﺗ ﺑﺎﻟﻠﻐﺔ ﰲ ﻭﻣﺸﻜﻠﻪ ﺑﺎﻟﺸ ﻟﻠﻘﺮﺁﻥ، ﺇﺫ ﻏ ﺳﺒﺤﺎﻧﻪ ﻳﻘﻮﻝ s t u n ،ﻪ)٢( ﱴﺣ ﻭﺭ ﺪ ﻓﻴﻪ ﺍﻟﺸﻌﺮ .  òÌÜÛa @ À @ ÐnÛa pb @ Þìy @ òîvy @a @@@@@@@ bèj’ ﻱ ﻋﻦ ﲨﺎﻋﺔ ﺯ ﺍﻻﺣﺘﺠﺎﺝ ﻭ ﻳﺐ ﺍﻟﻘﺮﺁﻥ ﻟﺸﻌﺮ ﺃﺻﻼ ﻌﺮ، ﻷﻥ ﺍﷲ ﻝﻮﻘﻳﻭ : o s ﺏ ﻭﻋﻤﻠﻮﺍ ﺑﻪ ﻥﺁﺮﻘﻟﺍ ﺪﺸﻨﻴﻓ ﺮ ﺑﻦ ﺍﻷﻧﺒﺎﺭﻱ ﱃ ﻋﺪﻡ ﺟﻮﺍﺯ ﺝ ﻋﻠﻰ ﻏﺮﻳﺐ ﻚ ﻻ ﳚﻌﻞ ﺍﻟ ﺍﻟﻘﺮﺁﻥ ﺑﺎﻟﺸﻌ ﻟﺰﺧﺮﻑ ، ٣: ﻭ ﻋﻠﻴﻪ ﺍﻷﺻﺤﺎﺏ ﺍ ﻥ ﻳﺴﺄﻝ ﻋﻦ ﺴﲑ«.) (٣ ﻧﻘﻞ ﺃﺑﻮ ﺑﻜﺮ ﻢ .ﺫﻫﺒﻮﺍ ﺇﱃ ﻢ ﻭﻫﻲ : ﻥﺇ ﺝﺎﺠﺘﺣﻻﺍ ) (١ : ﺑﻪ ﺇﻥ ﺫﻟﻚ ﺍﻟﻐﺮﻳﺐ ﻣﻦ y z n ﺍ ﻋ ﻎ ﳌﺎ ﺣﺾ ﺱ : »ﻪﻧﺃ ﻥﺎﻛ ﺪ ﺑﻪ ﻋﻠﻰ ﺍﻟﺘﻔﺴ ﺎﻴﻧﺎﺛ: ﻋﻠﻢ ﳍﻢ ﻢﻬﺠﺠﺣ ١- ﻥﺁﺮﻘﻠﻟ .) ﺍﻮﺟﻭ ﺍﳊﺮﻑ y ﻏﲑ ﺳﺎﺋﻎ ﻦﺑﺍ ﺱﺎﺒﻋ ﺪﻬﺸﺘﺴﻳ v w x ، ﻭﻟﻮ ﻛﺎﻥ ﺪ ﺑﺴﻨﺪﻩ ﻋﻦ ﺪ :ﻳﻌﲏ ﻛﺎﻥ ﻝ: o u v n ﺀﺍﺮﻌﺸﻟﺍ : ١٩٥ ﻭﻯ ﺃﺑﻮ ﻋﺒﻴﺪ ﻗﺎﻝ ﺃﺑﻮ ﻋﺒﻴﺪ ﺳﺒﺤﺎﻧﻪ ﻳﻘﻮﻝ s t u n ،ﻪ)٢( ﱴﺣ ﻭﺭ ﺪ ﻓﻴﻪ ﺍﻟﺸﻌﺮ . ﻌﺮ، ﻷﻥ ﺍﷲ ﻝﻮﻘﻳﻭ : o s ﺏ ﻭﻋﻤﻠﻮﺍ ﺑﻪ ﻥﺁﺮﻘﻟﺍ ﺪﺸﻨﻴﻓ ﺍﻟﻘﺮﺁﻥ ﺑﺎﻟﺸﻌ ﻟﺰﺧﺮﻑ ، ٣: ﻭ ﻋﻠﻴﻪ ﺍﻷﺻﺤﺎﺏ ﺍ ﻥ ﻳﺴﺄﻝ ﻋﻦ ﺴﲑ«.) (٣ ﺍﻟﻐﺮﻳﺐ ﻣﻦ y z n ﺍ ﻋ ﻎ ﳌﺎ ﺣﺾ ﺱ : »ﻪﻧﺃ ﻥﺎﻛ ﺪ ﺑﻪ ﻋﻠﻰ ﺍﻟﺘﻔﺴ ﺍﳊﺮﻑ y ﻏﲑ ﺳﺎﺋﻎ ﻦﺑﺍ ﺱﺎﺒﻋ ﺪﻬﺸﺘﺴﻳ v w x ، ﻭﻟﻮ ﻛﺎﻥ ﺪ ﺑﺴﻨﺪﻩ ﻋﻦ ﻝ: o u v n ﺀﺍﺮﻌﺸﻟﺍ : ١٩٥ ﻭﻯ ﺃﺑﻮ ﻋﺒﻴﺪ ﺳﺒﺤﺎﻧﻪ ﻳﻘﻮﻝ s t u n ،ﻪ)٢( ﱴﺣ ﻭﺭ ﻌﺮ، ﻷﻥ ﺍﷲ ﻝﻮﻘﻳﻭ : o s ﺏ ﻭﻋﻤﻠﻮﺍ ﺑﻪ ﺍﻟﻘﺮﺁﻥ ﺑﺎﻟﺸﻌ ﻟﺰﺧﺮﻑ ، ٣: ﻭ ﻋﻠﻴﻪ ﺍﻷﺻﺤﺎﺏ ﺍﻟﻐﺮﻳﺐ ﻣﻦ y z n ﺍ ﻋ ﻎ ﳌﺎ ﺣﺾ ﺍﳊﺮﻑ y ﻏﲑ ﺳﺎﺋﻎ )٢( ﺩ ﺍﳋﺎﻣﺲ ﺍﻟﻌﺪﺩ ﺣﻮﻟﻴﺔ . ١ ﻥﺎ :١/ ١١٩ . ٣ . •ﺣ ﻥﺎﻘﺗﻹﺍﻭ :١/١٩ ٠٠١ /١: ﺎﻘﺗﻹﺍﻭ ﻀﺎﺋﻞ ﺍﻟﻘﺮﺁﻥ :٤٣ ﺡ ﺍﻟﻮﻗﻒ :١/ ١٠٠ ﺡ ﺍﻟﻮﻗﻒ ﻭﺍﻻﺑﺘﺪﺍﺀ ﺭﺍﻥ ﺍﻟﺴﺎﺑﻘﺎﻥ، ﻭﻓﻀ )١( ﺡﺎﻀﻳﺇ )٢( ﺡﺎﻀﻳﺇ )٣( ﺭﺪﺼﳌﺍ © ª n ¨ ©ﱃ: o ¨ ﺩ ﺍﳋﺎﻣﺲ ﻼﻡ، ﻓﻬﻮ ﻻ ﻣﻦ ﺃﺧﺮﺟﻬﻢ ﻭﺃﻧﻪ ﻭﺻﻒ µ ¶ ¸ ¹ ﳑﻦ ﻳﻘﻮﻟﻮﻥ ﺍﻟﻌﺪﺩ ﻪ ﻛﻘﺒﻴﺢ ﺍﻟﻜﻼ ﻪﺒ ﻦﻣ ﺀﺎﻨﺜﺘﺳﺎﺑ ﺀﺍﺮﻌﺸ :٢٢٧ ، ³ ´ µ ﻋﻠﻰ ﻣﺜﺎﳍﻢ ﻜﻼﻡ، ﻭﻗﺒﻴﺤﻪ ﺗﺮﻯ ﺃﻧﻪ ﺒﻘﻋﺃ  à n ﺸﻟﺍ ° ± ² ³ ﺸﺮﻛﲔ ﻭﻣﻦ ﺣﻮﻟﻴﺔ ﻦﺴﺤﻛ ﻜﻟﺍ «.) (٢ ﺗ ﻋﻤﻮﻣﻪ، ﺃﻻ À Á  o ¬ ® ¯ ° ﻬﻢ ﺷﻌﺮﺍﺀ ﺍﳌﺸ :١/ ١٢٠ . •ﺣ ﻜﻼﻡ، ﺣﺴﻨﻪ ﺮﻩ ﳌﻀﻤﻨﺎﺗﻪ« ﻋ ﻟﻴﺲ ﻋﻠﻰ ¾ ¿ À ﻮﻣﲔ ﺑﻘﻮﻟﻪ: o - ٢٢٦ ، ﻬﻓ ١/ ١٠٠ ﻥﺎﻘﺗﻹﺍﻭ ١٥ .  òÌÜÛa @ À @ ÐnÛa pb @ Þìy @ òîvy @a @@@@@@@ bèj’ ﻧﻮﻉ ﻣﻦ ﺍﻟﻜ ﺍﺗﻪ، ﻭﺇﳕﺎ ﻳﻜﺮ ﺍﻟﺬﻡ ﻟﻠﺸﻌﺮﺍﺀ o ¼ ½ ¾ ﻟﺸﻌﺮﺍﺀ ﺍﳌﺬﻣﻮ n ﺀﺍﺮﻌﺸﻟﺍ : ٢٢٥ ﺡ ﺍﻟﻮﻗﻒ ﻭﺍﻻﺑﺘﺪﺍﺀ ﺍﻟﻘﺮﻃﱯ ١٥/ ٣١ : » ﺮﻌﺸﻟﺍ ﻳﻜﺮﻩ ﻟﺬ ﻥﺇﻭ ﺍ ﻪﻟﻮﻘﺑ : o ﺃﻭﻟﺌﻚ ﺍﻟ º n )١( ﺡﺎﻀﻳﺇ )٢( ﲑﺴﻔﺗ :١/ ١٢٠ . ﻥﺎ  òÌÜÛa @ À @ ÐnÛa pb @ Þìy @ òîvy @a @@@@@@@ bèj’  òÌÜÛa @ À @ ÐnÛa pb @ Þìy @ òîvy @a @@@@@@ bèj’ ﻟﻐﲑﻫﺎ ﻣﻦ ﻪﻟﻮﻗ » : ﻷ ﻥ ﺇﺫ ﻋﺮﺽ ﻟﻪ ﻭﺁﺩﺍﺑﻪ، ﺃﻭ ﺍﻟﺸﻌﺮ ﻣﺜﻞ « ). (٢ ﺇ ﻪﻟﻮﻗﻭ ( ﺎﳘﻮﳓﻭ . ﺩﺉ ﺍﻹﺳﻼﻡ ﺇﱃ ﺫﻡ ﻩﻇﺎﻫﺮ ﳝﺘﻠﺊ ﺷﻌﺮﺍ« ﻥﺎﻄﻴﺸﻟ «.)٣ ﻭ ﻣﻨﺎﻓﻴﺎ ﳌﺒﺎﺩ (  ﺎﳑ ﲑﺸﻳ ﻇ ﺧﲑ ﻣﻦ ﺃﻥ ﻭ :ﺃﻣﺴﻜﻮﺍ ﺍ ﻼﻡ ﻭﺃﻫﻠﻪ، ﺃﻭ ﺎ ﺍﻹﺳﻼﻡ.)١( ﺭﺳﻮﻝ ﺍﷲ ﻳ ﻢ ﻗﻴﺤﺎ ﹺ ﺮﻳﻪ ﺍ ﻥﺎﻄﻴﺸﻟﺍ ﺃﻭ. ﻌﺎﺩﻳﺎ ﻟﻺﺳﻼ ﺏ ﺍﻟﱵ ﻳﺮﻓﻀﻬﺎ ﺭ ﺍﳌﺮﻭﻱ ﻋﻦ ﻮﻑ ﺃﺣﺪﻛﻢ ﺪﺸ : »ﺍﻭﺬﺧ ﺍﺮﻌﺷ ﻌﻣ ﺏﺎﺒﺳﻷ ﺎﻣﺃﻭ ﺘﻠﺊ ﺟﻮ ﺮﻋﺎﺷ ﺸﻨﻳ ﻟﻐﲑﻫﺎ ﻣﻦ ﻪﻟﻮﻗ » : ﻷ ﻥ ﺇﺫ ﻋﺮﺽ ﻟﻪ ﻭﺁﺩﺍﺑﻪ، ﺃﻭ ﺍﻟﺸﻌﺮ ﻣﺜﻞ « ). (٢ ﺇ ﻪﻟﻮﻗﻭ ( ﺎﳘﻮﳓﻭ . ﺩﺉ ﺍﻹﺳﻼﻡ ﺇﱃ ﺫﻡ ﻩﻇﺎﻫﺮ ﳝﺘﻠﺊ ﺷﻌﺮﺍ« ﻥﺎﻄﻴﺸﻟ «.)٣ ﻭ ﻣﻨﺎﻓﻴﺎ ﳌﺒﺎﺩ (  ﺎﳑ ﲑﺸﻳ ﻇ ﺧﲑ ﻣﻦ ﺃﻥ ﻭ :ﺃﻣﺴﻜﻮﺍ ﺍ ﻼﻡ ﻭﺃﻫﻠﻪ، ﺃﻭ ﺎ ﺍﻹﺳﻼﻡ.)١( ﺭﺳﻮﻝ ﺍﷲ ﻳ ﻢ ﻗﻴﺤﺎ ﹺ ﺮﻳﻪ ﺍ ﻥﺎﻄﻴﺸﻟﺍ ﺃﻭ. ﻌﺎﺩﻳﺎ ﻟﻺﺳﻼ ﺏ ﺍﻟﱵ ﻳﺮﻓﻀﻬﺎ ﺭ ﺍﳌﺮﻭﻱ ﻋﻦ ﻮﻑ ﺃﺣﺪﻛﻢ ﺪﺸ : »ﺍﻭﺬﺧ ﺍﺮﻌ ﻌﻣ ﺏﺎﺒﺳﻷ ﺎﻣﺃﻭ ﻠﺊ ﺟﻮ ﺮﻋﺎ ﺸﻨﻳ ﺩ ﺍﳋﺎﻣﺲ ﻪ ﺭﺳﻮﻝ ﺍﷲ ﺤﻪ ﺇﺫ ﺑﻮﺏ ﺮﻌ(، )٥( ﻮﻬﻓ ﻛﺘﺎﺏ ﺍﻟﺸﻌﺮ : ﺍﻟﻌﺪﺩ ﻟﺬﻱ ﻳﻬﺠﺎ ﺑﻪ ﳍﻢ.) (٤ ﺭﻱ ﰲ ﺻﺤﻴﺤ ﺍﻹﻧﺴﺎﻥ ﺍﻟﺸﻌ ﺢﻴﺤﺻﻭ ﻢﻠﺴﻣ : ( . ﻫﻮ ﺍﻟﺸﻌﺮ ﺍﻟ ﳍ ﳍﺠﺎﺀ ﻏﺮﺿﺎ ﺍﻹﻣﺎﻡ ﺍﻟﺒﺨﺎﺭ ﺍ ﻟﻐﺎﻟﺐ ﻋﻠﻰ ﺏﺍﺩ ﻭ ٤٧/٤: /١٤٠ . . ١ ﷲ ﻭﺍﻟﻌﻠﻢ ﻭﺍﻟﻘﺮﺁﻥ ﺣﻮﻟﻴﺔ ﺮ ،ﻓﻴﻤﺎ ﺳﺒﻖ ﺟﻌﻠﻮﺍ ﻫﺬﺍ ﺍﳍ ﻣﺎ ﺃﺷﺎﺭ ﺇﻟﻴﻪ ﻩ ﺃﻥ ﻳﻜﻮﻥ ﺍ ﻒ :١/ ١٠٢ . ﺨﺎﺭﻱ: ﻛﺘﺎﺏ ﺍﻵ ﺔﻔﲢ ﺍ ﻷ ﻱﺫﻮﺣ ٨: / ﺍﳊﺪﻳﺚ ﺍﻟﺴﺎﺑﻖ . ﱯﻃﺮﻘﻟﺍ:١٣ /٥٣ ﻳﺼﺪﻩ ﻋﻦ ﺫﻛﺮ ﺍﷲ •ﺣ ﻪﻴﻄﻌﻳ.  òÌÜÛa @ À @ ÐnÛa pb @ Þìy @ òîvy @a @@@@@@@ bèj’ ﻪﻣ ﺮﻌﺸﻠﻟ ﺀﺍﺮﻌﺸ ﺟ ﻦﻳﺬﻟﺍ ﻣ ﻪ ﰲ ﺗﺄﻭﻳﻠﻪ )ﺑﺎﺏ ﻣﺎ ﻳﻜﺮ ٤١ ﻭﺇﻳﻀﺎﺡ ﺍﳌﻮﻗﻒ ﻠﻢ :ﺻﺤﻴﺢ ﺍﻟﺒﺨ ﺩﻭ :٤/ ٣٠٤ ﻭﲢ ٧١ ﻭﺗﻜﻤﻠﺘﻪ ﺑﻨﺤﻮ ١-١٠٣ ﺍ ﲑﺴﻔﺗﻭ ٧ ﻪﺘﻠﻤﻜﺗﻭ ﺣﱴ ) ﻳ ﰲ ﻣﺪﺡ ﻣﻦ ﻦ ﺃﻥ ﻳﺮﺍﺩ ﺑﺬ ﻤﻠﺴﻮ،ﻥ ﺸﻟﺍﻭ ﻥﻮﻜﻳ ﻪﺟﻭﻷﺍ ﻩ ﺍﳊﺪﻳﺚ) : ﱯﻃﺮﻘﻟﺍ ٦٤/ ٣١ : ﺟﻪ ﻱﺭﺎﺨﺒﻟﺍ ﻠﺴﻣﻭ ١٧ ﻦﻨﺳﻭ ﰊﺃ ﻭﺍﺩ ﻪ ﻢﻠﺴﻣ ٩٦٧ /٥: ﺡ ﻟﺍ ﻮ ﻗﻒ ٢٠/١: ﺢ ﻱﺭﺎﺨﺒﻟﺍ :٤/٧٤ ﰲ ﻁﺮﻔﻳﻭ ﻦﻜﳝﻭ  ﺴﳌﺍﻭ ﺪﻗﻭ ﻳ ﻋﻠﻰ ﻫﺬ )١( ﲑﺴﻔﺗ )٢( ﻪﺟﺮﺧﺃ ٥/٦٩ )٣( ﻪﺟﺮﺧﺃ )٤( ﺡﺎﻀﻳﺇ )٥( ﺢﻴﺤﺻ   ﺮﻌ(، )٥( ﻮﻬﻓ ﻛﺘﺎﺏ ﺍﻟﺸﻌﺮ : ﺍﻹﻧﺴﺎﻥ ﺍﻟﺸﻌ ﺢﻴﺤﺻﻭ ﻢﻠﺴﻣ : ( . ﺍ ﻟﻐﺎﻟﺐ ﻋﻠﻰ ﺏﺍﺩ ﻭ ٤٧/٤: /١٤٠ . . ١ ﷲ ﻭﺍﻟﻌﻠﻢ ﻭﺍﻟﻘﺮﺁﻥ ﻩ ﺃﻥ ﻳﻜﻮﻥ ﺍ ﻒ :١/ ١٠٢ . ﺨﺎﺭﻱ: ﻛﺘﺎﺏ ﺍﻵ ﺔﻔﲢ ﺍ ﻷ ﻱﺫﻮﺣ ٨: / ﺍﳊﺪﻳﺚ ﺍﻟﺴﺎﺑﻖ . ﱯﻃﺮﻘﻟﺍ:١٣ /٥٣ ﻳﺼﺪﻩ ﻋﻦ ﺫﻛﺮ ﺍﷲ )ﺑﺎﺏ ﻣﺎ ﻳﻜﺮ ٤١ ﻭﺇﻳﻀﺎﺡ ﺍﳌﻮﻗﻒ ﻠﻢ :ﺻﺤﻴﺢ ﺍﻟﺒﺨ ﺩﻭ :٤/ ٣٠٤ ﻭﲢ ٧١ ﻭﺗﻜﻤﻠﺘﻪ ﺑﻨﺤﻮ ١-١٠٣ ﺍ ﲑﺴﻔﺗﻭ ٧ ﻪﺘﻠﻤﻜﺗﻭ ﺣﱴ ) ﻩ ﺍﳊﺪﻳﺚ) : ﱯﻃﺮﻘﻟﺍ ٦٤/ ٣١ : ﺟﻪ ﻱﺭﺎﺨﺒﻟﺍ ﻠﺴﻣﻭ ١٧ ﻦﻨﺳﻭ ﰊﺃ ﻭﺍﺩ ﻪ ﻢﻠﺴﻣ ٩٦٧ /٥: ﺡ ﻟﺍ ﻮ ﻗﻒ ٢٠/١: ﺢ ﻱﺭﺎﺨﺒﻟﺍ :٤/٧٤ ﻋﻠﻰ ﻫﺬ )١( ﲑﺴﻔﺗ )٢( ﻪﺟﺮﺧﺃ ٥/٦٩ )٣( ﻪﺟﺮﺧﺃ )٤( ﺡﺎﻀﻳﺇ )٥( ﺢﻴﺤﺻ )ﺚﺑﺎ) : ﻛﺘﺎﺏ ﺍﻟﺸﻌﺮ : ﺢﻴﺤﺻﻭ ﻢﻠﺴﻣ : ( . ﺏﺍﺩ ﻭ ٤٧/٤: /١٤٠ . . ١ ﷲ ﻭﺍﻟﻌﻠﻢ ﻭﺍﻟﻘﺮﺁﻥ ﻒ :١/ ١٠٢ . ﺨﺎﺭﻱ: ﻛﺘﺎﺏ ﺍﻵ ﺔﻔﲢ ﺍ ﻷ ﻱﺫﻮﺣ ٨: / ﺍﳊﺪﻳﺚ ﺍﻟﺴﺎﺑﻖ . ﱯﻃﺮﻘﻟﺍ:١٣ /٥٣ ﻳﺼﺪﻩ ﻋﻦ ﺫﻛﺮ ﺍﷲ ٤١ ﻭﺇﻳﻀﺎﺡ ﺍﳌﻮﻗﻒ ﻠﻢ :ﺻﺤﻴﺢ ﺍﻟﺒﺨ ﺩﻭ :٤/ ٣٠٤ ﻭﲢ ٧١ ﻭﺗﻜﻤﻠﺘﻪ ﺑﻨﺤﻮ ١-١٠٣ ﺍ ﲑﺴﻔﺗﻭ ٧ ﻪﺘﻠﻤﻜﺗﻭ ﺣﱴ ) ﱯﻃﺮﻘﻟﺍ ٦٤/ ٣١ : ﺟﻪ ﻱﺭﺎﺨﺒﻟﺍ ﻠﺴﻣﻭ ١٧ ﻦﻨﺳﻭ ﰊﺃ ﻭﺍﺩ ﻪ ﻢﻠﺴﻣ ٩٦٧ /٥: ﺡ ﻟﺍ ﻮ ﻗﻒ ٢٠/١: ﺢ ﻱﺭﺎﺨﺒﻟﺍ :٤/٧٤ )١( ﲑﺴﻔﺗ )٢( ﻪﺟﺮﺧﺃ ٥/٦٩ )٣( ﻪﺟﺮﺧﺃ )٤( ﺡﺎﻀﻳﺇ )٥( ﺢﻴﺤﺻ  òÌÜÛa @ À @ ÐnÛa pb @ Þìy @ òîvy @a @@@@@@@ bèj’ ﻝﻮ ﷲﺍ ﺱﺪﻘﻟﺍ«.)٢( ﺫﺍﺑﺎ ﻋﻦ ﻮﺳﺭ ﺍ ﺃﻳﺪﻩ ﺑﺮﻭﺡ ﺍﳌﺸﺮﻛﲔ ﻭﺫ ﺃ ﺍﷲ، ﺍﻟﻠﻬﻢ ﺭﺍﺩﺍ ﺑﻪ ﻋﻠﻰ ﻋﻦ ﺭﺳﻮﻝ ﺭ ﺸﺪ ﺍﻟﺸﻌﺮ ﺴﺎﻥ :ﺃﺟﺐ ﺴﺎﻥ ﻭﻫﻮ ﻳﻨﺸ ﲔ : » ﺎﻳ ﺴﺣ ﺴﳊ ﲔﻤ . (٤)ﺴﻨﻪ ﺩ ﺍﳋﺎﻣﺲ ﲑﻩ ﺑﺎﻟﺘﻮﻗﻒ ﺤﺎﺑﺔ ﺃﻭ ﻣﻦ ﺮﻈﻨﻳﻭ : ﺳﻨﻦ ﺃﰊ ﻱ ﺃﻥ ﺍﻟﺸﺎﻋﺮ ﻮﻫ ﺭﻗﻢ )٥٢٢ (.  òÌÜÛa @ À @ ÐnÛa pb @ Þìy @ òîvy @a @@@@@@@ bèj’ ﺍﻟﻌﺪﺩ ﻣﻮﻥ ﰲ ﺗﻔﺴﲑ ﱰﻳﻞ ﻣﻦ ﺍﻟﺼﺤ ﱯﻃﺮ:١٣ / ١٥٣ ﺐ ﺻﺤﻴﺢ، ﻭﺭﻭﻱ ﻢﻠﺴﻣ :٥/ ١٧٦٧ ﺭ ﺔ ﺔﻋﺎﲨ ﻳﻣﺰﻠ ﻤﻦ ﺷﺎﻫﺪ ﺍﻟﺘﱰ ﻋﺒﻴﺪ، ﻭﺗﻔﺴﲑ ﺍﻟﻘﺮ ﺣﺪﻳﺚ ﺣﺴﻦ ﻏﺮﻳﺐ ﺮﻈﻨﻳﻭ : ﻣ ﺢﻴﺤﺻ ﺣﻮﻟﻴﺔ ﲟﻘﺘﻀﻰ ﺍﻟﻠﻐﺔ  ، ﻭﺃﻤﻋ ﻋ ﺍﻟﺘﻮﺟﻴﻪ ﻋﻦ ﺃﰊ :٨/ ١٣٩ ﺣ ﻝﺎﻗﻭ ﱯﻃﺮﻘﻟ :٣/ ١٥٣ ، •ﺣ ﻔﺴﲑ ﺍﻟﻘﺮﺁﻥ ﻒ ﻋﻦ ﺍﻟﻨﱯ -١٠٣ ﻭﻧﻘﻞ ﻫﺬﺍ . ٧ : ﲢﻔﺔ ﺍﻷﺣﻮﺫﻱ ١-١٢٨ ﻭﺗﻔﺴﲑ ﺍ ﻭﳑﻦ ﻣﻨﻊ ﺗﻔ ﻉﻮﻤﺴ، ﺑﺘﻮﻗﻴﻒ ﺍﻟﻮﻗﻒ :١/ ١٠٢- ٤/٣٠٤ . ﺢ ﻱﺭﺎﺨﺒﻟﺍ ٤/٤: ﻱﺬﻣﺮﺘﻟ ﻲﺋﺎﺴﻨﻟﺍﻭ: ﺑﻦ ﻣﺎﻟﻚ . ﻱﱪﻄﻟﺍ :١٩ / ١٢٧ ﺎﺜﻟﺎﺛ: ﻋﻠﻰ ﺍﳌﺴ )١( ﺡﺎﻀﻳﺇ ﺩﻭﺍﺩ :٤ )٢( ﺢﻴﺤﺻ )٣( ﺍﻟﺭﻭﺍﻩ ﻛﻌﺐ )٤( ﲑﺴﻔﺗ ﺮﻈﻨﻳﻭ : ﺳﻨﻦ ﺃﰊ ﻱ ﺃﻥ ﺍﻟﺸﺎﻋﺮ ﻮﻫ ﺭﻗﻢ )٥٢٢ (. ﱯﻃﺮ:١٣ / ١٥٣ ﺐ ﺻﺤﻴﺢ، ﻭﺭﻭﻱ ﻢﻠﺴﻣ :٥/ ١٧٦٧ ﺭ ﻋﺒﻴﺪ، ﻭﺗﻔﺴﲑ ﺍﻟﻘﺮ ﺣﺪﻳﺚ ﺣﺴﻦ ﻏﺮﻳﺐ ﺮﻈﻨﻳﻭ : ﻣ ﺢﻴﺤﺻ ﻋ ﺍﻟﺘﻮﺟﻴﻪ ﻋﻦ ﺃﰊ :٨/ ١٣٩ ﺣ ﻝﺎﻗﻭ ﱯﻃﺮﻘﻟ :٣/ ١٥٣ ، -١٠٣ ﻭﻧﻘﻞ ﻫﺬﺍ . ٧ : ﲢﻔﺔ ﺍﻷﺣﻮﺫﻱ ١-١٢٨ ﻭﺗﻔﺴﲑ ﺍ ﺍﻟﻮﻗﻒ :١/ ١٠٢- ٤/٣٠٤ . ﺢ ﻱﺭﺎﺨﺒﻟﺍ ٤/٤: ﻱﺬﻣﺮﺘﻟ ﻲﺋﺎﺴﻨﻟﺍﻭ: ﺑﻦ ﻣﺎﻟﻚ . ﻱﱪﻄﻟﺍ :١٩ / ١٢٧ )١( ﺡﺎﻀﻳﺇ ﺩﻭﺍﺩ :٤ )٢( ﺢﻴﺤﺻ )٣( ﺍﻟﺭﻭﺍﻩ ﻛﻌﺐ )٤( ﲑﺴﻔﺗ  òÌÜÛa @ À @ ÐnÛa pb @ Þìy @ òîvy @a @@@@@ bèj’ ﻔﺴﲑ ﺑﺎﻟﺮﺃﻱ ﻞﺟﻭ ﻪﻳﺃﺮﺑ ﻋﻦ ﻣﺜﻠﻪ ﻋﻦ ﺿﻲ ﺍﷲ ﻋﻨﻪ ٨٥ ، ﻓﻘﺎﻝ : ﻢ«.)٣( ﺎﻤﻛ ﻋﺒﺲ :١٣ ، ﺇﺫ ﻊ ﺇﱃ ﻪﺴﻔﻧ ﺱ ﻭﻋﻤﺮ ﺑﻦ ﻋﻦ ﺍﻟﺘﻔﺴﲑ ﻔﺴﺮ ﻟﻪ ﺇﱃ ﻴﻪ، ﻻﺣﺘﻴﺎﺝ ﻣﻦ ﻗﺒﻴﻞ ﺍﻟﺘﻔ ﻛﺘﺎﺏ ﺍﷲ ﻋﺰ ﻋ ﺃﻭ ﺍﻻﻣﺘﻨﺎﻉ ﻦ ﺃﰊ ﺑﻜﺮ ﺭﺿ Ï n ﺀﺎﺴﻨﻟﺍ : ﷲ ﻣﺎ ﱂ ﺃﻋﻠﻢ Á  n ﺒﻋ ﻷﺏ؟ ﰒ ﺭﺟﻊ ﺸﺔ ﻭﺍﺑﻦ ﻋﺒﺎﺱ ﺩﺭ ﺓ ﰲ ﺍﻟﻨﻬﻲ ﻻﺣﺘﻴﺎﺝ ﺍﳌﻔ ﻰ ﺃﺣﺪ ﻣﻌﻨﻴﻴﻪ ﺍﻟﻠﻐﻮﻱ ﻫﻮ ﻣﻦ ﻛ ﻗﺎﻝ ﰲ ﺃ ﺩ ﺍﻟﻨﻬﻲ ﻋﻨﻪ ﻣﺎ ﺭﻭﻱ ﻋﻦ Ë Ì Í Î Ï ﰲ ﻛﺘﺎﺏ ﺍﷲ ﻮﻟﻪ ﺗﻌﺎﱃ: o Á ﻓﻨﺎﻫﺎ، ﻓﻤﺎ ﺍﻷ ﻭﻱ ﻋﻦ ﻋﺎﺋﺸ ﻭﺍﻵﺛﺎﺭ ﺍﻟﻮﺍﺭ ﻔﺴﲑ ﺍﻟﻠﻔﻆ ﻞ ﺍﻟﻠﻔﻆ ﻋﻠﻰ ﻛﺎﻥ ﺍﻟﺘﻔﺴﲑ ﻪﻟ  : »ﻣ ﺍﺬ .  òÌÜÛa @ À @ ÐnÛa pb @ Þìy @ òîvy @a @@@@@@@ bèj’ ﺎﻤﻛﻭ ﺩﺭ ﺫﻟﻚ ﻣﻦﻒ، : ﱃo Ê Ë ﻘﻠﲏ ﺇﻥ ﻗﻠﺖ ﲑ)ﺃﺑ ( ﺎ ﻣﻦ ﻗﻮ ﻛﻬﺔ ﻗﺪ ﻋﺮﻓ ٤( ﺍﺬﻜﻫﻭ ﻭﺭ ﻩ ﺍﻷﺣﺎﺩﻳﺚ ﲑ ﺗﻔ: ﺎﺃﺣﺪﳘ ﺎﻤﻬﻴﻧﺎﺛﻭ :ﻞﲪ ﲔ.)١( ﺎﳌﻭ ﻛ ﻨﻬﻲ ﻋﻨﻪ ﻟﻮﻘﺑ ،)٢( ﻮﳓﻭ ﺬﻫ ﺤﺎﺑﺔ ﻭﺍﻟﺴﻠﻒ ﻣﻦ ﻗﻮﻟﻪ ﺗﻌﺎ ﺃﻱ ﺃﺭﺿﻲ ﻘﺗ ﻋﻪ ﻋﻦ ﺗﻔﺴﲑ ﻝ :ﻫﺬﻩ ﺍﻟﻔﺎﻛ ﻒ ﻳﺎ ﻋﻤﺮ.)٤ )٥( ﺍﻮﻟﱰﻓ ﺬﻫ ﻦﻣ ﲑﺴﻔﺘﻟﺍ ﺎﻥ ﺍﻟﻌﺮﺏ، ﻭ ﻬﻢ ﻣﻦ ﺍﻟﺘﺎﺑﻌﲔ ﻮﻉ ﻟﻮﺭﻭﺩ ﺍﻟﻨﻬ ﻓﻘﺪ ﺃﺧﻄﺄ « ﻦ ﻛﺒﺎﺭ ﺍﻟﺼﺤ ﻦﻋ ) ﺎﺘﻴﻘﻣ ( ﲰﺎﺀ ﺗﻈﻠﲏ، ﻭﺃ ﻦ ﻋﻤﺮ ﺍﻣﺘﻨﺎﻋ ﻠﻰ ﺍﳌﻨﱪ ﻓﻘﺎﻝ ﻥ ﻫﺬﺍ ﺍﻟﺘﻜﻠﻒ ﺰﻳﺰ ﻭﻏﲑﻫﻢ.) ﻋﻠﻰ ﻗﺴﻤﲔ ﰲ ﻣﻌﺮﻓﺔ ﻟﺴﺎ ﺃﺧﺬ ﻋﻨﻬ ﻓﻬﻮ ﳑﻨﻮ ﺏﺎﺻﺄﻓ ﲨﺎﻋﺔ ﻣﻦ ﺃ ﻧﻪ ﺳﺌﻞ »ﻱﺃ ﺎﲰ ﺭﻭﻱ ﻋﻦ ﻗﺮﺃﻫﺎ ﻋﻠ ﻝﺎﻘﻓ ﺇﻥ: ﻋﺒﺪ ﺍﻟﻌﺰ ﻋ ﻱﺃﺮﻟﺎﺑ ﰲ ﺮﺤﺒﺘﻟﺍ ﺩ ﺍﳋﺎﻣﺲ ،ﻦﻨﺳ ﺃﰊ ﺩﺍﻭﺩ : ﺮﺳﻼ ﺑﻠﻔﻆ ﺁﺧﺮ . ﺍﻟﻌﺪﺩ ﰊ ﺣﺰﻡ ﺗﻜﻠﻤﻮﺍ ﻓﻴﻪ ﺩ. ﻜﺔ ﻋﻦ ﺃﰊ ﺑﻜﺮ ﻣﺮ ﻭﻓﻴﻪ ﺳﻬﻴﻞ ﺑﻦ ﺃﰊ ﻆﻔﻠﻟﺍﻭ ﻷﰊ ﺩﺍﻭﺩ ﺭﻭﺍﻩ ﺍﺑﻦ ﺃﰊ ﺔﻜﻴﻠﻣ ﲑﺴ :١٠٣ . ﺣﻮﻟﻴﺔ ﻟﺘﺮﻣﺬﻱ :ﻭ ،ﻏﺮﻳﺐ ﺪﺋﺍﻮﻔﻟﺍ :٢/ ١٦٢ . ٢٢٠٠ (ﻝﺎﻗﻭ ﻭﺭ: ﻣﺔ ﰲ ﺃﺻﻮﻝ ﺍﻟﺘﻔﺴ •ﺣ :٢/ ١٦٤ . ﻭﺍﻟﻨﺴﺎﺋﻲ، ﻗﺎﻝ ﺍﻟﺘ ﻱ:٨/ ٢٧٩ ﻊﲨﻭ ٥/ ٢٩٢ ﺑﺮﻗﻢ )٠ ١/٣٧ -٣٨ ﺪﻘﻣﻭ ١٨ . . ﻥﰲ ﻋﻠﻮﻡ ﺍﻟﻘﺮﺁﻥ ﺑﻮ ﺩﺍﻭﺩ ﻭﺍﻟﺘﺮﻣﺬﻱ ٣ ، ﲢﻔﺔ ﺍﻷﺣﻮﺫﻱ ﻥﺎﳝﻹﺍ : ﻲﻘﻬﻴﺒﻟﺍ : :ﺗﻔﺴﲑ ﺍﻟﻄﱪﻱ : ﺏﺎﺘﻛ ﺍﳌﺒﺎﱐ :٨٤ ﺭ ﻖﺑﺎﺴﻟﺍ ٢٠٢ : . )١( ﻥﺎﻫﱪﻟﺍ )٢( ﺃﺑﺭﻭﺍﻩ ٣/١٩ ﺷﻌﺐ (٣) ﺮﻈﻨﻳﻭ: )٤( ﺔﻣﺪﻘﻣ )٥( ﺭﺪﺼﳌﺍ ،ﻦﻨﺳ ﺃﰊ ﺩﺍﻭﺩ : ﺮﺳﻼ ﺑﻠﻔﻆ ﺁﺧﺮ . ﰊ ﺣﺰﻡ ﺗﻜﻠﻤﻮﺍ ﻓﻴﻪ ﺩ. ﻜﺔ ﻋﻦ ﺃﰊ ﺑﻜﺮ ﻣﺮ ﻭﻓﻴﻪ ﺳﻬﻴﻞ ﺑﻦ ﺃﰊ ﻆﻔﻠﻟﺍﻭ ﻷﰊ ﺩﺍﻭﺩ ﺭﻭﺍﻩ ﺍﺑﻦ ﺃﰊ ﺔﻜﻴﻠﻣ ﲑﺴ :١٠٣ . ﻟﺘﺮﻣﺬﻱ :ﻭ ،ﻏﺮﻳﺐ ﺪﺋﺍﻮﻔﻟﺍ :٢/ ١٦٢ . ٢٢٠٠ (ﻝﺎﻗﻭ ﻭﺭ: ﻣﺔ ﰲ ﺃﺻﻮﻝ ﺍﻟﺘﻔﺴ :٢/ ١٦٤ . ﻭﺍﻟﻨﺴﺎﺋﻲ، ﻗﺎﻝ ﺍﻟﺘ ﻱ:٨/ ٢٧٩ ﻊﲨﻭ ٥/ ٢٩٢ ﺑﺮﻗﻢ )٠ ١/٣٧ -٣٨ ﺪﻘﻣﻭ ١٨ . . ﻥﰲ ﻋﻠﻮﻡ ﺍﻟﻘﺮﺁﻥ ﺑﻮ ﺩﺍﻭﺩ ﻭﺍﻟﺘﺮﻣﺬﻱ ٣ ، ﲢﻔﺔ ﺍﻷﺣﻮﺫﻱ ﻥﺎﳝﻹﺍ : ﻲﻘﻬﻴﺒﻟﺍ : :ﺗﻔﺴﲑ ﺍﻟﻄﱪﻱ : ﺏﺎﺘﻛ ﺍﳌﺒﺎﱐ :٨٤ ﺭ ﻖﺑﺎﺴﻟﺍ ٢٠٢ : . )١( ﻥﺎﻫﱪﻟﺍ )٢( ﺃﺑﺭﻭﺍﻩ ٣/١٩ ﺷﻌﺐ (٣) ﺮﻈﻨﻳﻭ: )٤( ﺔﻣﺪﻘﻣ )٥( ﺭﺪﺼﳌﺍ ،ﻦﻨﺳ ﺃﰊ ﺩﺍﻭﺩ : ﺮﺳﻼ ﺑﻠﻔﻆ ﺁﺧﺮ . ﰊ ﺣﺰﻡ ﺗﻜﻠﻤﻮﺍ ﻓﻴﻪ ﺩ. ﻜﺔ ﻋﻦ ﺃﰊ ﺑﻜﺮ ﻣﺮ ﻭﻓﻴﻪ ﺳﻬﻴﻞ ﺑﻦ ﺃﰊ ﻆﻔﻠﻟﺍﻭ ﻷﰊ ﺩﺍﻭﺩ ﺭﻭﺍﻩ ﺍﺑﻦ ﺃﰊ ﺔﻜﻴﻠﻣ ﲑﺴ :١٠٣ . ﻟﺘﺮﻣﺬﻱ :ﻭ ،ﻏﺮﻳﺐ ﺪﺋﺍﻮﻔﻟﺍ :٢/ ١٦٢ . ٢٢٠٠ (ﻝﺎﻗﻭ ﻭﺭ: ﻣﺔ ﰲ ﺃﺻﻮﻝ ﺍﻟﺘﻔﺴ :٢/ ١٦٤ . ﻭﺍﻟﻨﺴﺎﺋﻲ، ﻗﺎﻝ ﺍﻟﺘ ﻱ:٨/ ٢٧٩ ﻊﲨﻭ ٥/ ٢٩٢ ﺑﺮﻗﻢ )٠ ١/٣٧ -٣٨ ﺪﻘﻣﻭ ١٨ . .  òÌÜÛa @ À @ ÐnÛa pb @ Þìy @ òîvy @a @@@@@@@ bèj’ ﻥﰲ ﻋﻠﻮﻡ ﺍﻟﻘﺮﺁﻥ ﺑﻮ ﺩﺍﻭﺩ ﻭﺍﻟﺘﺮﻣﺬﻱ ٣ ، ﲢﻔﺔ ﺍﻷﺣﻮﺫﻱ ﻥﺎﳝﻹﺍ : ﻲﻘﻬﻴﺒﻟﺍ : :ﺗﻔﺴﲑ ﺍﻟﻄﱪﻱ : ﺏﺎﺘﻛ ﺍﳌﺒﺎﱐ :٨٤ ﺭ ﻖﺑﺎﺴﻟﺍ ٢٠٢ : . )١( ﻥﺎﻫﱪﻟﺍ )٢( ﺃﺑﺭﻭﺍﻩ ٣/١٩ ﺷﻌﺐ (٣) ﺮﻈﻨﻳﻭ: )٤( ﺔﻣﺪﻘﻣ )٥( ﺭﺪﺼﳌﺍ ﻋﻪ.) (١ ﻋ ﻥﻭ.)٢( ﺩ ﺍﳋﺎﻣﺲ ﺍﻟﻌﺪﺩ ١٦ﻭ١٦٨ . ﺮﻈ :ﻥﺎﻫﱪﻟﺍ :٢/٢ ﺣﻮﻟﻴﺔ ﻥﻭ:١/ ٢٥٧ ، ﻭﻳﻨﻈ •ﺣ :٢/ ١٦٨ . ﻭﺍﻟﺘﻔﺴﲑ ﻭﺍﳌﻔﺴﺮ ٢ . . ﻥﰲ ﻋﻠﻮﻡ ﺍﻟﻘﺮﺁﻥ ﻁﺎﺒﻨﺘﺳﻻﺍ ٢٤١ : ﻦﺑﺍ ﻋﻄﻴﺔ ٣٦٢ : ﱯﻃﺮﻘﻟﺍ ٣٣/١: )١( ﻥﺎﻫﱪﻟﺍ )٢( ﻝﻮﺻﺃ )٣( ﺔﻣﺪﻘﻣ )٤( ﲑﺴﻔﺗ  òÌÜÛa @ À @ ÐnÛa pb @ Þìy @ òîvy @a @@@@@ bèj’ ﺩ ﺍﳋﺎﻣﺲ ﺍﻟﻌﺪﺩ ﺴﲑ ﺑﺎﻟﻈﻦ .( ﺣﻮﻟﻴﺔ ﺼﻞ ﰲ )ﺗﺮﻙ ﺍﻟﺘﻔﺴ •ﺣ ﻢﻗﺮﺑ ) ٢١٩٩ ﻓﺼ ( ٢/ ١٦٢ . ﻥﺎﳝﻹﺍ ٢٩٢ /٥: ﰲﻥ ﻋﻠﻮﻡ ﺍﻟﻘﺮﺁﻥ : )ﺷﻌﺐ (١ )٢( ﻥﺎﻫﱪﻟﺍ ﺩ ﺍﳋﺎﻣﺲ ﺍﻟﻌﺪﺩ ﺴﲑ ﺑﺎﻟﻈﻦ .( ﺣﻮﻟﻴﺔ ﺼﻞ ﰲ )ﺗﺮﻙ ﺍﻟﺘﻔﺴ •ﺣ ﻢﻗﺮﺑ ) ٢١٩٩ ﻓﺼ ( ٢/ ١٦٢ . ﻥﺎﳝﻹﺍ ٢٩٢ /٥: ﰲﻥ ﻋﻠﻮﻡ ﺍﻟﻘﺮﺁﻥ : )ﺷﻌﺐ (١ )٢( ﻥﺎﻫﱪﻟﺍ  òÌÜÛa @ À @ ÐnÛa pb @ Þìy @ òîvy @a @@@@@@@ bèj’ ﺩ ﺍﳋﺎﻣﺲ ﺣﺪ ﻣﻦ ﺃﻫﻞ ﻄﻒ ﻣﻨﻪ ﻣﺎ ﻻ ﳜﻠﻮ ﺇﻣﺎ ﻃﻞ ﻗﻄﻌﺎ ﺃﻥ ﺎﻫﺪﺣﺃ ﺃﻧﻪ: ﻭﺫﻟﻚ ﻻ، ﻦﻣ ﻢﻬﺴﻔﻧﺃ ﺔ ﻭﺍﳌﻔﺴﺮﻳﻦ ﳉﻤﻊ ﺑﻴﻨﻬ،ﺎ ﻣﺎ ﺍﻟﻨﻬﻲ ﻓﺈﻧﻪ ﻓﻴﺘﺄﻭﻟﻪ ﻋﻠﻰ ﻻﺡ ﻟﻪ ﻣﻦ ﺭﺓ ﻳﻜﻮﻥ ﻣﻊ ،ﺍﻓﻖ ﻏﺮﺿﻪ ﺍﻟﻌﺪﺩ ﻻ ﳚﻬﻠﻪ ﺃﺣ ﻡ ﻧﻔﻴﺲ ﻧﻘﺘﻄ ﻥﺁﺮﻘ ﻱﺃﺮﻟﺎﺑ ﺮ ﺁﺧﺮ، ﻭﺑﺎﻃ ﻤﻌﻪ ،ﻟﻮﺟﻮﻩ ﺪﻨﺴﻣﻭ ،ﻪﻴﻟﺇ ﺍﺑﻦ ﻣﺴﻌﻮﺩ ﺃﻥ ﺍﻟﺼﺤﺎﺑﺔ ﻔﺔ ﻻ ﳝﻜﻦ ﺍﳉ ﻇﻬﺮ ﻟﻪ ﻭﺃﻣ . ﻪﻌﺒﻃ ،ﻩﺍﻮﻫﻭ ﻚ ﺍﳍﻮﻯ ﺎﳌ ﻪﻤﺼﺧ. ﺭﺎﺗﻭ ﺟﻪ ﺍﻟﺬﻱ ﻳﻮ ﺐ ﻫﺬﺍ ﺍﻟﻘﻮﻝ . ﺀ:١/ ٢٨٨ -١ ﺀ : . ٧٣/١ )١( ﺀﺎﻴﺣﻹﺍ )٢( ﺀﺎﻴﺣﻹﺍ  òÌÜÛa @ À @ ÐnÛa pb @ Þìy @ òîvy @a @@@@@@@ bèj’ ،ﺎ ﻓﺈﻥ ﺫﻟﻚ ﳊﺪﻳﺚ ﻡﻛﻼ ﻋﻦ ﺗﻔﺴﲑ ﻘﻟﺍ ﺍﳌﺮﺍﺩ ﺑﻪ ﺮﻣﺃ ﺇﻻ ﲟﺎ ﻳﺴﻤ ﻝ ﺍﷲ ﺍﺑﻦ ﻋﺒﺎﺱ ﻭﺍ  ، ﱐﺎﺜﻟﺍﻭ : ﻞﻳﻭﺎﻗﺃ ﻔﻠﺘﳐ ﰲ ﺍﳌﻌﲎ ﺎﲟ ﻴﻪ ﻣﻴﻞ ﻣﻦ ﻃ ﺿﻪ، ﻭﻟﻮﻻ ﺫﻟﻚ ﺧ ﺗﻠﺒﻴﺴﺎ ﻋﻠﻰ ﻬﻤﻪ ﺇﱃ ﺍﻟﻮﺟ ﱂ ﻳﺸﺮ ﺇﱃ ﺻﺎﺣﺐ ﺣﻮﻟﻴﺔ ﻭﻥ ﻣﺎ ﺎﻫﺍﻮﺳ ﻬﻲ ﺍﻟﻮﺍﺭﺩ ﳊﺎﺑ : ﻋ ﺇﻥ ﺍﻟﻨﻬﻲ ﻰ ﺍﻟﻨﻘﻞ، ﺃﻭ ﺪ ﰲ ﺍﻟﻘﺮﺁﻥ ﻋﺎ ﻣﻦ ﺭﺳﻮ ﺍ ﻣﺎ ﻣﺎ ﻳﻘﻮﻟﻪ ﺴﻤﻌﻮﻩ ﻣﻨﻪ ﻓﻘﺎﻟﻮﺍ ﻓﻴﻬﺎ ﻞ ﻣﻔﺴﺮ ﻗﺎﻝ ﻲﺀ ﺭﺃﻱ، ﻭﺇﻟﻴ ﺗﺼﺤﻴﺢ ﻏﺮﺿ ﻋﻠﻰ ﻭﺟﻮﻩ: ﺎﺤ ﻪﺘﻋﺪﺒﻟ ﺗﻭ ﳏﺘﻤﻠﺔ ﳝﻴﻞ ﻬﻓ ﻥﺁﺮﻘ:٢/ ١٧٠ ﱂﻭ •ﺣ ﺎ ﺍﳋﺎﺻﺔ ﺩﻭ ﰲ ﺗﻮﺟﻴﻪ ﺍﻟﻨﻬ ﺚ ﺑﺎﺧﺘﺼﺎﺭ ﻻﻗﺘﺼﺎﺭ ﻋﻠﻰ ﻻ ﻳﺘﻜﻠﻢ ﺃﺣﺪ ﻟﺫ ﻚ ﻣﺴﻤﻮﻋ ﺾ ﺍﻟﻘﺮﺁﻥ، ﺃﻣ ﻢ ﱂ ﻳﺴ ﻷ ﻌﺾ ﺍﻵﻳﺎﺕ، ﻓﺜﺒﺖ ﺃﻥ ﻛﻞ ﲔ: ﻥ ﻟﻪ ﰲ ﺍﻟﺸﻲ ﺗﺼ ﺘﺞ ﺑﻪ ﻋﻠﻰ ﻋ ﻭﻫﺬﺍ ﳚﺮﻱ ﻟﻌﻠﻢ، ﺗﺼﺤﻴﺤ ﳏ ﻛﺎﻧﺖ ﺍﻵﻳﺔ ﻟﱪﻫﺎﻥ ﰲ ﻋﻠﻮﻡ ﺍﻟﻘ ﺎﻓﺎﺕ ﺑﺼﻔﺎ ﻠﺴﺎﻥ.) (١ ﻡﺎﻣ ﰲ ﱄﺍﺰﻐﻟﺍ ﻮﺿﻮﻉ ﺍﻟﺒﺤﺚ ﻥ ﺍﳌﺮﺍﺩ ﺑﻪ ﺍ ﻻ ﳌﺮﺍﺩ ﺑﻪ ﺃﻥ ﺫ ﺃﻥ ﻳﻜﻮﻥ ﺇﻻ ﰲ ﺑﻌﺾ ﺴﲑ ﺑﺎﻟﺮﺃﻱ، ﰲ ﺗﻔﺴﲑ ﺑﻌ ﲨﻴﻌﻬﺎ ﳏﺎﻝ، ﻰ ﺃﺣﺪ ﻭﺟﻬﲔ ﳘﺎ :ﺃﻥ ﻳﻜﻮﻥ ﻪ ﻭﻫﻮﺍﻩ ﻟﻴﺤﺘ ﺫﻟﻚ ﺍﳌﻌﲎ ﻭ ، ﻥﻮﻜﻳ ﻣ ﻊ ﻌﻟﺍ ﻛ ﻭﻟﻜﻦ ﺇﺫﺍ ﻱﱪﻄﻟﺍ :١/٣٣ ﺍﻭ ﻮﺻﻮﳌﺍﻭ ﺍﻟﻌﻠﻢ ﺑﺎﻟﻠ ﺎﻣﻺﻟﻭ ﻳﺘﻌﻠﻖ ﲟﻮ ﺃﻥ ﻳﻜﻮﻥ ﻳﻜﻮﻥ ﺍﳌ ﻁﺮﺘﺸﻳ ﻳﺼﺎﺩﻑ ﻓﻬﻮ ﺗﻔﺴ ﺍﻮﻔﻠﺘﺧﺍ ﲨ ﻉﺎﲰﻭ ﻳﱰﻝ ﻋﻠﻰ ﳘﺪﺣﺃ ﻭﻓﻖ ﺭﺃﻳﻪ ﺫ ﻥﺁﺮﻘﻟﺍ ﻓﺓﺭﺎﺘ ،ﻞﻬﳉﺍ )١( ﲑﺴﻔﺗ ﷲ  ،ﻪ ¡ ¢ £ ¤ ﻝ: o ¡ ﺩ ﺍﳋﺎﻣﺲ ﻊﲨ ،ﻥﺁﺮﻘﻟﺍ ﺪ ﻻ ﻳﺼﻴﺐ ﻭﻫﻮ ﻻ ﳝﻨﻊ ﻜﺮ ﺭﺿﻲ ﺍﷲ ، ﺃﻭﻝ ﺍﻷﻣﺮ ﻌﻤﺮ ﺃﻳﻀﺎ ﺇﺫ ﻋﻦ ﺗﻔﺴﲑﻫﺎ ﺎﻳﻮﻐ .ﺮﺴﻓﻭ ﻞ ﻣﺎ ﺻﺎﺩﻑ ﲑﻩ ﺃﻧﻪ ﺍﻟﺘﺒﺲ P Q n ﻣﻦ ﺃﺻﺤﺎﺏ ، ﻣﻦ ﻛﻨﺎﻧﺔ ﻣﺎ ﺍﳊﺮﺟﺔ؟ ﺍﻟﻌﺪﺩ ﻝ ﺍﻷﻣﺮ ﻋﻦ ﲨ ﺍﻟﺘﻔﺴﲑ، ﻭﻗﺪ ﺎ ﺳﺌﻞ ﻋﻨﻪ، ﺻﺎﺭ ﺃﺑﻮ ﺑﻜ ﻋﻦ ﺗﻔﺴﲑﻫﺎ ﻛﻤﺎ ﺣﺼﻞ ﻟﻌ ﻋ ﻋﻠﻴﻪ ﻭﺍﻣﺘﻨﻊ ﺃﻧﻪ ﻓﺴﺮﻫﺎ ﻟﻐ ،ﺏﺮﻌﻟﺍ ﻞﺜﻣ ﺍﻟﻄﱪﻱ ﻭﻏﲑ N O P ﻣ ﻛﺎﻥ ﻋﻨﺪﻩ ﺑﻐﻮﱐ ﺭﺟﻼ ﻋﻤﺮ :ﻳﺎ ﻓﱴ ﻦ ﺗﺮﺩﺩﻩ ﺃﻭﻝ ﻰ ﻣﺬﻫﺒﻪ ﰲ ﻪﻤﻠﻋ ﻫﻮ ﲟﺎ ﺏ.)١( ﺍﺬﳍﻭ ﻋ ﻪ ﺍﻻﻣﺘﻨﺎﻉ ﻨﺎ ﻟﺬﻟﻚ .ﻭﻛ ﻱ ﻋ ﻞﻜﺷﺃ ،ﻚ ﻣﻨﻪ ﻊﻣ ﺘﺠﺎ ﻟﻪ ﺑﻜﻼﻡ ﺍ ﺝ ﺍﺑﻦ ﺟﺮﻳﺮ K L M N ﻛ ﺃ ﺑﻌﺾ ﻣﻦ ﻝ ﻋﻤﺮ : »ﺃ ﻋ ﻪ، ﻓﻘﺎﻝ ﻟﻪ ١٠٧ .  òÌÜÛa @ À @ ÐnÛa pb @ Þìy @ òîvy @a @@@@@@@ bèj’ ﺣﻮﻟﻴﺔ ﻷﰊ ﺑﻜﺮ ﻣﻦ ﻣﺎﻣﺎ ﻳﺒﲎ ﻰﻠﻋ ﻨﻊ ﻣﻨﻪ ﻟﻌﺪﻡ ﻫﺎ ﻣﻦ ﺍﻷﺳﺒﺎ ﺃﻥ ﺭﻭﻱ ﻋﻨﻪ ﺎﻫﲑ ﺎﻤﻛ ﺎﻨﻠﺜﻣ o  n ﺬﻟﺍ ﺍﺳﺘﺤﺴﻦ ﺫﻟﻚ ﺎ ﻳﻈﻬﺮ ﻟﻪ ﳏﺘﺠ ، ﻓﻘﺪ ﺃﺧﺮﺝ ﱃ: o J K ﺢ ﺍﻟﺮﺍﺀ، ﻭﻗﺮﺃ ﺍﻟﺮﺍﺀ ، ﻓﻘﺎﻝ ﻗﺎﻝ :ﻓﺄﺗﻮﻩ ﺑﻪ ﲑﺴﻔﺘﻟ :١٠٦ -٧ •ﺣ ﺜﺎﻝ ﻣﺎ ﺍﺗﻔﻖ ﻋﻦ ﺍﻟﻔﺘﻴﺎ . ﻲ ﻻ ﳚﻌﻞ ﺇ ﳝﺘﻨﻊ ﻣﻦ ﺍﻣﺘﻨ ﺑﻪ، ﺃﻭ ﻟﻨﺤﻮﻫ ،ﺔﻟﻼ)٢( ﺪﻌﺑ ﺃ ﻅ ﺃﺧﺮﻯ ﻏﲑ ﻣﻌﲎ ﻟﻔﻈﺔ : ﺍ ﺍﺑﻦ ﻋﺒﺎﺱ ﻇﺎﻔﻟﹰ ﺎ ﺃﺧﺮ ﲟﺎ ﺟ ﲣ ، ﻭﺎ(ﻑﻮ، ﻣﻦ ﻗﻮﻟﻪ ﺗﻌﺎ ﻫﺎ ﻋﻤﺮ ﺑﻔﺘﺢ ﺎﺟﹺ ﺮ (ﺮﺴﻜﺑ ﻗ ،ﻜﻦ ﻣﺪﳉﻴﺎ ﻣﻘﺪﻣﺔ ﰲ ﺃﺻﻮﻝ ﺍﻟ ٢ . ﻣﻨﻪ، ﻣﺜﺎﻟﻪ ﻣﺜ ﻋ ﺮﺩﺩ ﺑﻌﻀﻬﻢ ﺭﻏﺒﺔ ﻣﻨﻪ ﻟﻜﻲ ﺮﺍﺩ .ﻭﺇﻣﺎ ﺃﻥ ﻥ ﻏﲑﻩ ﻋﺎﳌﺎ ﺗﻔﺴﲑ ﺍﻟﻜﻼ ﰲ ﺗﻔﺴﲑ ﺃﻟﻔﺎﻅ ﻦ ﻋﺒﺎﺱ ﻋﻦ ﻭﳌﺎ ﻓﺴﺮﻫﺎ ﻟﻪ ﺿﻲ ﺍﷲ ﻋﻨﻪ ﺃﻟ ﱵﻤﻠﻛ)ﺟﺮﺣ ﲎ )ﺍﳊﺮﺝ ( ١ ، ﻓﻘﺪ ﻗﺮﺃﻫ  )ﹺ ﹰ ﺣﺮ ﺎﻘﻴﺿ ﻩ ﺭﺍﻋﻴﺎ، ﻭﻟﻴﻜ ﻱﱪﻄﻟﺍ :١/٣٩ ﻣﻭ ﻱﱪﻄﻟﺍ ٤٧٢ /١: ﳌﺍ ﻞﻜﺸ ﻭﻣﺜﺎﻝ ﺗﺮ ﻭﺇﻣﺎ ﺍﳊﻖ ﺍﳌﺮ ﺃﻥ ﻳﻜﻮﻥ ﻋﻨﻪ ﺇﱃ ﰲ ﺍﺬﻛﻭ ﺳﺄﻝ ﺍﺑﻦ ﻭ ،ﻪﻳﺃﺮﺑ ﻋﻤﺮ ﺭﺿ ﻛ ﻟﻪ ﻣﻊ ﻋﻠﻴﻪ ﻣﻌﲎ ﻡﺎﻌﻧﻷﺍ : ١٢٥ ﺍﻟﻨﱯ  ﻩﻮﻠﻌﺟﺍﻭ )١( ﲑﺴﻔﺗ )٢( ﲑﺴﻔﺗ ﺎ ﻋ ﻛ ﻠﻋ ﻪﻤﻠﻋ ﻫﻮ ﲟﺎ ﺏ.)١( ﺍﺬﳍﻭ ﻋ ﻪ ﺍﻻﻣﺘﻨﺎﻉ ﻨﺎ ﻟﺬﻟﻚ .ﻭﻛ ﻱ ﻋ ﻞﻜﺷﺃ ﻨﻊ ﻣﻨﻪ ﻟﻌﺪﻡ ﻫﺎ ﻣﻦ ﺍﻷﺳﺒﺎ ﺃﻥ ﺭﻭﻱ ﻋﻨﻪ ﺎﻫﲑ ﺎﻤﻛ ﺎﻨﻠﺜﻣ o  n ﺬﻟﺍ ﳝﺘﻨﻊ ﻣﻦ ﺍﻣﺘﻨ ﺑﻪ، ﺃﻭ ﻟﻨﺤﻮﻫ ،ﺔﻟﻼ)٢( ﺪﻌﺑ ﺃ ﻅ ﺃﺧﺮﻯ ﻏﲑ ﻣﻌﲎ ﻟﻔﻈﺔ : ﻥﺃ ﺎﳌﺎ ﻼﻜﻟ ﻅﺎﻔﻟ ﻦﻋ o  n ﺍ ﺔﻈ:  òÌÜÛa @ À @ ÐnÛa pb @ Þìy @ òîvy @a @@@@@@@ bèj’ ١٥٤ ﺩ ﺍﳋﺎﻣﺲ ﺭﺍﻋﻴﺔ، ﻭﻻ ﻴﻪ ﺷﻲﺀ ﻣﻦ ﻠﻰ ﺍﳌﻨﱪ ﻋﻦ ،ﺦ ﻣﻦ ﻫﺬﻳﻞ ﺍﻟﻌﺮﺏ ﺫﻟﻚ ﻦﹶ ﻔﺴﻟ ﻓﻴﻪ ﺗﻔﺴﲑ ١١-١١١ ، ﻭﰲ ﻮ ﰲ ﻟﺴﺎﻥ ﺍﻟﻌﺮﺏ ﻧﻘﻼ ﻋﻦ ﺻﺤﺎﺡ ﻟ ﻌﺠﻼﻥ ﺍﻟﻨﻬﺪﻱ . ﰲ ﹺﻱ ﻭﺎﻀﻴﺒﻟﺍ ﻩﺍﺰ ﺝ ﺍﻟﻌﺮﻭﺱ : ﺓﺩﺎﻣ ﻑ ﺍﻟﺴﲑ(، ﻳﻨﻈﺮ : ﻲِ ﺴﺎ ﺍﻟﻘ ﺩﺮﺒﺗ . ﻲﺴﻘﻟﺍ ﺐ . ﻥﺎﺴﻟ ﺍﻟﻌﺪﺩ ﻳﺼﻞ ﺇﻟﻴﻬﺎ ، ﻻ ﻳﺼﻞ ﺇﻟﻴ ﺳﺄﻝ ﻭﻫﻮ ﻋﻠ ٤ ، ﻓﻘﺎﻡ ﺷﻴﺦ ﺍ ﻓﻬﻞ ﺗﻌﺮﻑ ﻧﺎﻗﺘﻪﻒ : ﻟﺍ ﺔﻌﺒﻨﻟﺍ ﺩﻮﻋ ﳉﺎﻫﻠﻴﺔ، ﻓﺈﻥ ﺎﳘ . ﲑ ﺍﻟﻘﺮﻃﱯ:٠/ ٠١ ﺍﻟﺒﻴﺖ ﻟﺰﻫﲑ، ﻭﻫﻮ ﻧﻘ ﻋﺰﺍﻩ ﻟﺬﻱ ﺍﻟﺮﻣﺔ ﻭ ﻋﺒﺪ ﺍﻟﻠﻴﺚ ﻦﺑﻟﺍ ﺎﻝ ﺍﻟﺰﺑﻴﺪﻱ : »ﺰﻋﻭ ﻱﹺ ﻫﺬﺍ ﺍﻟﺮﻭ «.  òÌÜÛa @ À @ ÐnÛa pb @ Þìy @ òîvy @a @@@@@@@ bèj’ ﻥﺎﺴﻟ ﺍﻟﻌﺪﺩ ﺳﺄﻝ ﻭﻫﻮ ﻋﻠ ٤ ، ﻓﻘﺎﻡ ﺷﻴﺦ ﺍ ﻓﻬﻞ ﺗﻌﺮﻑ ﻧﺎﻗﺘﻪﻒ : ﻟﺍ ﺔﻌﺒﻨﻟﺍ ﺩﻮﻋ ﳉﺎﻫﻠﻴﺔ، ﻓﺈﻥ ﺎﳘ . ﲑ ﺍﻟﻘﺮﻃﱯ:٠/ ٠١ ﺍﻟﺒﻴﺖ ﻟﺰﻫﲑ، ﻭﻫﻮ ﻧﻘ ﻋﺰﺍﻩ ﻟﺬﻱ ﺍﻟﺮﻣﺔ ﻭ ﻋﺒﺪ ﺍﻟﻠﻴﺚ ﻦﺑﻟﺍ ﺎﻝ ﺍﻟﺰﺑﻴﺪﻱ : »ﺰﻋﻭ ﻱﹺ ﻫﺬﺍ ﺍﻟﺮﻭ «. ﺝﺎﺗ ﺃﻳﻀﺎ ﺑﻠﻔﻆ ﲣﻮﻑ ) ﺗ ﺍﳊﺪﻳﺪﺓ ﺍﻟﱵﻦ ﺐﹸ ﺧﺸ ﺓﺪﻳﺪﹶ ﻫﺬﻩ ﺍﳊ ﺍﷲ ﻋﻨﻪ » : | n ﻞﺤﻨﻟﺍ ٧: ﺎﻝ ﻟﻪ ﻋﻤﺮ: ﺍﳍﺬﱄ ﻳﺼﻒ ﻑﻮﺨﻛﻤﺎ ﺗ ﻧﻜﻢ ﺷﻌﺮ ﺍﳉ ﻜﻲ ﻭﺍﻟﺜﻌﻠﱯ ﻭﻏﲑﳘ ﻑ:٢/ ٤١١ ﲑﺴﻔﺗﻭ ﻭﻧﺴﺐ ﺍﻟﺰﳐﺸﺮﻱ ﻋ ﻭﰲ ﻣﻮﺿﻊ ﻣﻨﻪ ﺎ، ﻭﻫﻮ ﻟﻌﺒﺪ ﺍﷲ ﻭﺃ ﻲﻟﺎﻤﱡ ﹴ ﺍﻟﺜ ﻢﺣﺍﺰﻣ .ﻗ ﺎ ﹰ ﻋﻠﻰ ﹴ ﻟﻪ ﻗﺼﻴﺪﺓ ﻞ ﻦ(. ﻭﺭﻭﻱ ﺍﻟﺒﻴﺖ ﺓ)ﺧﻮﻑ (. ﻭﻦﹶ ﻔﺴﻟﺍ ﻫ ﹸ ﻞﻛﹾ ﻛﻤﺎ ﺗﺄﱠﺺ ﻘ ﺣﻮﻟﻴﺔ ﻥ ﻋﻤﺮ ﺭﺿﻲ y z { | ﺍﻟﺘﻨﻘﺺ، ﻓﻘﺎ ﺮﻧﺎ ﺃﺑﻮ ﻛﺒﲑ ﹰ ﺍﺩﹺ ﺮﹶ ﹰ ﻗ ﺎﻜ ...ﻛ ﻋﻠﻴﻜﻢ ﺑﺪﻳﻮ ﺍ ﻜﻧ ٨١ ﻭﻧﻘﻠﻪ ﻋﻦ ﻣﻜ ﺔﻴ:٢٧١ ، ﺍﻟﻜﺸﺎﻑ ﻛﺘﺎﺏ ﺍﻟﺘﻔﺴﲑ . ﻭ ﻭ ،ﻫﺮﻱ ﻭﺍﻟﺰﺟﺎﺝ ﻩ ﳍﻤﺎ :ﺎﻤﹶﻬ ﻭﻟﻴﺲ ﻟ ﺮﻟﺍ ﻣ ﹺ ﻦﻪ ﻻﺑﹶﻧ ﺃ ﺔﻳﹺ ﻭﺍ ﹴ ﻞﻳﹶ ﺬﹺ ﻫ ﺮﻌ ﺩﻳﻮﺍﻥ ﺷ ﻣﺶ، ﻣﺎﺩﺓ ) ﻦﻔﺳ ﺔﻏ:١/ ١٢٦ ، ﻣﺎﺩﺓ ﺎ ﺍﻟﻘﺴﻲ . ﻱﹶ ﺃ: ﱠ ﻘﻨﺗ •ﺣ ﻦ ﺍﳌﺴﻴﺐ ﺃﻥ ﻮﻟﻪ ﺗﻌﺎﱃ: o ﺨﻮﻑ ﻋﻨﺪﻧﺎ: ﻌﻢ، ﻗﺎﻝ ﺷﺎﻋ ﺮ ﻜﹸ ﻣﻨﻬﺎ ﺗﺎﻣ ﻞﺣ ﻋ ﻳﻬﺎ ﺍﻟﻨﺎﺱ ﻢﻬﻣﻼ«. )٢( ﺗﻔﺴﲑ ﺍﻟﻘﺮﻃﱯ:/٧ ١ ، ﻣﻘﺪﻣﺔ ﺍﺑﻦ ﻋﻄﻴ ﻱﺭﺎﺨﺒﻟﺍ : ٣/٩٩ ﺒﻞ ﻧﻘﻠﻪ ﻋﻦ ﺍﻷﺯﻫ ﻲ ﺑﻌﺪ ﺃﻥ ﻧﻘﻞ ﻋﺰﻩ ﺎﺩﻤ ﺣﺔﻤﺟﺮ ﰲ ﺗ ﰲ ﺪﹺ ﺟﹶ ﻭﱂ ﺃﻲﹶﻟ ﺬ ﺏ :/ ٣١ ٢٠٩ ﺎﻫ ﺮﻱ :ﺃﺳﺎﺱ ﺍﻟﺒﻼﻏ ﺎﺮ ﻳﺼﻨﻊ ﻣﻦ ﻋﻴﺪﺍ ﺳ ﻦﻋ ﻌﻴﺪ ﺑﻦ ﲣﻮﻑ (ﻣﻦ ﻗﻮ ﺬﻩ ﻟﻐﺘﻨﺎ، ﺍﻟﺘﺨ ﺭﻫﺎ؟، ﻗﺎﻝ:ﻧﻌ ﺣﺮﻟﺍ ﻑﻮﺨﺗ ﺮﻤﻋ ﻳﺎ ﺃﻳ : ﻭﻣﻌﺎﱐ ﻛﻼ، ﻱﱪﻄﻟﺍ :٨/٢٨ ﺗﻭ ﻱﱪﻄﻟﺍ :١٤ / ١١٣ ﻚ ﻳﻨﻈﺮ :ﺻﺤﻴﺢ )ﺧﻮﻑ ( ﻻﺑﻦ ﻣﻘﺒ ،ﻱﺮ ﻲﹺ ﺎﻏﺎﻧﻗﺎﻝ ﺍﻟﺼ ﻲﹺ ﻧﺎﹶ ﻏَ ﻷﺍ ﺐﺣﺎﺻ ﺬﹸ ﹴ ﺍﳍ ﲑﹺ ﺒﹶ ﹶﰊ ﻛ ﹺﱃ ﺃ ﻩ ﺇ ﻑ .(ﻭﻟﺴﺎﻥ ﺍﻟﻌﺮﺏ ﺔﻏﻼﺒﻟﺍ : ﺮﺸﳐﺰﻟﺍ : ﻧﻮﻉ ﻣﻦ ﺍﻟﺸﺠﺮ . ٩٩ / ٩ : ﺀﺎﺟﻭ ﻣﻌﲎ )ﲣ ﻝﺎﻘﻓ :ﺬﻫ ﰲ ﺃﺷﻌﺎﺭ ﻝﺎﻘﻓ ،ﻢﻜﺑﺎﺘﻛ )١( ﲑﺴﻔﺗ )٢( ﲑﺴﻔﺗ ﳓﻮ ﺫﻟﻚ ﻣﺎﺩﺓ ) ﺮﻫﻮﳉﺍ ﻯﻭﺭﻭ ﻩﹺ ﲑِ ﺴﹾ ﻔﺗ ﺧﻮﻑ ) ﺱﺎﺳﺃ ﺔﻌﺒﻨﻟﺍﻭ ﺏﺮﻌﻟﺍ ١١-١١١ ، ﻭﰲ ﻮ ﰲ ﻟﺴﺎﻥ ﺍﻟﻌﺮﺏ ﻧﻘﻼ ﻋﻦ ﺻﺤﺎﺡ ﻟ ﻌﺠﻼﻥ ﺍﻟﻨﻬﺪﻱ . ﰲ ﹺﻱ ﻭﺎﻀﻴﺒﻟﺍ ﻩﺍﺰ ﺝ ﺍﻟﻌﺮﻭﺱ : ﺓﺩﺎﻣ ﻑ ﺍﻟﺴﲑ(، ﻳﻨﻈﺮ : ﻲِ ﺴﺎ ﺍﻟﻘ ﺩﺮﺒﺗ . ﻲﺴﻘﻟﺍ ﺐ . ﻥﺎﺴﻟ ﺎﳘ .  òÌÜÛa @ À @ ÐnÛa pb @ Þìy @ òîvy @a @@@@@@@ bèj’ ﺝﺎﺗ ﺃﻳﻀﺎ ﺑﻠﻔﻆ ﲣﻮﻑ ) ﺗ ﺍﳊﺪﻳﺪﺓ ﺍﻟﱵﻦ ﺐﹸ ﺧﺸ ﺓﺪﻳﺪﹶ ﻫﺬﻩ ﺍﳊ ﺠﺎﺭ ﺍﻟﱵ ﻻ ﻗﻠﺐ ﺍﳌﻨﺎﻓﻖ ﺍﷲ ﻋﻨﻪ » : | n ﻞﺤﻨﻟﺍ ٧: ﺎﻝ ﻟﻪ ﻋﻤﺮ: ﺍﳍﺬﱄ ﻳﺼﻒ ﻑﻮﺨﻛﻤﺎ ﺗ ﻧﻜﻢ ﺷﻌﺮ ﺍﳉ ﻜﻲ ﻭﺍﻟﺜﻌﻠﱯ ﻭﻏﲑﳘ ﻑ:٢/ ٤١١ ﲑﺴﻔﺗﻭ ﻭﻧﺴﺐ ﺍﻟﺰﳐﺸﺮﻱ ﻋ ﻭﰲ ﻣﻮﺿﻊ ﻣﻨﻪ ﺎ، ﻭﻫﻮ ﻟﻌﺒﺪ ﺍﷲ ﻭﺃ ﻲﻟﺎﻤﱡ ﹴ ﺍﻟﺜ ﻢﺣﺍﺰﻣ .ﻗ ﺎ ﹰ ﻋﻠﻰ ﹴ ﻟﻪ ﻗﺼﻴﺪﺓ ﻞ ﻦ(. ﻭﺭﻭﻱ ﺍﻟﺒﻴﺖ ﺓ)ﺧﻮﻑ (. ﻭﻦﹶ ﻔﺴﻟﺍ ﻫ ﹸ ﻞﻛﹾ ﻛﻤﺎ ﺗﺄﱠﺺ ﻘ ﺣﻮﻟﻴﺔ ﻮﻥ ﺑﲔ ﺍﻷﺷﺠ ﺮﻤﻋ :ﻚﻟﺬﻛ ﻥ ﻋﻤﺮ ﺭﺿﻲ y z { | ﺍﻟﺘﻨﻘﺺ، ﻓﻘﺎ ﺮﻧﺎ ﺃﺑﻮ ﻛﺒﲑ ﹰ ﺍﺩﹺ ﺮﹶ ﹰ ﻗ ﺎﻜ ...ﻛ ﻋﻠﻴﻜﻢ ﺑﺪﻳﻮ ﺍ ﻜﻧ ٨١ ﻭﻧﻘﻠﻪ ﻋﻦ ﻣﻜ ﺔﻴ:٢٧١ ، ﺍﻟﻜﺸﺎﻑ ﻛﺘﺎﺏ ﺍﻟﺘﻔﺴﲑ . ﻭ ﻭ ،ﻫﺮﻱ ﻭﺍﻟﺰﺟﺎﺝ ﻩ ﳍﻤﺎ :ﺎﻤﹶﻬ ﻭﻟﻴﺲ ﻟ ﺮﻟﺍ ﻣ ﹺ ﻦﻪ ﻻﺑﹶﻧ ﺃ ﺔﻳﹺ ﻭﺍ ﹴ ﻞﻳﹶ ﺬﹺ ﻫ ﺮﻌ ﺩﻳﻮﺍﻥ ﺷ ﻣﺶ، ﻣﺎﺩﺓ ) ﻦﻔﺳ ﺔﻏ:١/ ١٢٦ ، ﻣﺎﺩﺓ ﺎ ﺍﻟﻘﺴﻲ . ﻱﹶ ﺃ: ﱠ ﻘﻨﺗ •ﺣ À ﻟﺸﺠﺮﺓ ﺗﻜﻮ ﻝﺎﻗ : ﻋ ﻝﺎﻘﻓ ﻦ ﺍﳌﺴﻴﺐ ﺃﻥ ﻮﻟﻪ ﺗﻌﺎﱃ: o ﺨﻮﻑ ﻋﻨﺪﻧﺎ: ﻌﻢ، ﻗﺎﻝ ﺷﺎﻋ ﺮ ﻜﹸ ﻣﻨﻬﺎ ﺗﺎﻣ ﻞﺣ ﻋ ﻳﻬﺎ ﺍﻟﻨﺎﺱ ﻢﻬﻣﻼ«. )٢( ﺗﻔﺴﲑ ﺍﻟﻘﺮﻃﱯ:/٧ ١ ، ﻣﻘﺪﻣﺔ ﺍﺑﻦ ﻋﻄﻴ ﻱﺭﺎﺨﺒﻟﺍ : ٣/٩٩ ﺒﻞ ﻧﻘﻠﻪ ﻋﻦ ﺍﻷﺯﻫ ﻲ ﺑﻌﺪ ﺃﻥ ﻧﻘﻞ ﻋﺰﻩ ﺎﺩﻤ ﺣﺔﻤﺟﺮ ﰲ ﺗ ﰲ ﺪﹺ ﺟﹶ ﻭﱂ ﺃﻲﹶﻟ ﺬ ﺏ :/ ٣١ ٢٠٩ ﺎﻫ ﺮﻱ :ﺃﺳﺎﺱ ﺍﻟﺒﻼﻏ ﺎﺮ ﻳﺼﻨﻊ ﻣﻦ ﻋﻴﺪﺍ ﳊﺮﺟﺔ ﻓﻴﻨﺎ :ﺍ ﻭﻻ ﺷﻲﺀ، ) (١ ﺳ ﻦﻋ ﻌﻴﺪ ﺑﻦ ﲣﻮﻑ (ﻣﻦ ﻗﻮ ﺬﻩ ﻟﻐﺘﻨﺎ، ﺍﻟﺘﺨ ﺭﻫﺎ؟، ﻗﺎﻝ:ﻧﻌ ﺣﺮﻟﺍ ﻑﻮﺨﺗ ﺮﻤﻋ ﻳﺎ ﺃﻳ : ﻭﻣﻌﺎﱐ ﻛﻼ، ﻱﱪﻄﻟﺍ :٨/٢٨ ﺗﻭ ﻱﱪﻄﻟﺍ :١٤ / ١١٣ ﻚ ﻳﻨﻈﺮ :ﺻﺤﻴﺢ )ﺧﻮﻑ ( ﻻﺑﻦ ﻣﻘﺒ ،ﻱﺮ ﻲﹺ ﺎﻏﺎﻧﻗﺎﻝ ﺍﻟﺼ ﻲﹺ ﻧﺎﹶ ﻏَ ﻷﺍ ﺐﺣﺎﺻ ﺬﹸ ﹴ ﺍﳍ ﲑﹺ ﺒﹶ ﹶﰊ ﻛ ﹺﱃ ﺃ ﻩ ﺇ ﻑ .(ﻭﻟﺴﺎﻥ ﺍﻟﻌﺮﺏ ﺔﻏﻼﺒﻟﺍ : ﺮﺸﳐﺰﻟﺍ : ﻧﻮﻉ ﻣﻦ ﺍﻟﺸﺠﺮ . ٩٩ / ٩ : è ﻝﺎﻗ ﺍﳊ : ،ﺔﻴﺸﺣﻭ ﲑﳋﺍ «.) ﺀﺎﺟﻭ ﻣﻌﲎ )ﲣ ﻝﺎﻘﻓ :ﺬﻫ ﰲ ﺃﺷﻌﺎﺭ ﻝﺎﻘﻓ ،ﻢﻜﺑﺎﺘﻛ )١( ﲑﺴﻔﺗ )٢( ﲑﺴﻔﺗ ﳓﻮ ﺫﻟﻚ ﻣﺎﺩﺓ ) ﺮﻫﻮﳉﺍ ﻯﻭﺭﻭ ﻩﹺ ﲑِ ﺴﹾ ﻔﺗ ﺧﻮﻑ ) ﺱﺎﺳﺃ ﺔﻌﺒﻨﻟﺍﻭ ﺏﺮﻌﻟﺍ ﺩ ﺍﳋﺎﻣﺲ ﻠﻰ ﺍﳌﻨﱪ ﻋﻦ ،ﺦ ﻣﻦ ﻫﺬﻳﻞ ﺍﻟﻌﺮﺏ ﺫﻟﻚ ﻦﹶ ﻔﺴﻟ ﻓﻴﻪ ﺗﻔﺴﲑ ١١-١١١ ، ﻭﰲ ﻮ ﰲ ﻟﺴﺎﻥ ﺍﻟﻌﺮﺏ ﻧﻘﻼ ﻋﻦ ﺻﺤﺎﺡ ﻟ ﻌﺠﻼﻥ ﺍﻟﻨﻬﺪﻱ . ﰲ ﹺﻱ ﻭﺎﻀﻴﺒﻟﺍ ﻩﺍﺰ ﺝ ﺍﻟﻌﺮﻭﺱ : ﺓﺩﺎﻣ ﻑ ﺍﻟﺴﲑ(، ﻳﻨﻈﺮ : ﻲِ ﺴﺎ ﺍﻟﻘ ﺩﺮﺒﺗ . ﻲﺴﻘﻟﺍ ﺐ .  òÌÜÛa @ À @ ÐnÛa pb @ Þìy @ òîvy @a @@@@@@@ bèj’ ﲑ ﺍﻟﻘﺮﻃﱯ:٠/ ٠١ ﺍﻟﺒﻴﺖ ﻟﺰﻫﲑ، ﻭﻫﻮ ﻧﻘ ﻋﺰﺍﻩ ﻟﺬﻱ ﺍﻟﺮﻣﺔ ﻭ ﻋﺒﺪ ﺍﻟﻠﻴﺚ ﻦﺑﻟﺍ ﺎﻝ ﺍﻟﺰﺑﻴﺪﻱ : »ﺰﻋﻭ ﻱﹺ ﻫﺬﺍ ﺍﻟﺮﻭ «. ﺝﺎﺗ ﺃﻳﻀﺎ ﺑﻠﻔﻆ ﲣﻮﻑ ) ﺗ ﺍﳊﺪﻳﺪﺓ ﺍﻟﱵﻦ ﺐﹸ ﺧﺸ ﺓﺪﻳﺪﹶ ﻫﺬﻩ ﺍﳊ ﻜﻲ ﻭﺍﻟﺜﻌﻠﱯ ﻭﻏﲑﳘ ﻑ:٢/ ٤١١ ﲑﺴﻔﺗﻭ ﻭﻧﺴﺐ ﺍﻟﺰﳐﺸﺮﻱ ﻋ ﻭﰲ ﻣﻮﺿﻊ ﻣﻨﻪ ﺎ، ﻭﻫﻮ ﻟﻌﺒﺪ ﺍﷲ ﻭﺃ ﻲﻟﺎﻤﱡ ﹴ ﺍﻟﺜ ﻢﺣﺍﺰﻣ .ﻗ ﺎ ﹰ ﻋﻠﻰ ﹴ ﻟﻪ ﻗﺼﻴﺪﺓ ﻞ ﻦ(. ﻭﺭﻭﻱ ﺍﻟﺒﻴﺖ ﺓ)ﺧﻮﻑ (. ﻭﻦﹶ ﻔﺴﻟﺍ ﻫ ﹸ ﻞﻛﹾ ﻛﻤﺎ ﺗﺄﱠﺺ ﻘ ٨١ ﻭﻧﻘﻠﻪ ﻋﻦ ﻣﻜ ﺔﻴ:٢٧١ ، ﺍﻟﻜﺸﺎﻑ ﻛﺘﺎﺏ ﺍﻟﺘﻔﺴﲑ . ﻭ ﻭ ،ﻫﺮﻱ ﻭﺍﻟﺰﺟﺎﺝ ﻩ ﳍﻤﺎ :ﺎﻤﹶﻬ ﻭﻟﻴﺲ ﻟ ﺮﻟﺍ ﻣ ﹺ ﻦﻪ ﻻﺑﹶﻧ ﺃ ﺔﻳﹺ ﻭﺍ ﹴ ﻞﻳﹶ ﺬﹺ ﻫ ﺮﻌ ﺩﻳﻮﺍﻥ ﺷ ﻣﺶ، ﻣﺎﺩﺓ ) ﻦﻔﺳ ﺔﻏ:١/ ١٢٦ ، ﻣﺎﺩﺓ ﺎ ﺍﻟﻘﺴﻲ . ﻱﹶ ﺃ: ﱠ ﻘﻨﺗ ﺗﻔﺴﲑ ﺍﻟﻘﺮﻃﱯ:/٧ ١ ، ﻣﻘﺪﻣﺔ ﺍﺑﻦ ﻋﻄﻴ ﻱﺭﺎﺨﺒﻟﺍ : ٣/٩٩ ﺒﻞ ﻧﻘﻠﻪ ﻋﻦ ﺍﻷﺯﻫ ﻲ ﺑﻌﺪ ﺃﻥ ﻧﻘﻞ ﻋﺰﻩ ﺎﺩﻤ ﺣﺔﻤﺟﺮ ﰲ ﺗ ﰲ ﺪﹺ ﺟﹶ ﻭﱂ ﺃﻲﹶﻟ ﺬ ﺏ :/ ٣١ ٢٠٩ ﺎﻫ ﺮﻱ :ﺃﺳﺎﺱ ﺍﻟﺒﻼﻏ ﺎﺮ ﻳﺼﻨﻊ ﻣﻦ ﻋﻴﺪﺍ ﻱﱪﻄﻟﺍ :٨/٢٨ ﺗﻭ ﻱﱪﻄﻟﺍ :١٤ / ١١٣ ﻚ ﻳﻨﻈﺮ :ﺻﺤﻴﺢ )ﺧﻮﻑ ( ﻻﺑﻦ ﻣﻘﺒ ،ﻱﺮ ﻲﹺ ﺎﻏﺎﻧﻗﺎﻝ ﺍﻟﺼ ﻲﹺ ﻧﺎﹶ ﻏَ ﻷﺍ ﺐﺣﺎﺻ ﺬﹸ ﹴ ﺍﳍ ﲑﹺ ﺒﹶ ﹶﰊ ﻛ ﹺﱃ ﺃ ﻩ ﺇ ﻑ .(ﻭﻟﺴﺎﻥ ﺍﻟﻌﺮﺏ ﺔﻏﻼﺒﻟﺍ : ﺮﺸﳐﺰﻟﺍ : ﻧﻮﻉ ﻣﻦ ﺍﻟﺸﺠﺮ . ٩٩ / ٩ : )١( ﲑﺴﻔﺗ )٢( ﲑﺴﻔﺗ ﳓﻮ ﺫﻟﻚ ﻣﺎﺩﺓ ) ﺮﻫﻮﳉﺍ ﻯﻭﺭﻭ ﻩﹺ ﲑِ ﺴﹾ ﻔﺗ ﺧﻮﻑ ) ﺱﺎﺳﺃ ﺔﻌﺒﻨﻟﺍﻭ ﺏﺮﻌﻟﺍ ١( ﺔﺸ)١ ﻫﻭ (٢) ،ﻪﺤ ﻪﺑ . (٢) ﻪﺑ . ١ •ﺣ òÌÜÛa @ À @ ÐnÛa ﻰ ﻫﺬﻩ ﺍﻟﺮﻭﺍ ﻣﻦ ﺍﳌﻌﺎﺭﺽ ﻲ ﱂ ﺗﺴﻠﻢ ﻣﻦ ﻣ ﻹﻣﺎﻡ ﺃﲪﺪ ﺗﻔﺴﲑ ﺍﻟﻘﺮﺁﻥ » ﺯﻮﳚ ﲑﺴﻔﺗ ﷲﺍ« .ﻝﺎﻗﻭ ﻣﺎﻡ ﺃﲪﺪ ﺭﺿ ﺀ ﺑﺮﻭﺍﻳﺔ ﺍﳌﺮﻭ ﻠﻘﻬﺎ ﺍﷲ ﺗﻌﺎﱃ o ² ³ ، ﺃﻱ : ﻞﻌﻓﺄﺳ ﻰ ﺍﻟﻠﻐﺔ«. ﺻﻮﻝ ﺍﲪﺪ :٥٨٠ ﻮﻝ ﺃﲪﺪ :٥٨١ . ١ ﺓﺩﻮﺴ :١٧٥-٧٦ pb @ Þìy @ òîvy @a ﻥ ﻣﺎ ﻳﺮﺩ ﻋﻠﻰ -ﺇﻥ ﺳﻠﻤﺖ ، ﻛﻴﻒ ﻭﻫﻲ ﻯﻭﺭ ﻋﻦ ﺍﻹ )١( » ﺯﻮﳚﻭ ﻲﻠﺒﻨﳊﺍ (٢) : ﺍ ﺃﲪﺪ ﺭﲪﻪ ﺍﻟﻠﻐﺔ ﻋﻨﺪ ﺍﻹ ﺪ ﻫﺬﺍ ﻣﺎ ﺟﺎ ﺎ ﺭﻭﺡ ﺧﻠﺃ ﻪﻧﺎﺤﺒﺳ : o ﹰ ﻱ ﻋﻠﻴﻚ ﺭﺯﻗﺎ ﻩ ﻋﻠﻰ ﻣﻘﺘﻀﻰ ﺓ :١٨٢ / ٢ ﺻﺃﻭ ﺪ :٢/ ٢٨١ ﻮﺻﺃﻭ ﺐ ﲑﻨﳌﺍ ٨٥/٢: ﺪ :٢/ ٢٨١ ﺴﳌﺍﻭ ﺪ :٢/ ٢٨١ . @@@@@@@ bèj’ ﻻﺇ ﻥﺃ ﺔﻠﺑﺎﻨﳊﺍ - ﻕﻼﻃﻹﺍ ﺪﻘﻓ ﺓﺩﻮﺴﳌﺍ : ﺏﺎﻄﳋﺍ ﻗﺪ ﻓﺴﺮ ﻰﻀﺘﻘﲟ ﺪﻀﻌﻳ ﺎﻫﺎﻨﻌﻣ : ﰲ ﻗﻮﻟﻪ ﻱﺮﺟﺄﺳ ﺃ ﻧﻪ ﻓﺴﺮﻩ )١( ﺓﺩﻮﺴﳌﺍ )٢( ﺪﻴﻬﻤﺘﻟﺍ )٣( ﺍﻟﻜﻮﻛﺐ )٤( ﺪﻴﻬﻤﺘﻟﺍ )٥( ﺪﻴﻬﻤﺘﻟﺍ  òÌÜÛa @ À @ ÐnÛa pb @ Þìy @ òîvy @a @@@@@@@ bèj’ ﻘﺪ ﺟﺎﺀ ﰲ « . ﻝﺎﻗﻭ ﻮﺑﺃ ﺷﻴﺨﻨﺎ ﻭﻗﺎﻝ: ﻔﺴﲑ ﺍﻟﻘﺮﺁﻥ ﷲﺍ ﺇﳕﺎ» (: ﺽ ﺍﷲ، ﻭﻗﺎﻝ ﻮﻝ ﺍﻟﺮﺟﻞ: ٥( »ﻩﺮﻫﺎﻇﻭ ﻋﻨﻪ ﺃﻭﻻ، ﻓﻘ ﻛﺮﻩ ﺷﻴﺨﻨﺎ« ﺷ ﻠﻐﺔ؛ ﺫﻛﺮﻩ »ﻻﻭ ﳛﺮﻡ ﺗﻔ ﻪﺑ« . ﺎﻝ ﰲ)ﺭﻭﺡ ﲰﺎﺀ ﺍﷲ ﻭﺃﺭﺽ ﰲ ﺍﻟﻠﻐﺔ، ﻳﻘﻮ ﻮ ﺍﳋﻄﺎﺏ:)٥ ﻣﻊ ﺍﳌﻨﻘﻮﻝ ﻀﻰ ﺍﻟﻠﻐﺔ، ﺫﻛ ﻰ ﻣﻘﺘﻀﻰ ﺍﻟﻠ ﻲﺣﻮﺘﻔﻟ:)٣( » ﺃﻭ ﻛﺜﺮ ﺃﺻﺤﺎﺑﻪ ﲪﻪ ﺍﷲ ﻪﻧﺃ ﺎﻗ : ﻋﺒﺪ ﺍﷲ ﲰﻭ ﻫﻮ ﺟﺎﺋﺰ ، «.)٤( ﻝﺎﻗ ﻮﺑﺃ ﻣﺎ ﻳﺘﻌﺎﺭﺽ ﻥ ﻋﻠﻰ ﻣﻘﺘﻀ ﲑ ﺍﻟﻘﺮﺁﻥ ﻋﻠﻰ ﺍﺑﻦ ﺍﻟﻨﺠﺎﺭ ﺍﻟ ﻭ ﺿﻲ ﺍﷲ ﻋﻨﻪ ﻭﺯﻱ ﻋﻨﻪ ﺭﲪ ﱃ، ﻛﻤﺎ ﻳﻘﺎﻝ ³ n ٦٤ : ﻃﻪ ، ﹰ ﻞ ﻟﻚ ﺧﲑﺍ« ﻣ ﻹﻣﺎﻡ ﺃﲪﺪ ﺗﻔﺴﲑ ﺍﻟﻘﺮﺁﻥ » ﺯﻮﳚ ﲑﺴﻔﺗ ﷲﺍ« .ﻝﺎﻗﻭ ﻣﺎﻡ ﺃﲪﺪ ﺭﺿ ﺀ ﺑﺮﻭﺍﻳﺔ ﺍﳌﺮﻭ ﻠﻘﻬﺎ ﺍﷲ ﺗﻌﺎﱃ o ² ³ ، ﺃﻱ : ﻞﻌﻓﺄﺳ ﻰ ﺍﻟﻠﻐﺔ«. ﻯﻭﺭ ﻋﻦ ﺍﻹ )١( » ﺯﻮﳚﻭ ﻲﻠﺒﻨﳊﺍ (٢) : ﺍ ﺃﲪﺪ ﺭﲪﻪ ﺍﻟﻠﻐﺔ ﻋﻨﺪ ﺍﻹ ﺪ ﻫﺬﺍ ﻣﺎ ﺟﺎ ﺎ ﺭﻭﺡ ﺧﻠﺃ ﻪﻧﺎﺤﺒﺳ : o ﹰ ﻱ ﻋﻠﻴﻚ ﺭﺯﻗﺎ ﻩ ﻋﻠﻰ ﻣﻘﺘﻀﻰ ﺪﻘﻓ ﺓﺩﻮ: ﺏﺎﻄ ﺮﺴﻓ ﻰﻀ ﺪﻀﻌ ﺎﻫﺎ: ﻪﻟﻮﻗ ﻱﺮﺟ ﻩﺮﺴﻓ « . ﻝﺎﻗﻭ ﻮﺑﺃ ﺷﻴﺨﻨﺎ ﻭﻗﺎﻝ: ﻔﺴﲑ ﺍﻟﻘﺮﺁﻥ ﷲﺍ ﺇﳕﺎ» (: ﺽ ﺍﷲ، ﻭﻗﺎﻝ ﻮﻝ ﺍﻟﺮﺟﻞ: ٥( »ﻩﺮﻫﺎﻇﻭ ﻛﺮﻩ ﺷﻴﺨﻨﺎ« ﺷ ﻠﻐﺔ؛ ﺫﻛﺮﻩ »ﻻﻭ ﳛﺮﻡ ﺗﻔ ﻪﺑ« . (٢) ﻪﺑ . ﺩ ﺍﳋﺎﻣﺲ ﺍﻟﻌﺪﺩ ﺣﻮﻟﻴﺔ ﲑ . •ﺣ ٠١ ﻛﺘﺎﺏ ﺍﻟﺘﻔﺴﲑ . ٠٢ ﺢ ﺍﻟﺒﺨﺎﺭﻱ:١٠/٣ ﺏﺎﺘﻛ ﺍﳌﺒﺎﱐ ٣: )١( ﺢﻴﺤﺻ )٢( ﺔﻣﺪﻘﻣ  òÌÜÛa @ À @ ÐnÛa pb @ Þìy @ òîvy @a @@@@@@@ bèj’  òÌÜÛa @ À @ ÐnÛa pb @ Þìy @ òîvy @a @@@@@@ bèj’ ﻀﺎ. ) (٤ ١ . ﻝﻮﻘ: ١/٢٣ﻭ١٧٧ ٨١٤ ﻭﻣﻨﺎﻫﺞ ﺍﻟﻌﻘﻮ ﻥﻭﺮﺴ :١/ ٢٥٨ . ﺤﻮﻝ : . ٥١ ٤١ ، ﺍﻟﺘﻠﻮﻳﺢ : ١/٨ ٥ ﻭﺍﻟﺘﻔﺴﲑ ﻭﺍﳌﻔﺴ ٢٩٦ ﻭﺇﺭﺷﺎﺩ ﺍﻟﻔﺤ ﻡﺎ :ﻦﺑﺍ ﻡﺰﺣ: ٣/ ﻥ :ﲏﻳﻮﳉﺍ : ١/١٥ ﻝ :ﻕ / ١ﺟـ ١ ﻝﻮﻘﻌﻟﺍ . ٣٢/١: )١( ﺎﻜﺣﻹﺍ )٢( ﻥﺎﻫﱪﻟﺍ )٣( ﻝﻮﺼﶈﺍ )٤( ﺞﻫﺎﻨﻣ ﻦ.) (١  òÌÜÛa @ À @ ÐnÛa pb @ Þìy @ òîvy @a @@@@@@@ bèj’ ﺸﻌﺮ؟ ﻓﻘﺎﻝ: ﺪ ﰲﻧﻘﻞ ﺍ ﺘﺎﺏ ﺍﻟﺼﻼﺓ ﻣﺮ ﺃﻧﻪ ﺃﺑﺪﻱ ﺑﺸﻲﺀ ﻣﻦ ﺍﻟﺸ ﻊﻨﳌ« )ﻭﻧ. (١ ﺍﻟﺘﻤﺎﻡ (ﰲ ﻛﺘ ﻘﺎ، ﻓﻜﻞ ﺍﻷﻣ ﺑ ﻞ ﺍﻟﺮﺟﻞ ﻟﻪ ﺬﺍ ﻳﻘﺘﻀﻲ ﺍﳌﻨ ﰊ ﻳﻌﻠﻰ ﰲ)ﺍ ﺪﻡ ﺍﳉﻮﺍﺯ.)(٢ ﲏﻊﻨﳌﺍ ﻘﻠﻄﻣ ﺍﻟﻘﺮﺁﻥ ﻳﺘﻤﺜﻞ : ﻭﻇﺎﻫﺮ ﻫﺬ ﺑﻦ ﺍﻟﻘﺎﺿﻲ ﺃﰊ ﻭﺻﺤﺢ ﻋﺪ ﻌﺠﺒﲏ (ﻻ ﻳﻌﲏ ﻪﻧ ﺳﺌﻞ ﻋﻦ ﻭﺃﺑﻮ ﺍﻟﻄﻴﺐ ﺑﻦ ﺍﺑﻦ ﺣﺴﲔ ﺪﲪ ،ﰲ ﺫﻟﻚ ﺃﲪﺪ) :ﻻ ﻳﻌ ﺎﻡ ﺃﲪﺪ: »ﺃ ﲏ .ﻗﺎﻝ ﻫﻮ ﻋﻦ ﻲﺿﺎﻘﻟﺍ ﻋﻦ ﺍﻹﻣﺎﻡ ﲪﺃ ﺮﻫ ﺃﻥ ﻗﻮﻝﺃ ﻦ ﺍﻹﻣﺎ ﻻ ﲏﺒﺠﻌﻳ ﺓﺩﻮﺴ ﻳﺍﻭﲔﺘ ﺎﻈﻟﺍﻭ ﺩ ﺍﳋﺎﻣﺲ ﺍﻟﻌﺪﺩ ﺓﺩﻮﺴ :١٧٥ . ﺣﻮﻟﻴﺔ ﻰﻠ :٢/ ٦٠٥ ﺴﳌﺍﻭ . •ﺣ ﻋﻦ ﺍﻟﻌﺪﺓ ﻷﰊ ﻳﻌﻠ ﺐ ﺍﳌﻨﲑ :٨٥١ /٢ ﺪ :٢/ ٢٨١ ﻪﻠﻘﻧﻭ ﺓ :١٧٦ ﻭﺍﻟﻜﻮﻛﺐ )١( ﺪﻴﻬﻤﺘﻟﺍ )٢( ﺓﺩﻮﺴﳌﺍ ١٥٩ ﺩ ﺍﳋﺎﻣﺲ ﺍﶈﻘﻘﲔ ﻣﻦ ﻊ ﻋﻨﺪﻩ ﻋﻠﻰ ﻘﺪ ﺟﺎﺀ ﰲ « . ﻝﺎﻗﻭ ﻮﺑﺃ ﺷﻴﺨﻨﺎ ﻭﻗﺎﻝ: ﻔﺴﲑ ﺍﻟﻘﺮﺁﻥ ﷲﺍ ﺇﳕﺎ» (: ﺽ ﺍﷲ، ﻭﻗﺎﻝ ﻮﻝ ﺍﻟﺮﺟﻞ: ٥( »ﻩﺮﻫﺎﻇﻭ ﺍﻟﻌﺪﺩ ﻈﻬﺮﻩ ﺑﻌﺾ ﺎ ﺍﳌﻨﻊﺕ ﺇﻓﺎﺩ ﻋﻨﻪ ﺃﻭﻻ، ﻓﻘ ﻛﺮﻩ ﺷﻴﺨﻨﺎ« ﺷ ﻠﻐﺔ؛ ﺫﻛﺮﻩ »ﻻﻭ ﳛﺮﻡ ﺗﻔ ﻪﺑ« . ﺎﻝ ﰲ)ﺭﻭﺡ ﲰﺎﺀ ﺍﷲ ﻭﺃﺭﺽ ﰲ ﺍﻟﻠﻐﺔ، ﻳﻘﻮ ﻮ ﺍﳋﻄﺎﺏ:)٥ ﻊﻨ ﺎﻤﻛ ﻈﺘﺳﺍ ﺴﻠﻢ ﰲ ﺛﺒﻮﺕ . ﻣﻊ ﺍﳌﻨﻘﻮﻝ ﻀﻰ ﺍﻟﻠﻐﺔ، ﺫﻛ ﻰ ﻣﻘﺘﻀﻰ ﺍﻟﻠ ﻲﺣﻮﺘﻔﻟ:)٣( » ﺃﻭ ﻛﺜﺮ ﺃﺻﺤﺎﺑﻪ ﲪﻪ ﺍﷲ ﻪﻧﺃ ﺎﻗ : ﻋﺒﺪ ﺍﷲ ﲰﻭ ﻫﻮ ﺟﺎﺋﺰ ، «.)٤( ﻝﺎﻗ ﻮﺑﺃ ﺣﻮﻟﻴﺔ  ﺍﻳﺔ ﺍﳌﻔﻴﺪﺓ ﺍﳌﻨ ﺽ- ﺎ ﱂﻓﺈ ﺴﺗ ﻦ ﺍﳌﻌﺎﺭﺽ!؟ ﻣﺎ ﻳﺘﻌﺎﺭﺽ ﻥ ﻋﻠﻰ ﻣﻘﺘﻀ ﲑ ﺍﻟﻘﺮﺁﻥ ﻋﻠﻰ ﺍﺑﻦ ﺍﻟﻨﺠﺎﺭ ﺍﻟ ﻭ ﺿﻲ ﺍﷲ ﻋﻨﻪ ﻭﺯﻱ ﻋﻨﻪ ﺭﲪ ﱃ، ﻛﻤﺎ ﻳﻘﺎﻝ ³ n ٦٤ : ﻃﻪ ، ﹰ ﻞ ﻟﻚ ﺧﲑﺍ« ١٠ . . . (٢) ﻪﺑ . ﺎﻝ ﰲ)ﺭﻭﺡ ﲰﺎﺀ ﺍﷲ ﻭﺃﺭﺽ ﰲ ﺍﻟﻠﻐﺔ، ﻳﻘﻮ ﻮ ﺍﳋﻄﺎﺏ:)٥ ﻀﻰ ﺍﻟﻠﻐﺔ، ﺫﻛ ﻰ ﻣﻘﺘﻀﻰ ﺍﻟﻠ ﻲﺣﻮﺘﻔﻟ:)٣( » ﺃﻭ ﻛﺜﺮ ﺃﺻﺤﺎﺑﻪ ﲪﻪ ﺍﷲ ﻪﻧﺃ ﺎﻗ : ﻋﺒﺪ ﺍﷲ ﲰﻭ ﻫﻮ ﺟﺎﺋﺰ ، «.)٤( ﻝﺎﻗ ﻮﺑﺃ ﻥ ﻋﻠﻰ ﻣﻘﺘﻀ ﲑ ﺍﻟﻘﺮﺁﻥ ﻋﻠﻰ ﺍﺑﻦ ﺍﻟﻨﺠﺎﺭ ﺍﻟ ﻭ ﺿﻲ ﺍﷲ ﻋﻨﻪ ﻭﺯﻱ ﻋﻨﻪ ﺭﲪ ﱃ، ﻛﻤﺎ ﻳﻘﺎﻝ ³ n ٦٤ : ﻃﻪ ، ﹰ ﻞ ﻟﻚ ﺧﲑﺍ« ﺗﻔﺴﲑ ﺍﻟﻘﺮﺁﻥ » ﺯﻮﳚ ﲑﺴﻔﺗ ﷲﺍ« .ﻝﺎﻗﻭ ﻣﺎﻡ ﺃﲪﺪ ﺭﺿ ﺀ ﺑﺮﻭﺍﻳﺔ ﺍﳌﺮﻭ ﻠﻘﻬﺎ ﺍﷲ ﺗﻌﺎﱃ o ² ³ ، ﺃﻱ : ﻞﻌﻓﺄﺳ ﻰ ﺍﻟﻠﻐﺔ«. )١( » ﺯﻮﳚﻭ ﻲﻠﺒﻨﳊﺍ (٢) : ﺍ ﺃﲪﺪ ﺭﲪﻪ ﺍﻟﻠﻐﺔ ﻋﻨﺪ ﺍﻹ ﺪ ﻫﺬﺍ ﻣﺎ ﺟﺎ ﺎ ﺭﻭﺡ ﺧﻠﺃ ﻪﻧﺎﺤﺒﺳ : o ﹰ ﻱ ﻋﻠﻴﻚ ﺭﺯﻗﺎ ﻩ ﻋﻠﻰ ﻣﻘﺘﻀﻰ ﺓﺩﻮ: ﺏﺎﻄ ﺮﺴﻓ ﻰﻀ ﺪﻀﻌ ﺎﻫ: ﻪﻟﻮﻗ ﻱﺮﺟ ﻩﺮﺴﻓ ١٠ . . . ١ ﺻﻮﻝ ﺍﲪﺪ :٥٨٠ ﻮﻝ ﺃﲪﺪ :٥٨١ . ١ ﺓﺩﻮﺴ :١٧٥-٧٦ ﺓ :١٨٢ / ٢ ﺻﺃﻭ ﺪ :٢/ ٢٨١ ﻮﺻﺃﻭ ﺐ ﲑﻨﳌﺍ ٨٥/٢: ﺪ :٢/ ٢٨١ ﺴﳌﺍﻭ ﺪ :٢/ ٢٨١ . )١( ﺓﺩﻮﺴﳌﺍ )٢( ﺪﻴﻬﻤﺘﻟﺍ )٣( ﺍﻟﻜﻮﻛﺐ )٤( ﺪﻴﻬﻤﺘﻟﺍ )٥( ﺪﻴﻬﻤﺘﻟﺍ  òÌÜÛa @ À @ ÐnÛa pb @ Þìy @ òîvy @a @@@@@@ bèj’ ﺎ ﻼ ﻳﻜﺘﻔﻰ ﺍﺟﺤﺔ ﻋﻠﻴﻬﺎ ﻛﺬﻟﻚ ﻓﻬﻲ ﻫﻮ ﺍﳌﺮﻭﻱ، ﺍﻭ ﻳﺔ ﺍﳉﻮﺍﺯ ( ﻭﻣﻨﻬﺎ ﺃﻥ: ﺍﳉﺒﻞ ﺃﺣﺪ ﺍﻟﻠﻐﺔ ﻛﺜﲑﺍ، ﻧﻘﻠﻨﺎ ﻧﺺ »ﻪﻨﻋﻭ- ﻦﻋ ﺍﻟﺘﻌﺎﺭﺽ، ﻼﻓ ﻴﺪﺓ ﺍﳉﻮﺍﺯ ﺭﺍ ﻛ ﺮﻯ ﻟﻴﺴﺖ ،ﻫﺎ ﺩﻟﻴﻞ ﺁﺧﺮ ﺬﻩ ﺍﻟﺮﻭﺍﻳﺔ)ﺭﻭ ﻛﺜﺮ ﺍﻟﺴﻠﻒ. ﻝ ﺍﺑﻦ ﻗﺎﺿﻲ ﺴﲑ ﲟﻘﺘﻀﻰ ﺏ«.)٣( ﺪﻗﻭ ﻲﺣﻮﺘﻔﻟﺍ : » ﻮﻥ ﰲ ﻣﺮﺗﺒﺔ ﻭﺍﻳﺔ ﻋﻨﻪ ﺍﳌﻔﻴ ﳊﻜﻢ، ﻭﺍﻷﺧﺮ ﳉﻮﺍﺯ ﻳﻌﻀﺪﻫ ﻱ ﺗﻀﻤﻨﺘﻪ ﻫﺬ ﺍﻟﻌﻤﻞ ﻋﻨﺪ ﺍﻛ ٢( ﺍﺬﻟﻭ ﻝﻮﻘﻳ ﺠﺎﺝ ﰲ ﺍﻟﺘﻔﺴ ﻰ ﻟﻐﺔ ﺍﻟﻌﺮﺏ ﹰ ﻣﺎ ﻗﺎﻟﻪ ﺎﻀ ﺩﻟﻴﻞ«.) (٤ ﰲ ﺍﻟﺮﺗﺒﺔ ﺗﻜﻮ ﻛﻴﻒ ﻭﺃﻥ ﺍﻟﺮﻭ ﻟﺘﺼﺮﻳﺢ ﺑﺎﳊ :ﺃﻥ ﺭﻭﺍﻳﺔ ﺍﳉ ﺍﳊﻜﻢ ﺍﻟﺬﻱ ﺮﻑ، ﻭﻋﻠﻴﻪ ﺭﻮ ﻪﺑﺎﺤﺻﺃ .) ﻋﺒﺎﺱ ﺍﻻﺣﺘﺠ ﺴﲑﻩ ﲟﻘﺘﻀﻰ ﳑﺎ ﻳﺪﻝ ﻟﻪ ﺃﻳﻀ ﺩ ﻠﻐﺔ ﻣﻦ ﻏﲑ ﰲ ﺇﺫﺍ ﺗﺴﺎﻭﺕ ﺎﺎﺤﺟ.)١( ﻛ ﳉﻮﺍﺯ ﻓﻴﻬﺎ ﺍ ﺎﻬﻴﻠﻋ .ﺎﻬﻨﻣﻭ: .ﺎﻬﻨﻣﻭ ﺃﻥ: ﻉ ﻭﺍﻟﻠﻐﺔ ﺮﻌﻟﺍﻭ ﺯ ﻋﻠﻴﻬﺎ ﻮﻬﲨ ﻮﻝ ﻋ ﻋﻦ ﺍﺑﻦ ﺠﻴﻓ ﻮﺯ ﺗﻔﺴ ﻚ ﺃﻳﻀﺎ، ﻭﳑﺎ ﲑﻩ ﲟﻘﺘﻀﻰ ﺍﻟﻠ ﺇ ﻥ ﺍﻟﺮﻭﺍﻳﺎﺕ ﺘﺪﻝ ﺩﻭﻥ ﺭﺟ ﻩﻮ : ﻥﺃ ﺭﻭﺍﻳﺔ ﺍﳉ ﻓﺘﻘﺪﻡ ﺗﻠﻚ ﻋ ﻋﻤﻠﻪ ﺑﺬﻟﻚ. ﺮﻯ) . (١ (٢) ﻪﺑ . ﺷﻬﺮ ﰲ ﺍﻟﺸﺮﻉ ﳌﻔﻴﺪﺓ ﺍﳉﻮﺍﺯ ﺔﻠﺑﺎﻨ: »ﻮﻘﻨﳌﺍ ﻘﺮﺁﻥ ﻋﺮﰊ، ﻲ ﺍﳌﻔﻴﺪ ﻟﺬﻟﻚ ﻻ ﳚﻮﺯ ﲑﺴﻔﺗ ﺃﻳﻪ، ﻻﻥ ﻦ ﺍﳌﺴﺘ ﻦ ﻭﺟﻮ ﺎﻬﻨﻣ: ،ﺔﻠﻤﺘ ﻨﻪ ﻣﻦ ﻮ ﺍﻷﺷﻬ ﺮﻭﺍﻳﺔ ﺍ ﻤﺔ ﺍﳊﻨ ﻥﻷ ﻘﻟﺍ ﻲﺣﻮﺘﻔ ﺪﲪ- ﻻ ﺎ ﻼ ﻳﻜﺘﻔﻰ ﺍﺟﺤﺔ ﻋﻠﻴﻬﺎ ﻛﺬﻟﻚ ﻓﻬﻲ ﻫﻮ ﺍﳌﺮﻭﻱ، ﺍﻭ ﻳﺔ ﺍﳉﻮﺍﺯ ( ﻭﻣﻨﻬﺎ ﺃﻥ: ﺍﳉﺒﻞ ﺃﺣﺪ ﺍﻟﻠﻐﺔ ﻛﺜﲑﺍ، ﻧﻘﻠﻨﺎ ﻧﺺ »ﻪﻨﻋﻭ- ﻦﻋ ﺍﻟﺘﻌﺎﺭﺽ، ﻼﻓ ﻴﺪﺓ ﺍﳉﻮﺍﺯ ﺭﺍ ﻛ ﺮﻯ ﻟﻴﺴﺖ ،ﻫﺎ ﺩﻟﻴﻞ ﺁﺧﺮ ﺬﻩ ﺍﻟﺮﻭﺍﻳﺔ)ﺭﻭ ﻛﺜﺮ ﺍﻟﺴﻠﻒ. ﻝ ﺍﺑﻦ ﻗﺎﺿﻲ ﺴﲑ ﲟﻘﺘﻀﻰ ﺏ«.)٣( ﺪﻗﻭ ﻲﺣﻮﺘﻔﻟﺍ : » ﻮﻥ ﰲ ﻣﺮﺗﺒﺔ ﻭﺍﻳﺔ ﻋﻨﻪ ﺍﳌﻔﻴ ﳊﻜﻢ، ﻭﺍﻷﺧﺮ ﳉﻮﺍﺯ ﻳﻌﻀﺪﻫ ﻱ ﺗﻀﻤﻨﺘﻪ ﻫﺬ ﺍﻟﻌﻤﻞ ﻋﻨﺪ ﺍﻛ ٢( ﺍﺬﻟﻭ ﻝﻮﻘﻳ ﺠﺎﺝ ﰲ ﺍﻟﺘﻔﺴ ﻰ ﻟﻐﺔ ﺍﻟﻌﺮﺏ ﹰ ﻣﺎ ﻗﺎﻟﻪ ﺎﻀ ﺩﻟﻴﻞ«.) (٤ ﰲ ﺍﻟﺮﺗﺒﺔ ﺗﻜﻮ ﻛﻴﻒ ﻭﺃﻥ ﺍﻟﺮﻭ ﻟﺘﺼﺮﻳﺢ ﺑﺎﳊ :ﺃﻥ ﺭﻭﺍﻳﺔ ﺍﳉ ﺍﳊﻜﻢ ﺍﻟﺬﻱ ﺮﻑ، ﻭﻋﻠﻴﻪ ﺭﻮ ﻪﺑﺎﺤﺻﺃ .) ﻋﺒﺎﺱ ﺍﻻﺣﺘﺠ ﺴﲑﻩ ﲟﻘﺘﻀﻰ ﳑﺎ ﻳﺪﻝ ﻟﻪ ﺃﻳﻀ ﺩ ﻠﻐﺔ ﻣﻦ ﻏﲑ ﰲ ﺇﺫﺍ ﺗﺴﺎﻭﺕ ﺎﺎﺤﺟ.)١( ﻛ ﳉﻮﺍﺯ ﻓﻴﻬﺎ ﺍ ﺎﻬﻴﻠﻋ .ﺎﻬﻨﻣﻭ: .ﺎﻬﻨﻣﻭ ﺃﻥ: ﻉ ﻭﺍﻟﻠﻐﺔ ﺮﻌﻟﺍﻭ ﺯ ﻋﻠﻴﻬﺎ ﻮﻬﲨ ﻮﻝ ﻋ ﻋﻦ ﺍﺑﻦ ﺠﻴﻓ ﻮﺯ ﺗﻔﺴ ﻚ ﺃﻳﻀﺎ، ﻭﳑﺎ ﲑﻩ ﲟﻘﺘﻀﻰ ﺍﻟﻠ ﺇ ﻥ ﺍﻟﺮﻭﺍﻳﺎﺕ ﺘﺪﻝ ﺩﻭﻥ ﺭﺟ ﻩﻮ : ﻥﺃ ﺭﻭﺍﻳﺔ ﺍﳉ ﻓﺘﻘﺪﻡ ﺗﻠﻚ ﻋ ﻋﻤﻠﻪ ﺑﺬﻟﻚ. ﺷﻬﺮ ﰲ ﺍﻟﺸﺮﻉ ﳌﻔﻴﺪﺓ ﺍﳉﻮﺍﺯ ﺔﻠﺑﺎﻨ: »ﻮﻘﻨﳌﺍ ﻘﺮﺁﻥ ﻋﺮﰊ، ﻲ ﺍﳌﻔﻴﺪ ﻟﺬﻟﻚ ﻻ ﳚﻮﺯ ﲑﺴﻔﺗ ﻳﻪ، ﻻﻥ ﻦ ﺍﳌﺴﺘ ﻦ ﻭﺟﻮ ﺎﻬﻨﻣ: ،ﺔﻠﻤﺘ ﻪ ﻣﻦ ﻮ ﺍﻷﺷﻬ ﺮﻭﺍﻳﺔ ﺍ ﻤﺔ ﺍﳊﻨ ﻥﻷ ﻘﻟﺍ ﻲﺣﻮﺘﻔ ﺪﲪ- ﻻ ٢( ﻪﺑ.) . ﻱﺪﻣﻵﺍ ٠٥/٣: ٥١٣ ﻡﺎﻜﺣﻹﺍﻭ : -٢٧٩ . . ١ . ١ ﻥ : ﲏﻳﻮﳉﺍ ٣/١: ﻟﺍ ﻔ ﺤﻮﻝ ٨٧٢ : - ﺐ ﲑﻨﳌﺍ ٨٥/٢: ﺭ ﻖﺑﺎﺴﻟﺍ ٨٥/٢: )١( ﻥﺎﻫﱪﻟﺍ )٢( ﺩﺎﺷﺭﺇ )٣( ﺍﻟﻜﻮﻛﺐ )٤( ﺭﺪﺼﳌﺍ  òÌÜÛa @ À @ ÐnÛa pb @ Þìy @ òîvy @a @@@@@@@ bèj’ : »ﻝﻭ «.) (٢ ﺎﻬ« «.)ﺎﻬ« ﻐﻮﻱ ﺃﻥ ﺓﺪ.)٢( ﻪﺑﺍ.)٣( ﺩ ﺍﳋﺎﻣﺲ ﺍﻟﻌﺪﺩ ﻴﻪ ﻓﻴﻪ . ٢١٨ . ﻷﺩﺑﺎﺀ ﻭﱂ ﺍﻗﻒ ﻋﻠﻴﻪ . ٢٣ ﺭ ﺷﺎﻫﲔ :ﻭ ٧٢١ ٨ ﺣﻮﻟﻴﺔ ﻧﻘﻠﻪ ﻋﻦ ﻣﻌﺠﻢ ﺍﻷ ﺕ ﺍﳌﻔﺴﺮﻳﻦ :٦/٢ : ﻥ ﺩ. ﻋﺒﺪ ﺍﻟﺼﺒﻮﺭ •ﺣ ﻭﻱ ﺍﳉﻮﻳﲏ: ٥٨ ٢ ﺮﻈﻨﻳﻭ : ﻃﺒﻘﺎﺕ ٢١١ ﻭﺗﺎﺭﻳﺦ ﺍﻟﻘﺮﺁ ﰲ ﺍﻟﺘﻔﺴﲑ : ﻭﺎﺼﻟﺍ ﺍﻷﺩﺑﺎﺀ ٥٥/ ٦١ : ﺳﺖ :ﺍﺑﻦ ﺍﻟﻨﺪﱘ ٢: )١( ﺞﻫﺎﻨﻣ )٢( ﻢﺠﻌﻣ )٣( ﺳﺮﻬﻔﻟﺍ ﺩ ﺍﳋﺎﻣﺲ ﺍﻟﻌﺪﺩ ﻴﻪ ﻓﻴﻪ . ٢١٨ . ﻷﺩﺑﺎﺀ ﻭﱂ ﺍﻗﻒ ﻋﻠﻴﻪ . ٢٣ ﺭ ﺷﺎﻫﲔ :ﻭ ٧٢١ ٨ ﺣﻮﻟﻴﺔ ﻧﻘﻠﻪ ﻋﻦ ﻣﻌﺠﻢ ﺍﻷ ﺕ ﺍﳌﻔﺴﺮﻳﻦ :٦/٢ : ﻥ ﺩ. (٢) ﻪﺑ . ﻋﺒﺪ ﺍﻟﺼﺒﻮﺭ •ﺣ ﻭﻱ ﺍﳉﻮﻳﲏ: ٥٨ ٢ ﺮﻈﻨﻳﻭ : ﻃﺒﻘﺎﺕ ٢١١ ﻭﺗﺎﺭﻳﺦ ﺍﻟﻘﺮﺁ ﰲ ﺍﻟﺘﻔﺴﲑ : ﻭﺎﺼﻟﺍ ﺍﻷﺩﺑﺎﺀ ٥٥/ ٦١ : ﺳﺖ :ﺍﺑﻦ ﺍﻟﻨﺪﱘ ٢: )١( ﺞﻫﺎﻨﻣ )٢( ﻢﺠﻌﻣ )٣( ﺳﺮﻬﻔﻟﺍ ﺩ ﺍﳋﺎﻣﺲ ﺎﻡ ﺃﲪﺪ ﻣﻦ ،ﺓ ﻭﺍﻟﻠﻐﻮﻳﲔ ﺝ ﺑﺎﻟﻠﻐﺔ ﰲ ﻪ ﻋﻨﺪ ﺗﻮﻓﺮ ﺗﺄﻟﻴﻔﻪ ﻛﺘﺎﺑﻪ ﺮﻳﺐ ﺍﻟﻘﺮﺁﻥ ﺎﻬﺟﺎﺘﳛ ﻢﻬﻓ ﻜﺘﺎﺑﻪ ﺍﻵﻧﻒ ﻪ.)٢( ﺩﺪﺷﻭ ﻲ ﻭﺃﰊ ﺣﺎﰎ ﺑﺎﻟﺮﺃﻱ ﻣﺜﻞ ﻈﻦ ﺃﻥ ﺫﻟﻚ ﺍﻟﻌﺪﺩ ، ﻓﻤﻨﻌﻪ ﺍﻹﻣﺎ ﻝﺍﻮﻗﺃ ﺓﺎﺤﻨﻟﺍ ﺻﺤﺔ ﺍﻻﺣﺘﺠﺎ ﻚﻟﺫ ﻪﺘﺤﺻﻭ ﻋﺒﻴﺪﺓ ﻟﺪﻯ ﺮﺴﻔﻳ ﻓﻴﻪ ﻏﺮ ﺤﻮﻳﺔ ﺍﻟﱵ ﳛ ﺑﻌﺪ ﺗﺄﻟﻴﻔﻪ ﻟﻜ ﻭﺑﻴﺎﻥ ﻪﺌﻄﺧ ﺀ ﻭﺍﻷﺻﻤﻌﻲ ﻳﻔﺴﺮ ﺍﻟﻘﺮﺁﻥ ﻲ.)٤( ﺪﻗﻭ ﻈﻳ ١٧٥-١٧٦ . ﺎﻤﻴﺳ ﺫﺍﻮﺸﻟﺍ ﺍﻟﺼﺤﻴﺢ ﻻ ﺐ ﺇﱃ ﻡﺪﻋ ﺻ ﺫ ﻋﻤﻼ ﺟﻮﺍﺯ ﺀﺎﻤﻠﻌﻟﺍ ﰊﻷ ﺮﻳﺐ ﺍﻟﻘﺮﺁﻥ، ﺾ ﺍﳌﺴﺎﺋﻞ ﺍﻟﻨﺤ ﻤﺎﺀ ﻋﺼﺮﻩ ﺑ ﻪﺒﻳﺬ ﺪﻌﺑ ، ﺃﻣﺜﺎﻝ ﺍﻟﻔﺮﺍﺀ ﻳ ﻭﺻﻔﻮﻩ ﺑﺄﻧﻪ ﻦﻋ ﻲﻌﻤﺻﻷﺍ ٢٥٠ . ﻃﺒﻘﺎﺕ ﺍﻟﻨﺤﻮﻳﲔ: - . ٧١ ﺣﻮﻟﻴﺔ ﺳ ﻰ ﺑﻌﺾ، ﻻ ﺕ ﻫﻮ ﺍﻟﻨﻘﻞ . ﲪﺪ ﱂ ﻳﺬﻫﺐ ﻋﻨﻪ ﻗﻮﻻ ﻭﻋ ﺍ ﻧﻘﺪ ﺑﻌﺾ ﰲ ﺗﻔﺴﲑ ﻏﺮ ﺭﺓ ﺇﱃ ﺑﻌﺾ ﲔ ﺑﻌﺾ ﻋﻠﻤ ﺘﺎﺑﻪ ﻫﺬﺍ ﺇﻻ ،ﻭﻣﻨﻬﺠﻪ ﻓﻴﻪ ﻦﻳ، )٣( ﱴﺣ ﻭ ﻭﺍﻩ ﺍﻟﺘﻮﺯﻱ ﻋ :٢٤٣ -٢٤٤ ﻭ ٠ ﲔﻛﺰﺳ :١٧٦ ﻃﻭ ﺯ ﺍﻟﻘﺮﺁﻥ :٦١/١ •ﺣ ﺑﻌﻀﻬﺎ ﻋﻠﻰ ﻮﺕ ﺍﻟﻘﺮﺍﺀﺍﺕ ﻦ ﳏﻞ ﺍﻟﱰﺍﻉ ﺃﻥ ﺍﻹﻣﺎﻡ ﺃﲪ ﻋ ﺑﻞ ﺍﻟﺜﺎﺑﺖ ﻣﺎ ﺭﻭﻱ ﻣﻦ ﺃﻭﻝ ﻣﺼﻨﻒ ﺏ، ﻣﻊ ﺇﺷﺎﺭ ﺼﻮﻣﺔ ﺑﻴﻨﻪ ﻭﺑﲔ ﻬﻢ ﻗﺮﺍﺀﺓ ﻛﺘﺎ ﰲ ﻣﺴﻠﻜ ﻭ ﻪ ﻭﺍﳌﱪﺩ ﻭﺁﺧﺮﻳﻦ ﻦ ﺍﳌﱪﺩ ﻭﺭﻭ ﻟﺪﺭﺍﺳﺎﺕ ﺍﻟﻨﺤﻮﻳﺔ ﲔﻳ :١٦٧ . ﺯ ﺍﻟﻘﺮﺁﻥ :(ﺳ ﻓﺆﺍﺩ ﺪﻘﻣﻭ ﻣﺔ ﳏﻘﻖ ﳎﺎﺯ ﺕ ﺃﻭ ﻟﺘﺮﺟﻴﺢ ﻷﻥ ﻃﺮﻳﻖ ﺛﺒﻮ ﻀﺎ ﺧﺎﺭﺝ ﻋﻦ ﻫﺬﺍ ﻳﺘﺒﲔ ﻟﻨﺎ ﰲ ﺍﳉﻤ ،ﺔﻠ ﺔﻣﺯﻼﻟﺍ . ﺔﻬ ﺔﻴﻧﺎﺜﻟﺍ : ﻘﺮﺁﻥ(، ﻭﻫﻮ ﻭﻛﻼﻡ ﺍﻟﻌﺮﺏ ( ﺣﺪﺛﺖ ﺧﺼ ﻓﺤﺮﻡ ﺑﻌﻀﻬ ﺍﻟﻨﻜﲑ ﻋﻠﻴﻪ ﺝ ﺎﺤﻨﻟﺍﻭ ﺱ ﻭ ﺍﻟﺒﻐﺪﺍﺩﻱ ﻋﻦ ﱘﺮﻜﻟﺍ ﻩﺮﺛﺃﻭ ﰲ ﺍﻟ ﺕ ﺍﻟﻨﺤﻮﻳﲔ ﻭﺍﻟﻠﻐﻮﻳﲔ ﻖﻘﳏ ﻛﺘﺎﺏ ) ﺯﺎﳎ ﺑﻐﺪﺍﺩ :١٣ / ٢٥٥ ﺍﺮﻘﻟﺍ ﺕﺍﺀ ﻷ ،ﻚﻟﺫ ﻭﻫﺬﺍ ﺃﻳﻀ ﺪﻌﺑ ﻫ ﲑﺴﻔﺘﻟﺍ ﻪﻃﻭﺮﺷ ﻬﺒﺸﻟﺍ )ﳎﺎﺯ ﺍﻟﻘ ﺮﻌﺸﻟﺎﺑ ﲎﻌﳌﺍ .)١( ﺪﻗﻭ ،ﺮﻛﺬﻟﺍ ﻥﻭﺮﺧﺁ ﺝﺎﺟﺰﻟﺍﻭ ﻣﺎ ﺭﻭﺍﻩ )١( ﻥﺁﺮﻘﻟﺍ )٢( ﻃﺒﻘﺎﺕ )٣( ﺔﻣﺪﻘﻣ )٤( ﺦﻳﺭﺎﺗ ﺎﻣ ﻭﺃ ﺏ ﺔﻣﺯﻼﻟﺍ . ﺔﻬ ﺔﻴﻧﺎﺜﻟﺍ : ﻘﺮﺁﻥ(، ﻭﻫﻮ ﻭﻛﻼﻡ ﺍﻟﻌﺮﺏ ( ﻪﻃﻭﺮﺷ ﻬﺒﺸﻟﺍ )ﳎﺎﺯ ﺍﻟﻘ ﺮﻌﺸﻟﺎﺑ ﲎﻌﳌﺍ .)١( ﻪﺠﻬﻨﻣﻭ ﻦ، )٣( ﻭ ﻦ ﻱ)ﻱ . (٢ ﺩ ﺍﳋﺎﻣﺲ ﺍﻟﻌﺪﺩ ٦٢٣ /٢: ﻱ . ﻦﻳﺮﺴﻔﳌﺍ : ﺩﻭﺍﺪﻟﺍ ﺣﻮﻟﻴﺔ ﻦﻳ :٢/ ٣٢٧ . ١/ ٢٥٥ ﺒﻃﻭ ﻘﺎﺕ •ﺣ ١ ﻭﻃﺒﻘﺎﺕ ﺍﳌﻔﺴﺮﻳ ، ﺗﺎﺭﻳﺦ ﺑﻐﺪﺍﺩ ٣: ﺕ ﲔﻳﻮﺤﻨﻟﺍ ٥٧١ : ﺕ ﲔﻳﻮﺤﻨﻟﺍ : ١٧٦ )١( ﻃﺒﻘﺎﺕ )٢( ﻃﺒﻘﺎﺕ ﺩ ﺍﳋﺎﻣﺲ ﺍﻟﻌﺪﺩ ٦٢٣ /٢: ﻱ . ﻦﻳﺮﺴﻔﳌﺍ : ﺩﻭﺍﺪﻟﺍ ﺣﻮﻟﻴﺔ ﻦﻳ :٢/ ٣٢٧ . (٢) ﻪﺑ . ١/ ٢٥٥ ﺒﻃﻭ ﻘﺎﺕ •ﺣ ١ ﻭﻃﺒﻘﺎﺕ ﺍﳌﻔﺴﺮﻳ ، ﺗﺎﺭﻳﺦ ﺑﻐﺪﺍﺩ ٣: ﺕ ﲔﻳﻮﺤﻨﻟﺍ ٥٧١ : ﺕ ﲔﻳﻮﺤﻨﻟﺍ : ١٧٦ )١( ﻃﺒﻘﺎﺕ )٢( ﻃﺒﻘﺎﺕ ﺎﻬ.)١( ﺤﻪ.) (٢ (٣).ﺪﻩﻓ ﺩ ﺍﳋﺎﻣﺲ ﻮﺤﻨ ﰊﺮﻌﻟﺍ ٣٨ : ﺍﻟﻌﺪﺩ ١٧ . ﻭﺍﻟﻘﺮﺍﺀﺍﺕ ﰲ ﻨﻟﺍ ﺎﺯ ﳏﻘﻖ ﺍ ٧/١: ٢٥٥ . ٧٤٢ ﻭﺃﺛﺮ ﺍﻟﻘﺮﺁﻥ ﺣﻮﻟﻴﺔ ١٣/٢٥٥ ﺔﻣﺪﻘﻣﻭ ﻢ ﺍﻷﺩﺑﺎﺀ :٥/ ٦١ ﺔﻳ :١٢٤ ﻭ١٨٣ ﻭ ٧ •ﺣ ، ﺗﺎﺭﻳﺦ ﺑﻐﺪﺍﺩ ٣: -٨٤ ﺮﻈﻨﻳﻭ : ﻢﺠﻌﻣ ﺍﻟﺪﺭﺍﺳﺎﺕ ﺍﻟﻨﺤﻮﻳ / . ٩١ﻭ٥١ ﺕ ﲔﻳﻮﺤﻨﻟﺍ : ١٧٦ ﺎﺯﳏﻘﻖ ﺍ ٣٨ : - ﺍﻟﻜﺮﱘ ﻭﺃﺛﺮﻩ ﰲ ﳏﺔ ﺎﺯ ﻘﻖ ﺍ ١: / )١( ﻃﺒﻘﺎﺕ )٢( ﺔﻣﺪﻘﻣ )٣( ﻥﺁﺮﻘﻟﺍ ﺔﻣﺪﻘﻣﻭ ﺩ ﺍﳋﺎﻣﺲ ﻮﺤﻨ ﰊﺮﻌﻟﺍ ٣٨ : ﺍﻟﻌﺪﺩ ١٧ . ﻭﺍﻟﻘﺮﺍﺀﺍﺕ ﰲ ﻨﻟﺍ ﺎﺯ ﳏﻘﻖ ﺍ ٧/١: ٢٥٥ . ٧٤٢ ﻭﺃﺛﺮ ﺍﻟﻘﺮﺁﻥ ﺣﻮﻟﻴﺔ ١٣/٢٥٥ ﺔﻣﺪﻘﻣﻭ ﻢ ﺍﻷﺩﺑﺎﺀ :٥/ ٦١ ﺔﻳ :١٢٤ ﻭ١٨٣ ﻭ ٧ •ﺣ ، ﺗﺎﺭﻳﺦ ﺑﻐﺪﺍﺩ ٣: -٨٤ ﺮﻈﻨﻳﻭ : ﻢﺠﻌﻣ ﺍﻟﺪﺭﺍﺳﺎﺕ ﺍﻟﻨﺤﻮﻳ / . ٩١ﻭ٥١ ﺕ ﲔﻳﻮﺤﻨﻟﺍ : ١٧٦ ﺎﺯﳏﻘﻖ ﺍ ٣٨ : - ﺍﻟﻜﺮﱘ ﻭﺃﺛﺮﻩ ﰲ ﳏﺔ ﺎﺯ ﻘﻖ ﺍ ١: / )١( ﻃﺒﻘﺎﺕ )٢( ﺔﻣﺪﻘﻣ )٣( ﻥﺁﺮﻘﻟﺍ ﺔﻣﺪﻘﻣﻭ ﻮﺤﻨ ﰊﺮﻌﻟﺍ ٣: ١٧ . ﻭﺍﻟﻘﺮﺍﺀﺍﺕ ﰲ ﻨﻟﺍ ﺎﺯ ﳏﻘﻖ ﺍ ٧/١: ٢٥٥ . ٧٤٢ ﻭﺃﺛﺮ ﺍﻟﻘﺮﺁﻥ ١٣/٢٥٥ ﺔﻣﺪﻘﻣﻭ ﻢ ﺍﻷﺩﺑﺎﺀ :٥/ ٦١ ﺔﻳ :١٢٤ ﻭ١٨٣ ﻭ ٧ ، ﺗﺎﺭﻳﺦ ﺑﻐﺪﺍﺩ ٣: -٨٤ ﺮﻈﻨﻳﻭ : ﻢﺠﻌﻣ ﺍﻟﺪﺭﺍﺳﺎﺕ ﺍﻟﻨﺤﻮﻳ / . ٩١ﻭ٥١ ﺕ ﲔﻳﻮﺤﻨﻟﺍ : ١٧٦ ﺎﺯﳏﻘﻖ ﺍ ٣٨ : - ﺍﻟﻜﺮﱘ ﻭﺃﺛﺮﻩ ﰲ ﳏﺔ ﺎﺯ ﻘﻖ ﺍ ١: / )١( ﻃﺒﻘﺎﺕ )٢( ﺔﻣﺪﻘﻣ )٣( ﻥﺁﺮﻘﻟﺍ ﺔﻣﺪﻘﻣﻭ ﰊﺮﻌ :  òÌÜÛa @ À @ ÐnÛa pb @ Þìy @ òîvy @a @@@@@@ bèj’ ﺩ ﺍﳋﺎﻣﺲ ﰲ ،ﺻﺤﻴﺤﻪ ﻓﺘﺢ ﺍﻟﺒﺎﺭﻱ، ﺇﻻ ﺍﻟﺸﻲﺀ ﻦ ﺃﻥ ﺍﻹﻣﺎﻡ ﻪﺑﺎ)ﲑﺴﻔﺘﻟﺍ - ﻰﻠﻋ ﺔﻐﻠﻟﺍ ﻋﻬﺪ ﺍﻟﺘﺎﺑﻌﲔ ﻐﺔ، ﻓﺄﻧﻜﺮﻭﺍ «.)٤( ﺭﺎﺷﺃﻭ ﺪﻣﺔ ﺗﻔﺴﲑﻩ: . ٣ ﺍﻟﻌﺪﺩ ﰲ ﻱﺭﺎﺨﺒﻟﺍ ﻓ ﻦ ﺣﺠﺮ ﰲ ﻲ ﻋﻦ ﺍﻟﻌﺮﺏ ﲔ ﺍﻟﺬﻫﱯ ﻣﻦ ﺟﺎﺀﺪ ﰲ ﻛﺘﺎ ﺎ ﺍﺑﻦ ﻋﺒﺎﺱ- ﻋ ﻄﺮﻳﻘﺔ ﺇﱃ ﺎﺀ ﻭﺃﻫﻞ ﺍﻟﻠﻐ ﺻﻼ ﻟﻠﻘﺮﺁﻥ« ﻮﺭﻱ ﰲ ﻣﻘﺪ . ﻱﺩﻭﺍﺪﻟﺍ ٨٢/٢: ﻣﻨﻪ، ﻣﻨﻬﻢ: ﺍﻟﻐﺮﻳﺐ، ﻭﺍﺑﻦ ﻪﻧﺇ ﻻ ﻲﻜﳛ ﻕ«.) (٣ ﳏﻤﺪ ﲔﺴﺣ ﺍﻟﺘﻔﺴﲑ، ﻓﻘﺪ ﺑﺔ-ﻻ ﺳﻴﻤﺎ ﻤﺮﺕ ﻫﺬﻩ ﺍﻟﻄ ﻮﺭﻋﻲ ﺍﻟﻔﻘﻬﺎ ﻌﻠﺘﻢ ﺍﻟﺸﻌﺮ ﺃﺻ ﻹﻣﺎﻡ ﺍﻟﻨﻴﺴﺎﺑﻮ ﺸﺮ ﺇﱃ ﻏﲑﻩ. ﻃﺒﻘﺎﺕ ﺍﳌﻔﺴﺮﻳﻦ: ﺣﻮﻟﻴﺔ ﻣ ﻪ ﻭﺃﺧﺬﻭﺍ ﰲ ﺍﳌﺸﻜﻞ ﻭ ﲏﻳﺪﳌﺍ » : ﺩ : »ﻮﻫ ﻕﺪﺻ ﺷﺎﺭ ﺇﻟﻴﻬﺎ ﺩ. (٢) ﻪﺑ . ﺍ ﺝ ﺑﺎﻟﻠﻐﺔ ﰲ ﻋﺘﻤﺎﺩ ﺍﻟﺼﺤﺎ :»ﺪﻗﻭ ﻤﺘﺳﺍ ﺼﻮﻣﺔ ﺑﲔ ﻣﺘﻮ ﻌﻠﺘﻢ ﺫﻟﻚ ﺟﻌ ﻗﺎﻝ ﺑﺬﻟﻚ ﺍ :( ٦/١ ﻭﱂ ﻳﺸ ﺩﺍﺪﻐ:١٣ / ٢٥٢ ﻃﻭ •ﺣ ﻋﺘﻤﺪﻭﺍ ﻛﺘﺎﺑﻪ ﰲ ﻭﺍﺑﻦ ﻗﺘﻴﺒﺔ ﱴ ﻗﺎﻝ ﻋﻨﻪ ﻋﻨﻪ ﺃﺑﻮ ﺩﺍﻭﺩ ﻭﻫﻲ ﺍﻟﱵ ﺃﺷ ﻮﻥ ﺍﻻﺣﺘﺠﺎﺝ ﻛﻼﻣﻪ ﻋﻦ ﺍﻋ ﻘﺮﺁﻥ، ﻗﻮﻟﻪ: ﺣﺪﺛﺖ ﺼﺧ ﻭﻗﺎﻟﻮﺍ ﺇﻥ ﻌﻓ ﺺ ﺇﱃ ﺃﻥ ﳑﻦ ﺎﺋﺐ ﺍﻟﻔﺮﻗﺎﻥ: ١ ﺮﻈﻨﻳﻭ : ﺗﺎﺭﻳﺦ ﺑﻐ ٢ . . ٢٣ / . ٠٨ ﻌﻠﻤﺎﺀ ﻗﺪ ﺍﻋ ،ﻱ ﰲ ﺗﻔﺴﲑﻩ ﲑﺜﻛ ﺣﱴ . (١) ﺢ«.)٢( ﻝﺎﻗﻭ ﺔﻬ ﺔﺜﻟﺎﺜﻟﺍ : ﻭ ﺭﻱ ﳑﻦ ﳝﻨﻌﻮ ﻭﻥ (ﻛ ﻋﻘﺐ ﲑ ﻏﺮﻳﺐ ﺍﻟﻘ ﻬﻢ، ﺇﱃ ﺃﻥ ،ﻫﺬﻩ ﺍﻟﻄﺮﻳﻘﺔ ﺶ ﻫﺬﺍ ﺍﻟﻨﺺ ﺍﻟﻘﺮﺁﻥ ﻭﺭﻏﺎ ﺎﺯﳏﻘﻖ ﺍ :١/١٧ ﺑﻐﺪﺍﺩ ٧٥٢ / ٣١ : ﺕ ﻦﻳﺮﺴﻔﳌﺍ ٨/٢: ﲑ ﻥﻭﺮﺴﻔﳌﺍﻭ ١: / ﻛﺒﺎﺭ ﺍﻟﻌ ﻱﱪﻄﻟﺍﻭ ﻢﻫﲑﻏﻭ ﺢﻴﺤﺼﻟﺍ ﻬﺒﺸﻟﺍ ﺭﻮﺑﺎﺴﻴﻨﻟﺍ ﻭﺮﺴﻔﳌﺍﻭ ﰲ ﺗﻔﺴﲑ ﻭﻣﻦ ﻳﻠﻴﻬ ﻫ ﻢﻬﻴﻠﻋ ﰲ ﻫﺎﻣﺶ ) ﻏﺮﺍﺋﺐ )١( ﺔﻣﺪﻘﻣ )٢( ﺦﻳﺭﺎﺗ )٣( ﻃﺒﻘﺎﺕ )٤( ﲑﺴﻔﺘﻟﺍ ﺩ ﺍﳋﺎﻣﺲ ﰲ ،ﺻﺤﻴﺤﻪ ﻓﺘﺢ ﺍﻟﺒﺎﺭﻱ، ﺇﻻ ﺍﻟﺸﻲﺀ ﻦ ﺃﻥ ﺍﻹﻣﺎﻡ ﻪﺑﺎ)ﲑﺴﻔﺘﻟﺍ - ﻰﻠﻋ ﺔﻐﻠﻟﺍ ﻋﻬﺪ ﺍﻟﺘﺎﺑﻌﲔ ﻐﺔ، ﻓﺄﻧﻜﺮﻭﺍ «.)٤( ﺭﺎﺷﺃﻭ ﺪﻣﺔ ﺗﻔﺴﲑﻩ: . ٣ ﺍﻟﻌﺪﺩ ﰲ ﻱﺭﺎﺨﺒﻟﺍ ﻓ ﻦ ﺣﺠﺮ ﰲ ﻲ ﻋﻦ ﺍﻟﻌﺮﺏ ﲔ ﺍﻟﺬﻫﱯ ﻣﻦ ﺟﺎﺀﺪ ﰲ ﻛﺘﺎ ﺎ ﺍﺑﻦ ﻋﺒﺎﺱ- ﻋ ﻄﺮﻳﻘﺔ ﺇﱃ ﺎﺀ ﻭﺃﻫﻞ ﺍﻟﻠﻐ ﺻﻼ ﻟﻠﻘﺮﺁﻥ« ﻮﺭﻱ ﰲ ﻣﻘﺪ . ﻱﺩﻭﺍﺪﻟﺍ ٨٢/٢: ﻣﻨﻪ، ﻣﻨﻬﻢ: ﺍﻟﻐﺮﻳﺐ، ﻭﺍﺑﻦ ﻪﻧﺇ ﻻ ﻲﻜﳛ ﻕ«.) (٣ ﳏﻤﺪ ﲔﺴﺣ ﺍﻟﺘﻔﺴﲑ، ﻓﻘﺪ ﺑﺔ-ﻻ ﺳﻴﻤﺎ ﻤﺮﺕ ﻫﺬﻩ ﺍﻟﻄ ﻮﺭﻋﻲ ﺍﻟﻔﻘﻬﺎ ﻌﻠﺘﻢ ﺍﻟﺸﻌﺮ ﺃﺻ ﻹﻣﺎﻡ ﺍﻟﻨﻴﺴﺎﺑﻮ ﺸﺮ ﺇﱃ ﻏﲑﻩ. ﻃﺒﻘﺎﺕ ﺍﳌﻔﺴﺮﻳﻦ: ﺣﻮﻟﻴﺔ ﻣ ﻪ ﻭﺃﺧﺬﻭﺍ ﰲ ﺍﳌﺸﻜﻞ ﻭ ﲏﻳﺪﳌﺍ » : ﺩ : »ﻮﻫ ﻕﺪﺻ ﺷﺎﺭ ﺇﻟﻴﻬﺎ ﺩ. ﺍ ﺝ ﺑﺎﻟﻠﻐﺔ ﰲ ﻋﺘﻤﺎﺩ ﺍﻟﺼﺤﺎ :»ﺪﻗﻭ ﻤﺘﺳﺍ ﺼﻮﻣﺔ ﺑﲔ ﻣﺘﻮ ﻌﻠﺘﻢ ﺫﻟﻚ ﺟﻌ ﻗﺎﻝ ﺑﺬﻟﻚ ﺍ :( ٦/١ ﻭﱂ ﻳﺸ ﺩﺍﺪﻐ:١٣ / ٢٥٢ ﻃﻭ •ﺣ ﻋﺘﻤﺪﻭﺍ ﻛﺘﺎﺑﻪ ﰲ ﻭﺍﺑﻦ ﻗﺘﻴﺒﺔ ﱴ ﻗﺎﻝ ﻋﻨﻪ ﻋﻨﻪ ﺃﺑﻮ ﺩﺍﻭﺩ ﻭﻫﻲ ﺍﻟﱵ ﺃﺷ ﻮﻥ ﺍﻻﺣﺘﺠﺎﺝ ﻛﻼﻣﻪ ﻋﻦ ﺍﻋ ﻘﺮﺁﻥ، ﻗﻮﻟﻪ: ﺣﺪﺛﺖ ﺼﺧ ﻭﻗﺎﻟﻮﺍ ﺇﻥ ﻌﻓ ﺺ ﺇﱃ ﺃﻥ ﳑﻦ ﺎﺋﺐ ﺍﻟﻔﺮﻗﺎﻥ: ١ ﺮﻈﻨﻳﻭ : ﺗﺎﺭﻳﺦ ﺑﻐ ٢ . . ٢٣ / . ٠٨ ﻌﻠﻤﺎﺀ ﻗﺪ ﺍﻋ ،ﻱ ﰲ ﺗﻔﺴﲑﻩ ﲑﺜﻛ ﺣﱴ . (٢) ﻪﺑ . (١) ﺢ«.)٢( ﻝﺎﻗﻭ ﺔﻬ ﺔﺜﻟﺎﺜﻟﺍ : ﻭ ﺭﻱ ﳑﻦ ﳝﻨﻌﻮ ﻭﻥ (ﻛ ﻋﻘﺐ ﲑ ﻏﺮﻳﺐ ﺍﻟﻘ ﻬﻢ، ﺇﱃ ﺃﻥ ،ﻫﺬﻩ ﺍﻟﻄﺮﻳﻘﺔ ﺶ ﻫﺬﺍ ﺍﻟﻨﺺ ﺍﻟﻘﺮﺁﻥ ﻭﺭﻏﺎ ﺎﺯﳏﻘﻖ ﺍ :١/١٧ ﺑﻐﺪﺍﺩ ٧٥٢ / ٣١ : ﺕ ﻦﻳﺮﺴﻔﳌﺍ ٨/٢: ﲑ ﻥﻭﺮﺴﻔﳌﺍﻭ ١: / ﻛﺒﺎﺭ ﺍﻟﻌ ﻱﱪﻄﻟﺍﻭ ﻢﻫﲑﻏﻭ ﺢﻴﺤﺼﻟﺍ ﻬﺒﺸﻟﺍ ﺭﻮﺑﺎﺴﻴﻨﻟﺍ ﻭﺮﺴﻔﳌﺍﻭ ﰲ ﺗﻔﺴﲑ ﻭﻣﻦ ﻳﻠﻴﻬ ﻫ ﻢﻬﻴﻠﻋ ﰲ ﻫﺎﻣﺶ ) ﻏﺮﺍﺋﺐ )١( ﺔﻣﺪﻘﻣ )٢( ﺦﻳﺭﺎﺗ )٣( ﻃﺒﻘﺎﺕ )٤( ﲑﺴﻔﺘﻟﺍ ١٦٧ ﺩ ﺍﳋﺎﻣﺲ ﺟﻌﺔ ﻣﻘﺪﻣﺔ ﻩ، ﺗﺒﲔ ﺃﻥ ﻓﻴﻪ ﺗﺼﺮﻳﺢ ﺮﺁﻥ ﻭﺇﻋﺮﺍﺑﻪ ﻗﻮﻟﻪ ﺗﻌﺎﱃ : h i n ﻧﻪ ﰲ ﺣﻜﻢ ﹺ ﻞﺴﹾ ﻠﺴ (ﻣﻔﻌﻮﻝ ﻟﻪ ﺲ ﺑﻌﺮ ،ﰊ ﺻﺤﻴﺢ ﺔﻐﻠﻟ : ،ﺍﺑﻦ ﺩﺭﻳﺪ ﻢﻜﶈﺍ : ﹺﻳﺺ ﺮﺒﻟﺍﻭ ﻝ ﰲ ﻳﺎﻗﻮﺕ ﺑﻌﺪ ﺎﻬﻌﲨﺄﺑ، ﺃﻻ ﺗﺮﺍﻩ ﺭﺩ ﺍﻟﱪﻳﺺ( ﻥﺎﺴﻟ ،  ﺍﻟﻘﺪﺡﹾﺖ ﻘﹶ ﻔﺻ َ ﺍﳌﺎﺀﹶﺖ ﺍﻟﺮﻳﺢ ﻘﹶ ﻔﺻ ﹰ ﹰ ﻭﴰﺎﻻ ﻪ ﳝﻴﻨﺎﺘﹶﺒ ﻠﹶ ﻗ ﻦ ﺛﺎﺑﺖ. ﺍﻟﻌﺪﺩ ، ﻓﻠﺪﻯ ﻣﺮﺍ ﰲ ﻩﲑﺴﻔﺗ ﻰ ﺍﻟﻌﻜﺲ، ﺴﲑ ﻟﻐﺔ ﺍﻟﻘﺮ ﻚ ﻳﻘﻮﻝ ﰲ e f g h ﺏ ﺍﻟﺼﻴﺐ ﻷ ﹺ ﺍﻟﺴ ﻖﻴﺣﺮﻟﺎﺑ ﻖ ﺕﻮﻤﹾ ﻟﺍ ﺭﹶ ﺬﺣ( ﻮﺍ، ﺑﺪﻣﺸﻖ؛ ﻭﻟﻴﺲ ﺑﺪﻣﺸﻖ. ﲨﻬﺮﺓ ﺍﻟ ﺸﻖﻣ ﰲ ﺩﺮ، ﰲﻭ ﺮ ﺑﺪﻣﺸﻖ  «ﻝﺎﻗ ﺑ ﱪﻳﺺ ﺍﱘ ﺍﻟﻐﻮﻃﺔ ﺮ ﺩﻣﺸﻖ ﻣﻦ ﻭﺭ ﻮﹸ ﻔﺼﻴﹺﻧﺎﺀ ﻟ ﹺﱃ ﺇ ﺀ ﺇ، ﻲﻌﻤﺻَ ﻝ ﺍﻷ ، ﻭﺻ ﺍﺫﹺ ﺍﻟﺸﻲﺀ ﺇﻳﺢ ﺍﻟﺮ ﻬﺎ ﺍﻟﺒﻴﺖ ﳊﺴﺎﻥ ﺑﻦ ،ﻱﺭﻮﺑﺎﺴﻴﻨﻟﺍ ﻈﺎﻥ ﺍﳌﺴﺄﻟﺔ ﻴ ﻪ، ﺑﻞ ﻋﻠﻰ ﺎ ﰲ ﺗﻔﺴ ﺝ ، ﻓﻤﻦ ﺫﻟﻚ c d e ﺇﱃ ﺃﺻﺤﺎﺏ ﻖﱠ ﻔﺼﻯ ﻳﺩﺮﺑ ﻖﻔﺼ.)١( ﻭ)ﺣ ﻳﺺ : ﻣﻮﺿﻊ، ﻗﺎﻟﻮ ﺑ ﺮ ﹶﻰ، ﻭﻫﻮ ﻠﻌﹶ ﻓ ﺮﺏ :( ﺮﻬ ﻧﹺﻳﺺ ﺮﺑﻭ ) ﻗﻮﻟﻪ » ﻭﺍﻟﱪﻳﺺ ﺪﻻﻥ ﻋﻠﻰ ﺃﻥ ﺍﻟﱪ ﻥ ﻣﺎﺀ ﺑﺮﺩﻯ ﻭﻫﻮ ﺀﺎﻧﹺ ﻟﻪ ﻣﻦ ﺇﹶﻪ ﺣﻮ ﻘﹶ ﻔ ﱟ ﰲ ﻗﻮ ﻥﹺﱃ ﺩ ﱟ ﺇ ﻥﺩ ﻄﺮﺏ، ﹶﺖ ﻘﱠ ﻔﺻﻭ ﻫﺬﻩ ﺍﳌﺼﺎﺩﺭ ﲨﻴﻌﻬ ﺣﻮﻟﻴﺔ  ﳌﻨﺴﻮﺏ ﺇﱃ ﺳﺮﻳﻊ ﰲ ﻣﻈ ﱂ ﻳﺬﻫﺐ ﺇﻟﻴ ﻪ ﰲ ﺍﻻﺣﺘﺠﺎﺝ ﺑﻌﺾ ﻣﻨﻬﺎ ` a b ﰲ)ﻥﻮﻠﻌﳚ ( ﻢﻬﻴﻠ ... ﺀ ﺑﺮﺩﻯ ﻳﺼ ﻱﺮﻫﺯﻷﺍﻭ : ﹺ ﺮﺒﻟﺍﻭ ﻞﺻﻷﺍ . ﻯﺩﺮﺑ ﺟﺎﺀ ﰲ)ﻟﺴﺎﻥ ﺍﻟﻌﺮ ﰲ ﺍﳍﺎﻣﺶ ﻪﻟﻮﻘﺑ : ﻭﻫﺬﺍﻥ ﺍﻟﺸﻌﺮﺍﻥ ﻳﺪ ﻥ ﻓﺎﻧﻪ ﻳﻘﻮﻝ ﻳﺴﻘﻮﻥ ﹶ ﻔﺻﹶ ﹶﻪ ﻭﺃ ﻘﱠ ﻔﹶﻪ ﻭﺻ ﻘﹶ ﻔ ﺩ ﹸ ﺍﻟﺸﺮﺍﺏ ﻣﻦ ﻞﻳﻮ ﹶ ﺃ ﻖﻔﹶ ﻄﺼﺘ ﻱ: ﻄﻀﺗ )ﻖﻔﺻ (. ﻫ ﻭﻧﺴﺒﺖ •ﺣ òÌÜÛa @ À @ ÐnÛa ﻫﻮ ﲢﻘﻴﻖ ﺍﳌ ﺳ ﻊ ﺍﺳﺘﻄﺮﺍﺩ ﺑﺬﻟﻚ ﻭﱂ ﺴﲑﻩ ﻃﺎﻓﺢ ﺔ، ﻧﺸﲑ ﺇﱃ ^ _ ` ﰲ ﻮﻉ ﺍﻟﻀﻤﲑ ﺮﻌﺷ : ﻋﻠﺮﻳﺺ ﺍﻟﺒﺭﺩ ﻥ ﺍﳌﻌﲎ :ﻣﺎ ١. ﻗﺎﻝ ﺍﺑﻦ ﺩﺭﻳﺪ ، ﻭﺃﺣﺴﺒﻪ ﺭﻭﻣﻲ : ﻣﺎﺩﺓ ﺑﺮﺹ ﻭﺟ . (٢) ﻪﺑ . ﺣﺐ ﺍﳊﺎﺷﻴﺔ ﻋﻠﻴﻪ ﻛﻮﺭﻳﻦ ﻣﺎ ﻧﺼﻪ: ﻭ ﺺ؟ ﻭﻛﺬﻟﻚ ﺣﺴﺎﻥ ﺼﻔﻖ، ﻓﻴﻘﺎﻝ :ﹶ ﻔﺻ ﲢﻮﻴﻖﻔﺼْﺗﻪ ﻭﺍﻟﺘ ﻸ ﺘﻓ ﺭﺎﺠﺷَ ﻷﺍ ﻖﻔﺼﺗ ١٠ / ٢٠٠ ، ﻣﺎﺩﺓ ) pb @ Þìy @ òîvy @a ﻢ ﻟﺪﻳﻨﺎ ﻫﻨﺎ ﻭﺧﺎﲤﺘﻪ ﻣﻊ ﺭﻱ ﱂ ﻳﻘﻞ ﺠﺎﺝ ﺎ، ﻭﺗﻔﺴ ﱐﺎﻌﳌ ﺔﻠﻤﺘﶈﺍ [ \ ] ﺯﺎﺟﻭ ﻮﺟﺭ ﻗﺎﻝ ﺣﺴﺎﻥ،  ﻭﺭﻦﹶ ﻣ ﻥﻮﹸ ﻘﺴ )ﻖﻔﺼﻳ ﻷﻥ ( ﺐ ﻥﺁﺮﻘﻟﺍ ٧٦/١: ﹼﻤﺖ ﺑﻪ ﺍﻟﻌﺮﺏ ﻠﻜ ﺐ ﺍﻟﻠﻐﺔ :ﺍﻷﺯﻫﺮﻱ ﻣﺸﻖ .ﻭﻋﻠﻖ ﺻﺎﺣ ﺫﻟﻚ ﻭﺍﻟﺒﻴﺘﲔ ﺍﳌﺬﻛ ﺎﺭ ﺇﱃ ﺍﻟﱪﻳﺺ ﺍﻷ ﻭ .٥/٧: ﺃﻣﺎ ﻳﺼ ﻸﹺﺫﺍ ﻣ ﻪ ﺇﹾﺘ ﻘﹶ ﻔﺻﹶ ﻪ ﻭﺃﺘ ﻪﺘﱠ ﻔﺼﻓ ، ﺢﻳﺮﻟﺍﻭﺗ ﻪ .ﻟﺴﺎﻥ ﺍﻟﻌﺮﺏ : @@@@@@@ bèj’ ﻢﻬﳌﺍﻭ ﻩﲑﺴﻔﺗ ﺭﻮﺑﺎﺴﻴﻨﻟﺍ ﺎﺠﺘﺣﻻﺎﺑ ﻭﺑﻴﺎﻥ ﳌﺍ o Z [ ﺓﺮﻘﺒﻟﺍ ٩١ : ،ﺭﻮﻛﺬﳌﺍ ﺴﻳ ﹶ ﺫ ﱠ ﻛﺮ ﻪﻟﻮﻘﻛ : )١( ﻏﺮﺍﺋﺐ ﻭﻗﺪ ﺗﻜ ﺬﻳﺐﻭ ﻣﺪﺑ ﺮ ﺫ ﺮﻛﺫ ﻧﺴﺐ ﺏﺮﻌﻟﺍ ﺘﹾ ﻘﱠ ﻔﺻﻭ ﻪﺘﺑﺮﺿ ﻪﺗﺩﺩﺭﻭ ﺲ ﺑﻌﺮ ،ﰊ ﺻﺤﻴﺢ ﺔﻐﻠﻟ : ،ﺍﺑﻦ ﺩﺭﻳﺪ ﻢﻜﶈﺍ : ﹺﻳﺺ ﺮﺒﻟﺍﻭ ﻝ ﰲ ﻳﺎﻗﻮﺕ ﺑﻌﺪ ﺎﻬﻌﲨﺄﺑ، ﺃﻻ ﺗﺮﺍﻩ ﺭﺩ ﺍﻟﱪﻳﺺ( ﻥﺎﺴﻟ ،  ﺍﻟﻘﺪﺡﹾﺖ ﻘﹶ ﻔﺻ َ ﺍﳌﺎﺀﹶﺖ ﺍﻟﺮﻳﺢ ﻘﹶ ﻔﺻ ﹰ ﹰ ﻭﴰﺎﻻ ﻪ ﳝﻴﻨﺎﺘﹶﺒ ﻠﹶ ﻗ ﻦ ﺛﺎﺑﺖ. ﻮﺍ، ﺑﺪﻣﺸﻖ؛ ﻭﻟﻴﺲ ﺑﺪﻣﺸﻖ. ﲨﻬﺮﺓ ﺍﻟ ﺸﻖﻣ ﰲ ﺩﺮ، ﰲﻭ ﺮ ﺑﺪﻣﺸﻖ  «ﻝﺎﻗ ﺑ ﱪﻳﺺ ﺍﱘ ﺍﻟﻐﻮﻃﺔ ﺮ ﺩﻣﺸﻖ ﻣﻦ ﻭﺭ ﻮﹸ ﻔﺼﻴﹺﻧﺎﺀ ﻟ ﹺﱃ ﺇ ﺀ ﺇ، ﻲﻌﻤﺻَ ﻝ ﺍﻷ ، ﻭﺻ ﺍﺫﹺ ﺍﻟﺸﻲﺀ ﺇﻳﺢ ﺍﻟﺮ ﻬﺎ ﺍﻟﺒﻴﺖ ﳊﺴﺎﻥ ﺑﻦ ﻳﺺ : ﻣﻮﺿﻊ، ﻗﺎﻟﻮ ﺑ ﺮ ﹶﻰ، ﻭﻫﻮ ﻠﻌﹶ ﻓ ﺮﺏ :( ﺮﻬ ﻧﹺﻳﺺ ﺮﺑﻭ ) ﻗﻮﻟﻪ » ﻭﺍﻟﱪﻳﺺ ﺪﻻﻥ ﻋﻠﻰ ﺃﻥ ﺍﻟﱪ ﻥ ﻣﺎﺀ ﺑﺮﺩﻯ ﻭﻫﻮ ﺀﺎﻧﹺ ﻟﻪ ﻣﻦ ﺇﹶﻪ ﺣﻮ ﻘﹶ ﻔ ﱟ ﰲ ﻗﻮ ﻥﹺﱃ ﺩ ﱟ ﺇ ﻥﺩ ﻄﺮﺏ، ﹶﺖ ﻘﱠ ﻔﺻﻭ ﻫﺬﻩ ﺍﳌﺼﺎﺩﺭ ﲨﻴﻌﻬ ﻱﺮﻫﺯﻷﺍﻭ : ﹺ ﺮﺒﻟﺍﻭ ﻞﺻﻷﺍ . ﻯﺩﺮﺑ ﺟﺎﺀ ﰲ)ﻟﺴﺎﻥ ﺍﻟﻌﺮ ﰲ ﺍﳍﺎﻣﺶ ﻪﻟﻮﻘﺑ : ﻭﻫﺬﺍﻥ ﺍﻟﺸﻌﺮﺍﻥ ﻳﺪ ﻥ ﻓﺎﻧﻪ ﻳﻘﻮﻝ ﻳﺴﻘﻮﻥ ﹶ ﻔﺻﹶ ﹶﻪ ﻭﺃ ﻘﱠ ﻔﹶﻪ ﻭﺻ ﻘﹶ ﻔ ﺩ ﹸ ﺍﻟﺸﺮﺍﺏ ﻣﻦ ﻞﻳﻮ ﹶ ﺃ ﻖﻔﹶ ﻄﺼﺘ ﻱ: ﻄﻀﺗ )ﻖﻔﺻ (. ﻫ ﻭﻧﺴﺒﺖ ١. ﻗﺎﻝ ﺍﺑﻦ ﺩﺭﻳﺪ ، ﻭﺃﺣﺴﺒﻪ ﺭﻭﻣﻲ : ﻣﺎﺩﺓ ﺑﺮﺹ ﻭﺟ . (٢) ﻪﺑ . ﺣﺐ ﺍﳊﺎﺷﻴﺔ ﻋﻠﻴﻪ ﻛﻮﺭﻳﻦ ﻣﺎ ﻧﺼﻪ: ﻭ ﺺ؟ ﻭﻛﺬﻟﻚ ﺣﺴﺎﻥ ﺼﻔﻖ، ﻓﻴﻘﺎﻝ :ﹶ ﻔﺻ ﲢﻮﻴﻖﻔﺼْﺗﻪ ﻭﺍﻟﺘ ﻸ ﺘﻓ ﺭﺎﺠﺷَ ﻷﺍ ﻖﻔﺼﺗ ١٠ / ٢٠٠ ، ﻣﺎﺩﺓ ) ﺐ ﻥﺁﺮﻘﻟﺍ ٧٦/١: ﹼﻤﺖ ﺑﻪ ﺍﻟﻌﺮﺏ ﻠﻜ ﺐ ﺍﻟﻠﻐﺔ :ﺍﻷﺯﻫﺮﻱ ﻣﺸﻖ .ﻭﻋﻠﻖ ﺻﺎﺣ ﺫﻟﻚ ﻭﺍﻟﺒﻴﺘﲔ ﺍﳌﺬﻛ ﺎﺭ ﺇﱃ ﺍﻟﱪﻳﺺ ﺍﻷ ﻭ .٥/٧: ﺃﻣﺎ ﻳﺼ ﻸﹺﺫﺍ ﻣ ﻪ ﺇﹾﺘ ﻘﹶ ﻔﺻﹶ ﻪ ﻭﺃﺘ ﻪﺘﱠ ﻔﺼﻓ ، ﺢﻳﺮﻟﺍﻭﺗ ﻪ .ﻟﺴﺎﻥ ﺍﻟﻌﺮﺏ : )١( ﻏﺮﺍﺋﺐ ﻭﻗﺪ ﺗﻜ ﺬﻳﺐﻭ ﻣﺪﺑ ﺮ ﺫ ﺮﻛﺫ ﻧﺴﺐ ﺏﺮﻌﻟﺍ ﺘﹾ ﻘﱠ ﻔﺻﻭ ﻪﺘﺑﺮﺿ ﻪﺗﺩﺩﺭﻭ  òÌÜÛa @ À @ ÐnÛa pb @ Þìy @ òîvy @a @@@@@ bèj’ ﻣﺎﺮ.) (١ ﻘﺎﻕ ﺍﻟﺼﻼ «.) (٢ nﺓﺮﻘﺒﻟﺍ ٦٢ : ﻮﻥ ﺍﳌﺬﻛﻮ ﹺ ﺗﻜﺮ ﹺ ﺍﻟﻠﺌﻴﻢ ﻢﺘﺷ ﻝﺎﻗ ﻭﺍﺷﺘﻘ » : ﺎ ﻭﺍﺭﺗﺴﻢ . ﺮﻌﺷ : ﹺ ﱘﺪﺘﺴﻤﻛ ﻙ« r s t n ﺠﺐ ﺃﻥ ﻳﻜﻮ ﺷ ﻋﻦﹺﺽ ﺮﻋﺃ ﻣﻮﺿﻊ ﺁﺧﺮ ﺻﻠﻰ ﺎﻋﻠﻰ ﺩ ﻬﺎ ﻭﻗﻮﻣﺘﻬﺎ، ﺼﺎﻙﱠﻰ ﻋ ﻠﺻ o p q r ﻲﺀ ﺍﳊﻘﲑ، ﻓﻴﺠ ... ﹸ ﺃﻭ )ﺓﻼﺼﻟﺍ ﻣ ( ﰲ ﺎ ...ﺻﻭ ﺑﺎﻟﻨﺎﺭ ﺇﺫﺍ ﻟﻴﻨﺘﻬ ﻪ ... ﺎﻤﻓ m n o ﻞﻴﺜﻤﺘﻟ ﻲﺸﻟﺎﺑ ﻩﺧﺎﺭﹺ ﺍﺩ ﱘﺮﻜ ﻔﺴﲑ ﻟﻔﻈﺔ) ﻰﺸﻋ : ﺎﺍﻟﺮﻳﺢ ﰲ ﺩ ﺻﻠﻴﺖ ﺍﻟﻌﺼﺎ ﻣ ﹺ ﺮﻙ ﻪﻣﺪﺘﺳﺍﻭ o j k l m ﱃ ﳝﻨﻊ ﻣﻦ ﺘﻟﺍ ﻋ ﺮﻔﹾ ﻏ َ ﺍﻟﻜ ﺀﺍﺭﻮ ﻤﺎ ﺗﻌﺮﺽ ﻔﺘﻟ ﺪﻋﺎﺀ ﻗﺎﻝ ﻋﻷﺍ ﺎﻬﻠﺑﺎﻘﻓ ﻦﻣ ﻢﳍﻮﻗ ﺻ: ﹾ ﺑﺄﻣ ﻞﺠﻌﻓﻼ ﺗ ﻪﻟﻮﻗ ﱃﺎﻌﺗ : o »ﻥﺇ ﺍﷲ ﺗﻌﺎﱃ ﹾ ﻏﹶ ﺃﻭ ﻤﻨﻴﺣﻭ ﲟﻌﲎ ﺍﻟﺪ ﺎﻣﺇﻭ ﻣ ﻓ ﰲﻭ ﻗ ﻝﻮﻘﻳ : » »: ﻮﻝﺇ» ﺩ ﺍﳋﺎﻣﺲ ٤ /٦١٢ ، ﻣﺎﺩﺓ ﻴﻪﻠﺮﻩ ﻭﻳﹶﻣ ﺃ ﺮﺑﺪﻳ . ﺍﻟﻨﺎﺭ ﻟﺘﺴﺘﻘﻴﻢ ، ١/ ٣١٢ ، ﻭ ﻟﺴﺎﻥ ﲑﺜ ﺍﺪﺟ. ﻭﺍﻟﺒﻴﺖ ﺍﻟﻜﺘﺎﺏ ﻟﺴﻴﺒﻮﻳﻪ : ﺓﺃﺮﻣ، ُﺀﺎﻴﹾ ﻠﻌﹾ ﻟﺍﻭ : = ﺍﻟﻌﺪﺩ ،ﻟﺴﺎﻥ ﺍﻟﻌﺮﺏ : ٤ ﻱﹶ ﺃﺼﺎ ﻓﻼﻥﻠﻲ ﻋ ﺎ ﻋﻠﻰﻌﺼﺎ ﺇﺩﺍﺭ ﻱﺮﺸ :١/ ١٤٣ ﻭ ٨ ﻭ٩٦ ﻭﻫﻮ ﲑﺜﻛ ﺍ ﻭﻭﺭﺩ ﺍﻟﺒﻴﺖ ﰲ ﺪﻣ .ﻭﹸ ﺔﻴﻣ: ﺍﺳﻢ ﺍﻣ ( ﺏ،ﳊﺎﰎ ﺍﻟﻄﺎﺋﻲ ﻝﺎﻘﻳﻭ : ﻲﱢ ﻠﺼﹲ ﻳ ﻥﻼﻓ ﹰ ﺪﺍﻳﻭ .ﹸ ﺍﻟﻌ ﺔﻴﻠﺼﺗﻭ ﺱ ﺍﻟﺒﻼﻏﺔ : ﺸﳐﺰﻟﺍ ـﺟﻭ ٣٠ /٨ ﻭ ٩ ﻟﻨﻌﻤﺎﻥ ﺑﻦ ﺍﳌﻨﺬﺭ . ﺍﻷﺑﺪ ﺑﺪﻻ ﻣﻦ ﺍﻷﻣ ﺣﻮﻟﻴﺔ ﺖ ﰲ)ﻟﺴﺎﻥ ﺍﻟﻌﺮ ﺑﻦ ﺯﻫﲑ ﺍﻟﻌﺒﺴﻲ، ﻭﹰ ﺭ ﻭﻳﺪﺍﻬﺎ ﺭﺘﺤﺴ ﻲﻧﹶ ﺄﻫﺎ ﻛﺎﻟﺘﺮﻣ .ﺃﺳﺎ ﻪﻴﻓ ﺎﻀﻳﺃ : ١٩٩ ﻭ ﺎﻥ ﺎ ﺇﱃ ﺍ ﻳﻌﺘﺬﺭ ) ﺪﺼﻗ (. ﻭﺭﻭﻱ •ﺣ ١٦ .ﻭﻧﺴﺐ ﺍﻟﺒﻴﺖ . ١ ﺑ ﺍﻟﺒﻴﺖ ﻟﻘﻴﺲ ﺴﻤﹶ ﹶﻬﺎ ﻓ ﻔﻴﻘﹾ ﺜﺗ ﺕﺩﺭ ﻱﺃ: ﻣﹶ ﺃ ﻢﹶ ﻜﺣﹶ ﻣﺎ ﺃ ) ﺩﻭﻡ، ﻭﻋﺼﺎ (. (٢) ﻪﺑ . ١-١٩٧ ﺮﻈﻨﻳﻭ ﻓ ﻝ ﻗﺼﻴﺪﺗﻪ ﱵﻟﺍ ﺎﻛ ﺏ :/ ٣ ٣٥٣ ﺓﺩﺎﻣ ﺐ ﻥﺁﺮﻘﻟﺍ ٨٦/١: . ﺐ ﻥﺁﺮﻘﻟﺍ ٤٣/١: ﺎﺼﻌﻟﺍ ﺖ : ﺇﺫﺍ ﺃﺭ ﺍﻣﺘﻬﺎ: ﻲ ﻓﻴﻬﺎﹶﻧ ﺄﺘﻟﺍ ، : ١٢ / ٢١٢ ﺓﺩﺎﻣ ﺐ ﻥﺁﺮﻘﻟﺍ ٦٩/١: ﺍ ﻟﺬﺑﻴﺎﱐ، ﻭﻫﻮ ﺃﻭﻝ ١ ، ﻭﻟﺴﺎﻥ ﺍﻟﻌﺮﺏ )١( ﻏﺮﺍﺋﺐ ) ﺭﻮﻋ( )٢( ﻏﺮﺍﺋﺐ ﻭ ﺖﻴﻠﺻ ﺍﺪﺘﺳﺍﻭ ﺏﺮﻌﻟﺍ )٣( ﻏﺮﺍﺋﺐ ﺔﻐﺑﺎﻨﻠﻟ ١/٥٦ ٤ /٦١٢ ، ﻣﺎﺩﺓ ﻴﻪﻠﺮﻩ ﻭﻳﹶﻣ ﺃ ﺮﺑﺪﻳ . ﺍﻟﻨﺎﺭ ﻟﺘﺴﺘﻘﻴﻢ ، ١/ ٣١٢ ، ﻭ ﻟﺴﺎﻥ ﲑﺜ ﺍﺪﺟ. ﻭﺍﻟﺒﻴﺖ ﺍﻟﻜﺘﺎﺏ ﻟﺴﻴﺒﻮﻳﻪ : ﺓﺃﺮﻣ، ُﺀﺎﻴﹾ ﻠﻌﹾ ﻟﺍﻭ : = ،ﻟﺴﺎﻥ ﺍﻟﻌﺮﺏ : ٤ ﻱﹶ ﺃﺼﺎ ﻓﻼﻥﻠﻲ ﻋ ﺎ ﻋﻠﻰﻌﺼﺎ ﺇﺩﺍﺭ ﻱﺮﺸ :١/ ١٤٣ ﻭ ٨ ﻭ٩٦ ﻭﻫﻮ ﲑﺜﻛ ﺍ ﻭﻭﺭﺩ ﺍﻟﺒﻴﺖ ﰲ ﺪﻣ .ﻭﹸ ﺔﻴﻣ: ﺍﺳﻢ ﺍﻣ ( ﺏ،ﳊﺎﰎ ﺍﻟﻄﺎﺋﻲ ﻝﺎﻘﻳﻭ : ﻲﱢ ﻠﺼﹲ ﻳ ﻥﻼﻓ ﹰ ﺪﺍﻳﻭ .ﹸ ﺍﻟﻌ ﺔﻴﻠﺼﺗﻭ ﺱ ﺍﻟﺒﻼﻏﺔ : ﺸﳐﺰﻟﺍ ـﺟﻭ ٣٠ /٨ ﻭ ٩ ﻟﻨﻌﻤﺎﻥ ﺑﻦ ﺍﳌﻨﺬﺭ . ﺍﻷﺑﺪ ﺑﺪﻻ ﻣﻦ ﺍﻷﻣ ﺖ ﰲ)ﻟﺴﺎﻥ ﺍﻟﻌﺮ ﺑﻦ ﺯﻫﲑ ﺍﻟﻌﺒﺴﻲ، ﻭﹰ ﺭ ﻭﻳﺪﺍﻬﺎ ﺭﺘﺤﺴ ﻲﻧﹶ ﺄﻫﺎ ﻛﺎﻟﺘﺮﻣ .ﺃﺳﺎ ﻪﻴﻓ ﺎﻀﻳﺃ : ١٩٩ ﻭ ﺎﻥ ﺎ ﺇﱃ ﺍ ﻳﻌﺘﺬﺭ ) ﺪﺼﻗ (. ﻭﺭﻭﻱ ١٦ .ﻭﻧﺴﺐ ﺍﻟﺒﻴﺖ . ١ ﺑ ﺍﻟﺒﻴﺖ ﻟﻘﻴﺲ ﺴﻤﹶ ﹶﻬﺎ ﻓ ﻔﻴﻘﹾ ﺜﺗ ﺕﺩﺭ ﻱﺃ: ﻣﹶ ﺃ ﻢﹶ ﻜﺣﹶ ﻣﺎ ﺃ ) ﺩﻭﻡ، ﻭﻋﺼﺎ (. ١-١٩٧ ﺮﻈﻨﻳﻭ ﻓ ﻝ ﻗﺼﻴﺪﺗﻪ ﱵﻟﺍ ﺎﻛ ﺏ :/ ٣ ٣٥٣ ﺓﺩﺎﻣ ﺐ ﻥﺁﺮﻘﻟﺍ ٨٦/١: . ﺐ ﻥﺁﺮﻘﻟﺍ ٤٣/١: ﺎﺼﻌﻟﺍ ﺖ : ﺇﺫﺍ ﺃﺭ ﺍﻣﺘﻬﺎ: ﻲ ﻓﻴﻬﺎﹶﻧ ﺄﺘﻟﺍ ، : ١٢ / ٢١٢ ﺓﺩﺎﻣ ﺐ ﻥﺁﺮﻘﻟﺍ ٦٩/١: ﺍ ﻟﺬﺑﻴﺎﱐ، ﻭﻫﻮ ﺃﻭﻝ ١ ، ﻭﻟﺴﺎﻥ ﺍﻟﻌﺮﺏ )١( ﻏﺮﺍﺋﺐ ) ﺭﻮﻋ( )٢( ﻏﺮﺍﺋﺐ ﻭ ﺖﻴﻠﺻ ﺍﺪﺘﺳﺍﻭ ﺏﺮﻌﻟﺍ )٣( ﻏﺮﺍﺋﺐ ﺔﻐﺑﺎﻨﻠﻟ ١/٥٦  òÌÜÛa @ À @ ÐnÛa pb @ Þìy @ òîvy @a @@@@@@@ bèj’  òÌÜÛa @ À @ ÐnÛa pb @ Þìy @ òîvy @a @@@@@@ bèj’ ﰒ ﺍﻟﺘﻔﺴﲑ ﻼﺣﻪ، ﻭﺇﲤﺎﻡ ﻭﻣﻊ ﻛﻞ ﻣﺎ ، ﻓﺈﻥ ﺫﻟﻚ ،ﻑ ﺍﳌﻌﻠﻼﺕ ﻣﺎ ﳚﺐ ﺇﺻﻼ ﻻ،ﺕﺎﺿﺍﺮﺘﻋ ﺕ ﺍﳌﻌﻘﺪﺍﺕ ﺕ، ﻭﺍﻟﻮﻗﻮﻑ ﻣﻊ ﺇﺻﻼﺡ ﲑ ﺍﻟﻜﺒﲑ (ﻭﺍﻻ ﻯﻮﺳ ﺎﻴﺑﻷﺍ ﺕﺍﺀ ﺕﺍﱪﺘﻌﳌﺍ ،ﺕﺎﻳﻮﻨﻌﳌﺍﻭ ﺓﰲ ) ﲑﺴﻔﺘﻟﺍ ،ﺕﻼﻀﻌﳌﺍ ﻭﺃﺛﺒﺖ ﺀﺍﺮﻘﻟﺍ ﺚ ﺍﻟﻠﻔﻈﻴﺎﺕ ﳌﺴﺎﺋﻞ ﺍﳌﻮﺭﺩﺓ ﻣﻦ ﺍﳌﻮﺍﺿﻊ ﺮﻱ، ﻝﺎﻗ:» ﻋﻠﻰ ﺍﳌﺒﺎﺣﺚ ﺇﲤﺎﻣﻪ ﻣﻦ ﺍﳌ ﰲ)ﺍﻟﻜﺸﺎﻑ ( ﺮﺸﳐﺰﻟﺍﻭ ﻞﻤﺘﺸﳌ ﺎ ﻳﻨﺒﻐﻲ ﰲ ﺪﺟﻮ ﻤ ﺎ.)٢( ﻤ ﺩ ﺍﳋﺎﻣﺲ ﹺ ﻞﺒﺠﹾ ﹺ ﺍﻟ ﻞ، ﺕﻮﹾ ﻗﹶ ﺃ : ﺍﻟﻌﺪﺩ : ﻞﺻﹶ ﻲ ﺃ ﻓﹶﻊ ﻔﺗﺮﻤﹾ ﻟﺍ ﺪﻣﻷﺍﻭ : ﺮﻫﺪﻟﺍ . (٢) ﻪﺑ . ﹸ ﺔﻴﻟﺎﻌﹾ ﺽ ﺍﻟ، ﺪﻨﺴﻟﺍﻭ: ﺪﺑَ ﺍﻷﹶ ﻭ ﻞﺧﺩ ﺪﺣ ﻦ ﺣﻮﻟﻴﺔ ﹺ ﺍﻷﺭﺽ ﻲ ﺍﻷﺻﻞ ﻓ ﺎﻟﻒ: ﻦﻲ ﻣﺎﺿﹾﻤ ﻟﺍ •ﺣ ﻚﻟﹶ ﺬﹶ ﻛ، ﺀﺎﻴﹾ ﻠﻌﹾ ﻟﺍﻭ ﹸ ﺔﻴﻟﺎﺨﹾ ﺽ ﺍﻟ، َﻭﺎﺴﻟﺍ ﻮﺭﻱ : . ٦/١ ٢ . ﻢ ﻣﻮﺿﻊ، ﺪﻨﺴﻟﺍﻭ : ﹶﺖ ﻠﺧ، ُ ﺍﻷﺭﺽ ﺀﺍﻮﹶ ﻘﹾ ﻟﺍﻭ ﺐ ﻥﺁﺮﻘﻟﺍ : ﻮﺑﺎﺴﻴﻨﻟﺍ ﺐ ﻥﺁﺮﻘﻟﺍ ٤٢٢ /٣: =ﻢﺳﺍ ﻱﹶ ﺃ: ﺧ )١( ﻏﺮﺍﺋﺐ )٢( ﻏﺮﺍﺋﺐ ﹺ ﻞﺒﺠﹾ ﹺ ﺍﻟ ﻞ، ﺕﻮﹾ ﻗﹶ ﺃ : : ﻞﺻﹶ ﻲ ﺃ ﻓﹶﻊ ﻔﺗﺮﻤﹾ ﻟﺍ ﺪﻣﻷﺍﻭ : ﺮﻫﺪﻟﺍ . ﹸ ﺔﻴﻟﺎﻌﹾ ﺽ ﺍﻟ، ﺪﻨﺴﻟﺍﻭ: ﺪﺑَ ﺍﻷﹶ ﻭ ﻞﺧﺩ ﺪﺣ ﻦ ﹺ ﺍﻷﺭﺽ ﻲ ﺍﻷﺻﻞ ﻓ ﺎﻟﻒ: ﻦﻲ ﻣﺎﺿﹾﻤ ﻟﺍ ﻚﻟﹶ ﺬﹶ ﻛ، ﺀﺎﻴﹾ ﻠﻌﹾ ﻟﺍﻭ ﹸ ﺔﻴﻟﺎﺨﹾ ﺽ ﺍﻟ، َﻭﺎﺴﻟﺍ ﻮﺭﻱ : . ٦/١ ٢ . ﻢ ﻣﻮﺿﻊ، ﺪﻨﺴﻟﺍﻭ : ﹶﺖ ﻠﺧ، ُ ﺍﻷﺭﺽ ﺀﺍﻮﹶ ﻘﹾ ﻟﺍﻭ ﺐ ﻥﺁﺮﻘﻟﺍ : ﻮﺑﺎﺴﻴﻨﻟﺍ ﺐ ﻥﺁﺮﻘﻟﺍ ٤٢٢ /٣: =ﻢﺳﺍ ﻱﹶ ﺃ: ﺧ )١( ﻏﺮﺍﺋﺐ )٢( ﻏﺮﺍﺋﺐ ﹺ ﻞﺒﺠﹾ ﹺ ﺍﻟ ﻞ، ﺕﻮﹾ ﻗﹶ ﺃ : : ﻞﺻﹶ ﻲ ﺃ ﻓﹶﻊ ﻔﺗﺮﻤﹾ ﻟﺍ ﺪﻣﻷﺍﻭ : ﺮﻫﺪﻟﺍ . ﹸ ﺔﻴﻟﺎﻌﹾ ﺽ ﺍﻟ، ﺪﻨﺴﻟﺍﻭ: ﺪﺑَ ﺍﻷﹶ ﻭ ﻞﺧﺩ ﺪﺣ ﻦ ﹺ ﺍﻷﺭﺽ ﻲ ﺍﻷﺻﻞ ﻓ ﺎﻟﻒ: ﻦﻲ ﻣﺎﺿﹾﻤ ﻟﺍ ﻚﻟﹶ ﺬﹶ ﻛ، ﺀﺎﻴﹾ ﻠﻌﹾ ﻟﺍﻭ ﹸ ﺔﻴﻟﺎﺨﹾ ﺽ ﺍﻟ، َﻭﺎﺴﻟﺍ ﻮﺭﻱ : . ٦/١ ٢ . ﻢ ﻣﻮﺿﻊ، ﺪﻨﺴﻟﺍﻭ : ﹶﺖ ﻠﺧ، ُ ﺍﻷﺭﺽ ﺀﺍﻮﹶ ﻘﹾ ﻟﺍﻭ ﺐ ﻥﺁﺮﻘﻟﺍ : ﻮﺑﺎﺴﻴﻨﻟﺍ ﺐ ﻥﺁﺮﻘﻟﺍ ٤٢٢ /٣: =ﻢﺳﺍ ﻱﹶ ﺃ: ﺧ )١( ﻏﺮﺍﺋﺐ )٢( ﻏﺮﺍﺋﺐ  òÌÜÛa @ À @ ÐnÛa pb @ Þìy @ òîvy @a @@@@@@@ bèj’ » ﺔﻴﺑ« ). (١ ﻪ) :ﻮﻟﻪﺳ ﺩ ﺍﳋﺎﻣﺲ ﺍﻟﻌﺪﺩ ﺣﻮﻟﻴﺔ •ﺣ - .٧ ﺭ ﻖﺑﺎﺴﻟﺍ ٦/ ١: - )١( ﺭﺪﺼﳌﺍ ﺩ ﺍﳋﺎﻣﺲ ﻭﻫﺬﺍ ﺃﻣﺮ ﻻ ﱯ- )١( ﻰﻠﻋ ﻚ، ﻓﻬﻮ ﻗﺪ ﻠﻰ ﻣﻌﺎﻥ ﻗﺪ ﻋﻠﻰ ﻣﺎ ﳚﺪﻩ ﺮﺏ، ﻣﻊ ﺃﻥ ﻘﺎﻡ ﰲ ﳎﺎﻝ ﺔﻐﻠﻟﺍ ﺎﻬﻠﻟﺫﻭ ﺮ ﺍﻟﻨﺤﻮ ﰲ ﻟﻴﺎ ﻓﺈﻧﺎ ﻧﺮﻯ ﻝﻮﻘ : »ﺪﻗﻭ ﺍﳌﻌﺘﺰﻟﺔ ﻋﻦ ﺍﻟﻌﺪﺩ ،ﺔﺣﺎﺼﻔﻟﺍﻭ ﺍﻟﺸﻴﺦ ﺍﻟﺬﻫﱯ ﺿﻮﻋﻴﺘﻪ ﻛﺬﻟﻚ ﻞ ﺍﻷﻟﻔﺎﻅ ﻋﻠ ﻋ ﻳﻞ، ﺍﻋﺘﻤﺎﺩﺍ ﰲ ﻛﻼﻡ ﺍﻟﻌﺮ ﺫﺇ ﺍ ﻛﺎﻥ ﺍﳌﻘ ﻡ ﻱﺮﺸﳐﺰﻟﺍ ﻱﺮﺸﳐ ﺮﺨﺴﻳ ﻬﺎ ﻣﺒﺪﺃ ﺍﻋﺘﺰﺍﻟ ﱄ«.)٤( ﻮﻘﻳﻭ ﺪﻣﺔ ﻓﻜﺮﺓ ﺤﺎﻧﻪ ﻳﻮﻡ ﺔﻣﺎﻴﻘﻟﺍ . ﻣﻦ ﺍﻟﺼﺤﺔ ﲔ- ﻢﻬﻨﻣﻭ ﻭﻋﻠﻰ ﻣﻮﺿ ﺼﻮﺹ، ﻭﳛﻤﻞ ﻇﺎﻫﺮ ﰲ ﺍﻟﺘﺄﻭﻳﻞ ﺎﺩﺭﺓ ﺍﻟﻮﺭﻭﺩ ، ﻭﻻ ﺳﻴﻤﺎ :)٣(» ﻡﺪﺨﺘﺳﺍ ﺔﻳﻮ : »ﺸﳐﺰﻟﺍﻭ ﻫﺎ ﺃﻭ ﺗﺄﻭﻳﻠﻬ ﻌﺘﻘﺪ ﺍﻻﻋﺘﺰﺍﱄ ﺨﻴﻴﻞ ﰲ ﺧﺪ ﺅﻳﺔ ﺍﳌﺆﻣﻨﲔ ﷲ ﺳﺒﺤ ﺣﻮﻟﻴﺔ ﻟﺒﻴﺎﻥ ﺩ ﺎﻬﺘﺟﺭ ﺪﻦﻣ ﲔﺜﺣﺎﺒﻟﺍ ﻋﻠﻰ ﺍﳌﻌﺎﱐ، ﻷﻟﻔﺎﻅ ﻭﺍﻟﻨﺼ ﻇﺎ ﻭ ﺑﺘﻌﺴﻒ ﰲ ﻧﻔﺴﻬﺎ، ﻧﺎ ﺽ ﺍﳌﺮﺍﺩ ﻣﻨﻪ ﻱﻭﺎﺼﻟﺍ ﻪﻨﻋ: ﺮﳚﺎﺗﻪ ﺍﻟﻨﺤﻮ ﺔ ﳝﺲ ﻇﺎﻫﺮ ﻼ ﻟﻴﻨﺼﺮ ﺍﳌﻌ ﺘﻤﺜﻴﻞ ﻭﺍﻟﺘﺨ ﰲ ﺗﺄﻭﻳﻠﻪ ﻟﻠﻤﺮﺍﺩ ﺑﺮﺅ . •ﺣ ﻰﻠﻋ ﻪﻤﻈﻧ ﺒﻟ ﻻﺣﻈﻪ ﺍﻟﻌﺪﻳﺪ ﻝﻻﺪﺘﺳﻻﺍ ﺴﲑﻩ ﻟﺒﻌﺾ ﺍﻷ ﺑﺒﻌﺪ ﻛﺒﲑ، ﺃ ﻛﺎﻧﺖ ﻏﺮﻳﺒﺔ ،ﻨﺎﻩ ﻭﺍﺳﺘﻔﺎﺽ ﱄ)٢(. ﺍﻳﻘﻮﻝ ﰲ ﳎﺎﻝ ﲣﺮ ﺍ ﻛﺎﻧﺖ ﺍﻵﻳﺔ ﻌﺴﻔﺎ ﻣﺘﻤﺤﻼ ﺃﺳﻠﻮﺏ ﺍﻟﺘ /٤٤٥ -٤٤٧ . ٤/ ١٩٢ ﻭ٢٣٢ ﰲ ﲑ ﺍﻟﻘﺮﺁﻥ :٢٤١ . . . ﻋﻠﻴﻬﺎ، ﻓﺘﻘﺎﺱ ﻴﻪ، ﻭﻫﻮ ﻣﺎ ﺰﳐﺸﺮﻱ ﰲ ﺣﻴﺎﻧﺎ ﰲ ﺗﻔﺴ ﻬﺎ ﻆﻔﻠﻟﺍ ﻻﺇ ﻫﺪ ﳍﺎ ﻥﺇﻭ ﻛ ﺪ ﺍﺷﺘﻬﺮ ﻨﻌﻣ ﺒﻫﺬ ﻪ ﱄﺍﺰﺘﻋﻻﺍ ﻝ« .ﻝﻮﻘﻳﻭ ﻻﻋﺘﺰﺍﻝ، ﻓﺈﺫﺍ ﻱ ﳓﻮﻳﺎ ﻣﺘﻌ ﳐﺰﻟﺍ ﻱﺮﺸ «.) (٥ ﲑ ﻥﻭﺮﺴﻔﳌﺍﻭ ١: / ﺗﻔﺴﲑ ﺍﻟﻜﺸﺎﻑ : ﺍﻟﺰﳐﺸﺮﻱ ﰲ ﺗﻔﺴﲑ ﻱﺮﺸﳐﺰﻟﺍ :١٤٧ ﺭ ﻖﺑﺎﺴﻟﺍ ٦٤١ : . ﻋ ﺔﺠﳊﺍ ﺟﺪﺍﻝ ﻓﻴ ﻣﻨﻬﺞ ﺍﻟﺰ ﻳﻐﺮﺏ ﺣﺃ ﻻ ﳛﺘﻤﻠﻬ ﻣﻦ ﺷﻮﺍﻫ ﺍﻟﻠﻔﻆ ﻗﺪ ﻧﺼﺮﺓ ﻣﺬ ﻝﺍﺰﺘﻋﻼﻟ ﺔﻣﺪﺧ ﻻﺍ ﻱﺮﺸﳐﺰﻟﺍ ﻡﺪﺨﺘﺳﺍ ﺪﻴﺣﻮﺘﻟﺍ« )١( ﲑﺴﻔﺘﻟﺍ )٢( ﺮﻈﻨﻳ ﺗ: )٣( ﺞﻬﻨﻣ ﺍ )٤( ﺞﻬﻨﻣ ﺍ )٥( ﺭﺪﺼﳌﺍ ﱯ- )١( ﱯ ﻘﺎﻡ ﰲ ﳎﺎ ﺔﻐﻠﻟﺍ ﻬﻠﻟﺫﻭ ﺮ ﺍﻟﻨﺤﻮ ﻟﻴﺎ ﻓﺈﻧﺎ ﻧﺮ ﻝﻮﻘ : »ﻗﻭ ﺫﺇ ﺍ ﻛﺎﻥ ﺍﳌﻘ ﻡ ﻱﺮﺸﳐﺰﻟﺍ ﻱﺮﺸﳐ ﺮﺨﺴﻳ ﻬﺎ ﻣﺒﺪﺃ ﺍﻋﺘﺰﺍﻟ ﱄ«.)٤( ﻮﻘﻳﻭ ، ﻭﻻ ﺳﻴﻤﺎ :)٣(» ﻡﺪﺨﺘﺳﺍ ﺔﻳﻮ : »ﺸﳐﺰﻟﺍﻭ ﻫﺎ ﺃﻭ ﺗﺄﻭﻳﻠﻬ ﻌﺘﻘﺪ ﺍﻻﻋﺘﺰﺍﱄ ﺽ ﺍﳌﺮﺍﺩ ﻣﻨﻪ ﻱﻭﺎﺼﻟﺍ ﻪﻨﻋ: ﺮﳚﺎﺗﻪ ﺍﻟﻨﺤﻮ ﺔ ﳝﺲ ﻇﺎﻫﺮ ﻼ ﻟﻴﻨﺼﺮ ﺍﳌﻌ ،ﻨﺎﻩ ﻭﺍﺳﺘﻔﺎﺽ ﱄ)٢(. ﺍﻳﻘﻮﻝ ﰲ ﳎﺎﻝ ﲣﺮ ﺍ ﻛﺎﻧﺖ ﺍﻵﻳﺔ ﻌﺴﻔﺎ ﻣﺘﻤﺤﻼ ﺪ ﺍﺷﺘﻬﺮ ﻨﻌﻣ ﺒﻫﺬ ﻪ ﱄﺍﺰﺘﻋﻻﺍ ﻝ« .ﻝﻮﻘﻳﻭ ﻻﻋﺘﺰﺍﻝ، ﻓﺈﺫﺍ ﻱ ﳓﻮﻳﺎ ﻣﺘﻌ ﻆ ﻗﺪ ﺮﺓ ﻣﺬ ﻝﺍﺰﺘ ﺔﻣ ﻻﺍ ﻱﺮﺸﳐ ،)١( ﻙ ﻫﺒﻪ .« ﺩ ﺍﳋﺎﻣﺲ ﺍﻟﻌﺪﺩ ﺣﻮﻟﻴﺔ :٢٣٣ ﻭ٢٣٥ . ١٧٤-١٧٦ . ﻪ) :ﻮﻟﻪﺳ •ﺣ ﻟﺪﺭﺍﺳﺎﺕ ﺍﻟﻨﺤﻮﻳﺔ ﻭﻳﻨﻈﺮ ﻣﻨﻪ ﺃﻳﻀﺎ :٤ ﺍﻟﻜﺮﱘ ﻭﺃﺛﺮﻩ ﰲ ﺍﻟ ﻱﺮﺸﳐﺰﻟﺍ :٤١ ﻭ )١( ﻥﺁﺮﻘﻟﺍ )٢( ﺞﻬﻨﻣ ﺍ ﺩ ﺍﳋﺎﻣﺲ ﺍﻟﻌﺪﺩ ﺣﻮﻟﻴﺔ :٢٣٣ ﻭ٢٣٥ . ١٧٤-١٧٦ . •ﺣ ﻟﺪﺭﺍﺳﺎﺕ ﺍﻟﻨﺤﻮﻳﺔ ﻭﻳﻨﻈﺮ ﻣﻨﻪ ﺃﻳﻀﺎ :٤ ﺍﻟﻜﺮﱘ ﻭﺃﺛﺮﻩ ﰲ ﺍﻟ ﻱﺮﺸﳐﺰﻟﺍ :٤١ ﻭ )١( ﻥﺁﺮﻘﻟﺍ )٢( ﺞﻬﻨﻣ ﺍ ﺩ ﺍﳋﺎﻣﺲ ﺍﻟﻌﺪﺩ ﺣﻮﻟﻴﺔ :٢٣٣ ﻭ٢٣٥ . ١٧٤-١٧٦ . •ﺣ ﻟﺪﺭﺍﺳﺎﺕ ﺍﻟﻨﺤﻮﻳﺔ ﻭﻳﻨﻈﺮ ﻣﻨﻪ ﺃﻳﻀﺎ :٤ ﺍﻟﻜﺮﱘ ﻭﺃﺛﺮﻩ ﰲ ﺍﻟ ﻱﺮﺸﳐﺰﻟﺍ :٤١ ﻭ )١( ﻥﺁﺮﻘﻟﺍ )٢( ﺞﻬﻨﻣ ﺍ ١٧٣ òÌÜÛa @ À @ ÐnÛa pb @ Þìy @ òîvy @a @@@@@@@ bèj’  òÌÜÛa @ À @ ÐnÛa pb @ Þìy @ òîvy @a @@@@@@ bèj’ (. ﲔ)١ ،ﻪ)٢( )٢( ﺩ ﺍﳋﺎﻣﺲ ﺍﺗﺮ ﻭﺍﻵﺣﺎﺩ ﺎﻣﻌﺔ ﺑﻐﺪﺍﺩ، ﺳﻨﺔ ﺍﻟﻌﺪﺩ ﺮﻱ ﻓﻴﻪ ﺍﻟﺘﻮ ﻡﻮﻠ،ﺔﻴﻣﻼﺳﻹﺍ ﺎﺟ .ﻭ ،ﻞﻘﻨﻟﺍ ﺮﳚﻭ / ١١٧ . ﰲ ﳎﻠﺔ ﻛﻠﻴﺔ ﺍﻟﻌﻠ ﺞ ﺍﳌﻌﻘﻞ:٧٧١ /١ ﺣﻮﻟﻴﺔ ﻪ ﺍﻟﻌﻘﻞ ﻋﻠﻰ ١/٢٧٦ ﻭﺍﳌﺰﻫﺮ ١ ﻊ ﻭﺍﻟﻈﻦ، ﻣﻨﺸﻮﺭ ﻷﺍ ﺳﻨﻮﻱ، ﻭﻣﻨﺎﻫﺞ •ﺣ ﻣﺎ ﺭﻛﺐ ﻓﻴﻪ ﻝﻮﺼﶈﺍ :ﻕ ١ ﺝ ١ ﺍﻟﻘﺮﺁﻥ ﺑﲔ ﺍﻟﻘﻄﻊ ٩٢ ﺎﻳﺔ ﺍﻟﺴﻮﻝﻭ : ﻣ ﻭﺇﻥ ﻣﻨﻬﺎ ( ﰲ ﺗﻔﺼﻴﻞ ﺭﺃﻳﻬﻢ ﺍ: ﲝﺜﻨﺎ :ﺩﻻﻟﺔ ﺃﻟﻔﺎﻅ ١ .ﻡ ﻝ :٤٩٢ /١-٦٩ ،ﺔﻴﻠﻘﻌﻟﺍ ﺎﻀﻳﺃ .)٣( )١( ﻳﻨﻈﺮ ﰲ )٢( ﲝ ﺮﻈﻨﻳ ١٩٩٩ )٣( ﻝﻮﺼﶈﺍ ﺩ ﺍﳋﺎﻣﺲ ﺍﺗﺮ ﻭﺍﻵﺣﺎﺩ ﺎﻣﻌﺔ ﺑﻐﺪﺍﺩ، ﺳﻨﺔ ﺍﻟﻌﺪﺩ ﺮﻱ ﻓﻴﻪ ﺍﻟﺘﻮ ﻡﻮﻠ،ﺔﻴﻣﻼﺳﻹﺍ ﺎﺟ .ﻭ ،ﻞﻘﻨﻟﺍ ﺮﳚﻭ / ١١٧ . ﰲ ﳎﻠﺔ ﻛﻠﻴﺔ ﺍﻟﻌﻠ ﺞ ﺍﳌﻌﻘﻞ:٧٧١ /١ ﺣﻮﻟﻴﺔ ﻪ ﺍﻟﻌﻘﻞ ﻋﻠﻰ ١/٢٧٦ ﻭﺍﳌﺰﻫﺮ ١ ﻊ ﻭﺍﻟﻈﻦ، ﻣﻨﺸﻮﺭ ﻷﺍ ﺳﻨﻮﻱ، ﻭﻣﻨﺎﻫﺞ •ﺣ ﻣﺎ ﺭﻛﺐ ﻓﻴﻪ ﻝﻮﺼﶈﺍ :ﻕ ١ ﺝ ١ ﺍﻟﻘﺮﺁﻥ ﺑﲔ ﺍﻟﻘﻄﻊ ٩٢ ﺎﻳﺔ ﺍﻟﺴﻮﻝﻭ : ﻣ ﻭﺇﻥ ﻣﻨﻬﺎ ( ﰲ ﺗﻔﺼﻴﻞ ﺭﺃﻳﻬﻢ ﺍ: ﲝﺜﻨﺎ :ﺩﻻﻟﺔ ﺃﻟﻔﺎﻅ ١ .ﻡ ﻝ :٤٩٢ /١-٦٩ ،ﺔﻴﻠﻘﻌﻟﺍ ﺎﻀﻳﺃ .)٣( )١( ﻳﻨﻈﺮ ﰲ )٢( ﲝ ﺮﻈﻨﻳ ١٩٩٩ )٣( ﻝﻮﺼﶈﺍ ( ﺎﻀ.)٣( ﺓﱪ.)١( ﻪ   òÌÜÛa @ À @ ÐnÛa pb @ Þìy @ òîvy @a @@@@@@ bèj’  òÌÜÛa @ À @ ÐnÛa pb @ Þìy @ òîvy @a @@@@@@@ bèj’ ﺩ ﺍﳋﺎﻣﺲ ﺔ، ، ١ ١٩٧٨ . ، ١ ١٩٨٠ . ١٩٧٤ .ﻡ ﺭﻣﻀﺎﻥ، ﺩﻣﺸﻖ، ﺭﺍ ،ﺭﺎﺼﻧﻷﺍ ، ٢ ﻜﺘﺐ ،ﺍﻟﻌﺮﺑﻴﺔ ، ١ ١٩٧٩ . ١٩ . ﺩﺭﺍﻮﳌﺍ ﻟﻌﺒﺪ ﺍﷲ ﺍﻟﻌﺪﺩ ١ . ﻡ ﺩﺍﺭ ﺍﻟﻜﺘﺐ ﺔﻴﻓﺎﻘﺜﻟﺍ ﺮﰊ، ١٩٦٨ . ﺍﻵﻓﺎﻕ ﺍﳉﺪﻳﺪﺓ، ﺎﻣﻌﺔ ﻋﲔ ﴰﺲ، ٤ ﻳﻦ ﻋﺒﺪ ﻦﲪﺮﻟﺍ ﺭ ﺬﻳﺐ، ﺍﻟﻘﺎﻫﺮﺓ، ﺩ ﺮﺓ، ﺩﺍﺭ ﺇﺣﻴﺎﺀ ﺍﻟﻜ ،ﺪ، ﺩﺍﺭ ﺍﻟﻔﻜﺮ ٣ ، ﻣﻌﺔ ﺍﳌﻮﺻﻞ، ٩٨٩ ﺏ، ١٩٨٧ . ـ . ،، ١ﺪﱐ ١٩٨٥ . ﻥ، ﻭﺑﺬﻳﻠﻪ ﺃﻋﺬﺏ ﻊﺟﺍﺮﳌﺍﻭ ،ﱯﻠﳊﺍ ١٥٩ ، ٣ ﻟﻠﺒﺪﻱ، ﺍﻟﻜﻮﻳﺖ، ، ﺩﺍﺭﺓ ﺍﻻﲢﺎﺩ ﺍﻟﻌﺮ ﺎﻛﺮ، ﺑﲑﻭﺕ، ﺩﺍﺭ . ﺕ . ﻪ) :ﻮﻟﻪﺳ ﺩ ، ٢ ١٩٥٩ . ،ﺓﺮﻫﺎﻘﻟﺍ ، ﺮﺸﻧ ﺎﺟ ﻱ، ﲢﻘﻴﻖ ﳏﻲ ﺍﻟﺪ ﺩ . ﻋﺒﺪ ﺍﻟﻌﻈﻴﻢ ﺍﻟﺬ ﻀﻞ ﺇﺑﺮﺍﻫﻴﻢ، ﺍﻟﻘﺎﻫﺮ ﺴﻠﻔﻴﺔ، ﺩ. . ﺕ ﻂ ﻋﺒﺪ ﺍﻟﺮﲪﻦ ﳏﻤﺪ ﻳﺔ ﺩﺍﺭ ﺍﻟﻜﺘﺐ، ﺟﺎ . ١ ﺎﻣﺔ ﺍﳌﺼﺮﻳﺔ ﻟﻠﻜﺘﺎﺏ ﺍﻟﻘﻠﻢ، ﺩ . ﺕ . ، ١ﲑﻳﺔ، ١٣٠٦ ـﻫ ﻤﺸﺔ، ﺟﺪﺓ، ﺩﺍﺭ ﺍﳌﺪ ﻦ ﳏﻤﺪ ﺑﻦ ﺳﻠﻴﻤﺎﻥ ﺣﻮﻟﻴﺔ ﺔ ﺍﳌﺼﺎﺩﺭ ﻭ ﺓ، ﻣﺼﻄﻔﻰ ﺍﻟﺒﺎﰊ ﳏﻤﺪ ﲰﲑ ﳒﻴﺐ ﺍﻟ ﺑﻦ ﳏﻤﺪ، ﺍﻟﻘﺎﻫﺮﺓ ﻘﻴﻖ ﺃﲪﺪ ﳏﻤﺪ ﺷﺎ ،ﺮﺓ، ﺩﺍﺭ ﺍﳊﺪﻳﺚ ﻔﻜﺮ، ﺏ . . ﺕ ﻱ، ﺑﻐﺪﺍﺩ، ﺍﻟﺮﺍﺑﻄﺔ، ﺪﺒ ،ﺍﶈﺴﻦ ﺍﻟﺘﺮﻛﻲ ﺃﺑﻮ ﺑﻜﺮ ﺍﻷﻧﺒﺎﺭﻱ ،ﲏﻳﻮﳉﺍ ﻖﻴﻘﲢ ﻘﻴﻖ ﳏﻤﺪ ﺃﺑﻮ ﺍﻟﻔﻀ ﳌﻨﻮﺭﺓ، ﺍﳌﻜﺘﺒﺔ ﺍﻟﺴﻠ ﻟﻘﻠﻢ، ﺩ . . ﺕ ﺒﺎﺭﻛﻔﻮﺭﻱ، ﺿﺒﻂ ﺪ، ﺍﳌﻮﺻﻞ، ﻣﺪﻳﺮﻳ ، ﺩﺍﺭ ﺍﻟﻔﻜﺮ ٩٨٨ ﺍﻟﻘﺎﻫﺮﺓ، ﺍﳍﻴﺌﺔ ﺍﻟﻌﺎ ﻫﱯ، ﺑﲑﻭﺕ، ﺩﺍﺭ ﺯﺍﱐ، ﺍ ﻟﻘﺎﻫﺮﺓ، ﺍﳋﲑ ﻣﻔﻴﺪ ﳏﻤﺪ ﺃﺑﻮ ﻋﻤﺸ ﺪﺋﺍﻭﺰﻟﺍ : ﳏﻤﺪ ﺑﻦ •ﺣ ﺔﻤﺋﺎﻗ ﺍﻟﺴﻴﻮﻃﻲ، ﺍﻟﻘﺎﻫﺮ ﺍﻟﻨﺤﻮ ﺍﻟﻌﺮﰊ : ﺩ.ﳏ ﻜﺎﻡ :ﺍﻵﻣﺪﻱ ﻋﻠﻲ ﻜﺎﻡ :ﺍﺑﻦ ﺣﺰ ﻡ، ﲢﻘ ﺣﺎﻣﺪ ﺍﻟﻐﺰﺍﱄ، ﺍﻟﻘﺎﻫﺮ ﱐ، ﺍﻟﻘﺎﻫﺮﺓ، ﺩﺍﺭ ﺍﻟﻔ ﻋﻠﻲ ﻧﻘﻲ ﺍﳊﻴﺪﺭﻱ ﺪ :ﺩ. ﻋﺒﺪ ﺍﷲ ﺒﻋ ﰲ ﻛﺘﺎﺏ ﺍﷲ : ﻘﻪ :ﺇﻣﺎﻡ ﺍﳊﺮﻣﲔ ﻥ :ﺍﻟﺰﺭﻛﺸﻲ، ﲢﻘ ﻟﺒﻐﺪﺍﺩﻱ، ﺍﳌﺪﻳﻨﺔ ﺍﳌ ﺒﻮﺭ ﺷﺎﻫﲔ، ﺩﺍﺭ ﺍ ﺟﺎﻣﻊ ﺍﻟﺘﺮﻣﺬﻱ :ﺍﳌﺒ .ﳏﺴﻦ ﻋﺒﺪ ﺍﳊﻤﻴﺪ ﻥﺎﻴﺒﻟ :(ﺩ ، ﺕﻭﲑﺑ ﻷﺣﻜﺎﻡ ﺍﻟﻘﺮﺁﻥ (: . ﳏﻤﺪ ﺣﺴﲔ ﺍﻟﺬ ﺳﻌﺪ ﺍﻟﺪﻳﻦ ﺍﻟﺘﻔﺘﺎﺯ ﳊﻨﺒﻠﻲ، ﲢﻘﻴﻖ ﺩ .ﻣ ﻊ ﺍﻷﺻﻮﻝ ﻭﳎﻤﻊ ،ﺓﺭﻮﻨﳌ ١٩٦١ . ﻥﰲ ﻋﻠﻮﻡ ﺍﻟﻘﺮﺁﻥ، ﻥﺁﺮ ﻭﺍﻟﻘﺮﺍﺀﺍﺕ ﰲ ﰲﻡ ﺃﺻﻮﻝ ﺍﻷﺣﻜ ﻡﰲ ﺃﺻﻮﻝ ﺍﻷﺣﻜ ﻡﻮﻠﻋ ﻦﻳﺪﻟﺍ : ﺣ ﻮﺑﺃ ﻝﻮﺤﻔﻟﺍ :ﱐﺎﻛﻮﺸﻟﺍ ﻁﺎﺒﻨﺘﺳﻻﺍ :ﺪﻴﺴﻟﺍ ﻣﺬﻫﺐ ﺍﻹﻣﺎﻡ ﺃﲪﺪ ﺍﻟﻮﻗﻒ ﻭﺍﻻﺑﺘﺪﺍﺀ ﻭﻧﻴﺔ، ١٩٧١ . ﰲﺎﻥ ﺃﺻﻮﻝ ﺍﻟﻔﻘ ١٤ ـﻫ . ﰲﻥ ﻋﻠﻮﻡ ﺍﻟﻘﺮﺁﻥ ١٩ . ﺦﺩﺍﺪﻐﺑ : ﺍﳋﻄﻴﺐ ﺍ ﺦ ﻥﺁﺮﻘﻟﺍ : ﻋﺒﺪ ﺍﻟﺼﺒ ﻱﺫﻮﺣﻷﺍ ﺑﺮﺸﺡ ﺟ ﺗﻔﺴﲑ ﺍﻟﻘﺮﺁﻥ : ﺩ. ﲑ ﻱﱪﻄﻟﺍ ) ﺟﺎﻣﻊ ﺍﻟ ﲑ ﱯﻃﺮﻘﻟﺍ ) ﻊﻣﺎﳉﺍ ﲑ ﻥﻭﺮﺴﻔﳌﺍﻭ : ﺩ ﻋﻠﻰﺢ ﺢﻴﺿﻮﺘﻟﺍ : ﻴﺪ :ﺃﺑﻮ ﺍﳋﻄﺎﺏ ﺍﳊ ﺪﺋﺍﻮﻔﻟﺍ ﻣﻦ ﺟﺎﻣﻊ ﻢ ﺍﳌﺪﱐ، ﺍﳌﺪﻳﻨﺔ ﺍﳌﻨ ١- ﻥﺎﻘﺗﻹﺍ ٢ - ﺮﺛﺃ ﺮﻘﻟﺍ ٣ - ﻡﺎﻜﺣﻹﺍ ٤ - ﻡﺎﻜﺣﻹﺍ ٥ - ﺀﺎﻴﺣﺇ ﻋ ٦ - ﺩﺎﺷﺭﺇ ﺍ ٧ - ﻝﻮﺻﺃ ﺍ ٨ - ﻝﻮﺻﺃ ﻣ ٩ - ﺡﺎﻀﻳﺇ ﻭﺎﻌﺘﻟﺍ ١٠- ﺎﻫﱪﻟﺍ ٤٠٠ ١١- ﺎﻫﱪﻟﺍ ٩٥٧ ١٢- ﺦﻳﺭﺎﺗ ١٣- ﻳﺭﺎﺗﺦ ١٤- ﺔﻔﲢ ﺍ ١٥- ﺭﻮﻄﺗ ١٦- ﲑﺴﻔﺗ ١٧- ﲑﺴﻔﺗ ١٨- ﲑﺴﻔﺘﻟﺍ ١٩- ﺢﻳﻮﻠﺘﻟﺍ ٢٠- ﻴﻬﻤﺘﻟﺍ ٢١- ﻊﲨ ﻢﺷﺎﻫ  òÌÜÛa @ À @ ÐnÛa pb @ Þìy @ òîvy @a @@@@@@@ bèj’ ﺩ ﺍﳋﺎﻣﺲ ، ١ﻼﻝ ، ١٩٧٢ . ﻝﻼﻘﺘﺳ ،ﻯﱪﻜﻟﺍ ـ ) ﺔﻴﺷﺎﲝ ﲑﺴﻔﺗ ١٩٧٨ . ﺀ ،ﺍﻟﻜﺘﺐ ﺍﻟﻌﺮﺑﻴﺔ ، ١ ١٩٧٩ . ﺸﻖ، ﺩﺍﺭ ،ﺍﻟﻔﻜﺮ ﻬﻮﻝ(، ﻧﺸﺮ ﺁﺭﺛﺮ ﻤﺪ ﻋﻠﻲ ،ﺻﺒﻴﺢ ﺍﻟﻌﺪﺩ ١٩٨ . ﻪ) :ﻮﻟﻪﺳ ،ﺔﻴﻧﺎ ١٩٨٨ . ﺮ، ﻣﺼﺮ، ﺍﻻﺳﺘﻘﻼ ،ﻋﻤﺮ، ﺍﻟﻘﺎﻫﺮﺓ ﺳﻻﺍ ﻴﻤﻨﻴﺔ، ١٣٢١ ـﻫ ﺲ، ١٩٦٦ . ﺖ، ﺍﻟﻌﺼﺮﻳﺔ، ٨، ٢ ﺍﻟﻘﺎﻫﺮﺓ، ﺩﺍﺭ ﺀﺎﻴﺣﺇ ، ٢ﻟﺔ، ١٩٨١ . ﻳﺎﺽ، ﺍﻟﻔﺮﺩﻭﺱ، ﻈﻬﺮ ﺍ ﻟﺴﻘﺎﺀ، ﺩﻣﺸ ﺐ ﺍﻟﻌﺮﺑﻴﺔ، ﺩ..ﺕ ١٩ . ﻢ ﺍﳌﻌﺎﱐ ﳌﺆﻟﻒ ﻬﳎ ﺩ، ﺩ. . ﺕ ﺸﻲ، ﺍﻟﻘﺎﻫﺮﺓ، ﳏﻤ ،ﻄﻔﻰ ﺍﻟﺒﺎﰊ ٨٨ ، ١ ﺍﻟﺪﺍﺭ ﺍﳌﺼﺮﻳﺔ ﺍﻟﻠﺒﻨﺎ ﺖ ﻣﻨﲑ، ١٩٨٦ . ﻌﺮﺑﻴﺔ، ﺩ ، ﺕ . ﻴﻖ ﻋﻠﻰ ﳏﻤﺪ ﻋﻤﺮ ﻋ ﲢﻘﻴﻖ ﻋﻠﻲ ﳏﻤﺪ ﺑﻮﺭﻱ، ﻣﺼﺮ، ﺍﳌﻴ ﲑﻭﺕ، ﺩﺍﺭ ﺍﻷﻧﺪﻟﺲ ﺳﺎﱂ ﻣﻜﺮﻡ، ﺍﻟﻜﻮﻳﺖ ، ﻓﺆﺍﺩ ﻋﺒﺪ ﺍﻟﺒﺎﻗﻲ ﺕ، ﻣﺆﺳﺴﺔ ﺍﻟﺮﺳﺎﻟ ﺟﺎﺑﺮ ﺍﻟﻌﻠﻮﺍﱐ، ﺍﻟﺮﻳ ﻡ، ﲢﻘﻴﻖ ﳏﻤﺪ ﻣﻈ ﺓ، ﺩﺍﺭ ﺃﺣﻴﺎﺀ ﺍﻟﻜﺘﺐ ﻫﺮﺓ، ﺍﳌﺪﱐ، ٩٦٤ . ﺕ. ﺎﺏ ﺍﳌﺒﺎﱐ ﰲ ﻧﻈﻢ ﳏﻤﻮﺩ ﻧﺼﺎﺭ، ﺑﻐﺪﺍ ﺩ ﺑﻦ ﺍﳊﺴﻦ ﺍﻟﺒﺪﺧﺸ ﺣﻮﻟﻴﺔ ﻛﺮ، ﺍﻟﻘﺎﻫﺮﺓ، ﻣﺼﻄ ،ﺠﺴﺘﺎﱐ، ﺍﻟﻘﺎﻫﺮﺓ ﺩ، ﻣﻄﺒﻌﺔ ﻭﺃﻭﻓﺴﺖ ﺭ ﺇﺣﻴﺎﺀ ﺍﻟﻜﺘﺐ ﺍﻟﻌ ﻋﻠﻲ ﺍﻟﺪﺍﻭﺩﻱ، ﲢﻘﻴ ﲢ ،ﳊﺴﻦ ﺍﻟﺰﺑﻴﺪﻱ ﻦ ﺑﻦ ﳏﻤﺪ ﺍﻟﻨﻴﺴﺎ ﻔﺪﺍﺀ ﺍﺑﻦ ﻛﺜﲑ، ﺑﲑ ﺔ، ﺩ . . ﺕ ﺔ :ﺩ. ﺳﺎ ﻋﺒﺪ ﺍﻟﻌﺎﻝ ﻜﺮ ، ١٩٨٣ . ﺒﻠﻲ، ﲢﻘﻴﻖ ﳏﻤﺪ ﺆﺍﺩ ﺳﺰﻛﲔ، ﺑﲑﻭﺕ ﺯﻱ، ﲢﻘﻴﻖ ﺩ. ﻪﻃ ﺪﲪﺃ : ﺍﺑﻦ ﺍﻟﻠﺤﺎﻡ ﻭﺁﺧﺮﻭﻥ، ﺍﻟﻘﺎﻫﺮ ﻦ ﻋﺒﺪ ﺍﳊﻤﻴﺪ، ﺍﻟﻘﺎﻫ ﺩﺍﺭ ﺍﳌﺴﺘﺸﺮﻕ، ﺩ ﻋﻄﻴﺔ، ﻭﻣﻘﺪﻣﺔ ﻛﺘﺎ ١ . ﳏ ﻴﻖ، ﳏﻤﻮﺩ ﳏﻤﺪ ﻱﻭﺎﻀﻴﺒﻠ :ﺪﻤﳏ •ﺣ ﻖ ﺃﲪﺪ ﳏﻤﺪ ﺷﺎﻛ ﺑﻦ ﺍﻷﺷﻌﺚ ﺍﻟﺴﺠ ﺷﻴﺔ ﺍﻟﺴﻨﺪﻱ، ﺑﻐﺪﺍﺩ ﺭﺍﺩ ، ﺍﳊﺪﻳﺚ ﻭﺩﺍﺭ ﻋ ﺲ ﺍﻟﺪﻳﻦ ﳏﻤﺪ ﺑﻦ ﻮﻳﲔ :ﳏﻤﺪ ﺑﻦ ﺍﳊ ﺐ ﺍﻟﻔﺮﻗﺎﻥ :ﺍﳊﺴﻦ ﻋﻤﺎﺩ ﺍﻟﺪﻳﻦ ﺃﰊ ﺍﻟﻔ ﺍﻟﻘﺎﻫﺮﺓ، ﺍﻻﺳﺘﻘﺎﻣﺔ ﺍﻟﺪﺭﺍﺳﺎﺕ ﺍﻟﻨﺤﻮﻳﺔ ﺍﻟﻘﺎﻫﺮﺓ ، ﺩﺍﺭ ﺍﻟﻔﻜ ﺠﺎﺭ ﺍﻟﻔﺘﻮﺣﻲ ﺍﳊﻨﺒ ﲢﻘﻴﻖ ﺩ، .ﳏﻤﺪ ﻓﺆ ﻪﻘﻔﻟﺍ : ﺍﻟﻔﺨﺮ ﺍﻟﺮﺍﺯ ﻔﻘﻪ ﻋﻠﻰ ﻣﺬﻫﺐ ﻖ ﳏﻤﺪ ﺟﺎﺩ ﺍﳌﻮﱄ ﻖ ﳏﻤﺪ ﳏﻲ ﺍﻟﺪﻳﻦ ،ﺍﳊﻤﻮﻱ، ﺑﲑﻭﺕ ﺁﻥ) :ﻋ ﻣﻘﺪﻣﺔ ﺍﺑﻦ ﺒﺔ ﺍﳋﺎﳒﻲ، ١٩٥٤ ﲑ :ﺍﺑﻦ ﺗﻴﻤﻴﺔ، ﲢﻘﻴﻖ ﻣﻨﻬﺎﺝ ﺍﻟﻮﺻﻮﻝ ﻟﻠ ﺔﻟ : ،ﻲﻌﻓﺎﺸﻟﺍ ﻖﻴﻘﲢ ﰊﺃ ﺩﻭﺍﺩ : ﻥﺎﻤﻴﻠﺳ ﺢ ﻱﺭﺎﺨﺒﻟﺍ ﲝﺎﺷﻴ : ﺢ ﻢﻠﺴﻣ : ،ﺍﻟﻘﺎﻫﺮﺓ ﺕ ﻦﻳﺮﺴﻔﳌﺍ ﴰﺲ : ﺕ ﺍﻟﻨﺤﻮﻳﲔ ﻭﺍﻟﻠﻐﻮ ١٩٧٢ . ﺐ ﻥﺁﺮﻘﻟﺍ ﻭ ﻏﺎﺋﺐﺭ ﻱﱪ . ( ﻞ ﻥﺁﺮﻘﻟﺍ : ﻆﻓﺎﳊﺍ ﺳﺖ : ﺍﺑﻦ ﺍﻟﻨﺪﱘ، ﻥ ﱘﺮﻜﻟﺍ ﻭﺃﺛﺮﻩ ﰲ ﺎﻑ : ،ﻱﺮﺸﳐﺰﻟﺍ ﺍ ﻛﺐ ﲑﻨﳌﺍ : ﺍﺑﻦ ﺍﻟﻨﺠ ١٩٥٧ . . ﻥﺁﺮﻘﻟ : ،ﺃﺑﻮ ﻋﺒﻴﺪﺓ ﻝﻮﰲ ﻋﻠﻢ ﺃﺻﻮﻝ ﰲﺼﺮ ﺃﺻﻮﻝ ﺍﻟﻔ ،ﻭﻓﺴﺖ ١٩٨٠ . ﺮ : ،ﻲﻃﻮﻴﺴﻟﺍ ﻖﻴﻘﲢ ﺓﺩ :ﻝﺁ ﺗﻴﻤﻴﺔ، ﲢﻴﻖ ﻢ ﺀﺎﺑﺩﻷﺍ : ﺍ ﺕﻮﻗﺎﻳ ﺘﺎﻥ ﰲ ﻋﻠﻮﻡ ﺍﻟﻘﺮﺁ ﻱ، ﺍﻟﻘﺎﻫﺮﺓ، ﻣﻜﺘﺒﺔ ﰲﺔ ﺃﺻﻮﻝ ﺍﻟﺘﻔﺴﲑ ﺞ ﻝﻮﻘﻌﻟﺍ ﻣ ﺡﺮﺷ ﺕ . ﻪ) :ﻮﻟﻪﺳ ٢٢- ﻟﺎﺳﺮﻟﺍ ٢٣- ﻦﻨﺳﺃ ٢٤- ﺢﻴﺤﺻ ٢٥- ﺢﻴﺤﺻ ٦٢- ﻃﺒﻘﺎﺕ ٧٢- ﻃﺒﻘﺎﺕ ٢، ١ ٨٢-ﻏﺮﺍﺋﺐ ﱪﻄﻟﺍ ٢٩- ﻞﺋﺎﻀﻓ ٣٠- ﺳﺮﻬﻔﻟﺍ ٣١- ﻥﺁﺮﻘﻟﺍ ٣٢- ﺎﺸﻜﻟﺍ ٣٣- ﻛﻮﻜﻟﺍ ٧، ١ ٣٤- ﺯﺎﳎﺍ ٣٥- ﻮﺼﶈﺍ ٣٦- ﺼﺘﺨﳌﺍ ﻭﻷﺎﺑ ٣٧- ﺮﻫﺰﳌﺍ ٣٨- ﺩﻮﺴﳌﺍ ٣٩- ﻢﺠﻌﻣ ٤٠- ﺘﻣﺪﻘﻣ ﺮﻔﺟ ٤١- ﺔﻣﺪﻘﻣ ٤٢- ﺞﻫﺎﻨﻣ ﺩ.ﺕ ﺩ ﺍﳋﺎﻣﺲ ، ١ﻼﻝ ، ١٩٧٢ . ﻝﻼﻘﺘﺳ ﻯﱪﻜﻟﺍ ـ ) ﺔﻴﺷﺎﲝ ﺴﻔﺗ ١٩٧٨ . ﺀ ﺍﻟﻜﺘﺐ ﺍﻟﻌﺮﺑﻴﺔ ، ١ ١٩٧٩ . ﺸﻖ، ﺩﺍﺭ ﺮﻜﻔﻟﺍ ﻬﻮﻝ(، ﻧﺸﺮ ﺁﺭﺛ ﻤﺪ ﻋﻠﻲ ﺢﻴﺒﺻ ﺍﻟﻌﺪﺩ ١٩٨ . ،ﺔﻴﻧﺎ ١٩٨٨ . ﺮ، ﻣﺼﺮ، ﺍﻻﺳﺘﻘﻼ ،ﻋﻤﺮ، ﺍﻟﻘﺎﻫﺮﺓ ﺳﻻﺍ ﻴﻤﻨﻴﺔ، ١٣٢١ ـﻫ ﺲ، ١٩٦٦ . ﺖ، ﺍﻟﻌﺼﺮﻳﺔ، ٨، ٢ ﺍﻟﻘﺎﻫﺮﺓ، ﺩﺍﺭ ﺀﺎﻴﺣﺇ ، ٢ﻟﺔ، ١٩٨١ . ﻳﺎﺽ، ﺍﻟﻔﺮﺩﻭﺱ، ﻈﻬﺮ ﺍ ﻟﺴﻘﺎﺀ، ﺩﻣﺸ ﺐ ﺍﻟﻌﺮﺑﻴﺔ، ﺩ..ﺕ ١٩ . ﻢ ﺍﳌﻌﺎﱐ ﳌﺆﻟﻒ ﻬﳎ ﺩ، ﺩ. . ﺕ ﺸﻲ، ﺍﻟﻘﺎﻫﺮﺓ، ﳏﻤ ،ﻄﻔﻰ ﺍﻟﺒﺎﰊ ٨٨ ، ١ ﺍﻟﺪﺍﺭ ﺍﳌﺼﺮﻳﺔ ﺍﻟﻠﺒﻨﺎ ﺖ ﻣﻨﲑ، ١٩٨٦ . ﻌﺮﺑﻴﺔ، ﺩ ، ﺕ . ﻴﻖ ﻋﻠﻰ ﳏﻤﺪ ﻋﻤﺮ ﻋ ﲢﻘﻴﻖ ﻋﻠﻲ ﳏﻤﺪ ﺑﻮﺭﻱ، ﻣﺼﺮ، ﺍﳌﻴ ﲑﻭﺕ، ﺩﺍﺭ ﺍﻷﻧﺪﻟﺲ ﺳﺎﱂ ﻣﻜﺮﻡ، ﺍﻟﻜﻮﻳﺖ ، ﻓﺆﺍﺩ ﻋﺒﺪ ﺍﻟﺒﺎﻗﻲ ﺕ، ﻣﺆﺳﺴﺔ ﺍﻟﺮﺳﺎﻟ ﺟﺎﺑﺮ ﺍﻟﻌﻠﻮﺍﱐ، ﺍﻟﺮﻳ ﻡ، ﲢﻘﻴﻖ ﳏﻤﺪ ﻣﻈ ﺓ، ﺩﺍﺭ ﺃﺣﻴﺎﺀ ﺍﻟﻜﺘﺐ ﻫﺮﺓ، ﺍﳌﺪﱐ، ٩٦٤ . ﺕ. ﺎﺏ ﺍﳌﺒﺎﱐ ﰲ ﻧﻈﻢ ﳏﻤﻮﺩ ﻧﺼﺎﺭ، ﺑﻐﺪﺍ ﺩ ﺑﻦ ﺍﳊﺴﻦ ﺍﻟﺒﺪﺧﺸ ﺣﻮﻟﻴﺔ ﻛﺮ، ﺍﻟﻘﺎﻫﺮﺓ، ﻣﺼﻄ ،ﺠﺴﺘﺎﱐ، ﺍﻟﻘﺎﻫﺮﺓ ﺩ، ﻣﻄﺒﻌﺔ ﻭﺃﻭﻓﺴﺖ ﺭ ﺇﺣﻴﺎﺀ ﺍﻟﻜﺘﺐ ﺍﻟﻌ ﻋﻠﻲ ﺍﻟﺪﺍﻭﺩﻱ، ﲢﻘﻴ ﲢ ،ﳊﺴﻦ ﺍﻟﺰﺑﻴﺪﻱ ﻦ ﺑﻦ ﳏﻤﺪ ﺍﻟﻨﻴﺴﺎ ﻔﺪﺍﺀ ﺍﺑﻦ ﻛﺜﲑ، ﺑﲑ ﺔ، ﺩ . . ﺕ ﺔ :ﺩ. ﺳﺎ ﻋﺒﺪ ﺍﻟﻌﺎﻝ ﻜﺮ ، ١٩٨٣ . ﺒﻠﻲ، ﲢﻘﻴﻖ ﳏﻤﺪ ﺆﺍﺩ ﺳﺰﻛﲔ، ﺑﲑﻭﺕ ﺯﻱ، ﲢﻘﻴﻖ ﺩ. ﻪﻃ ﺪﲪﺃ : ﺍﺑﻦ ﺍﻟﻠﺤﺎﻡ ﻭﺁﺧﺮﻭﻥ، ﺍﻟﻘﺎﻫﺮ ﻦ ﻋﺒﺪ ﺍﳊﻤﻴﺪ، ﺍﻟﻘﺎﻫ ﺩﺍﺭ ﺍﳌﺴﺘﺸﺮﻕ، ﺩ ﻋﻄﻴﺔ، ﻭﻣﻘﺪﻣﺔ ﻛﺘﺎ ١ . ﳏ ﻴﻖ، ﳏﻤﻮﺩ ﳏﻤﺪ ﻱﻭﺎﻀﻴﺒﻠ :ﺪﻤﳏ •ﺣ ﻖ ﺃﲪﺪ ﳏﻤﺪ ﺷﺎﻛ ﺑﻦ ﺍﻷﺷﻌﺚ ﺍﻟﺴﺠ ﺷﻴﺔ ﺍﻟﺴﻨﺪﻱ، ﺑﻐﺪﺍﺩ ﺭﺍﺩ ، ﺍﳊﺪﻳﺚ ﻭﺩﺍﺭ ﻋ ﺲ ﺍﻟﺪﻳﻦ ﳏﻤﺪ ﺑﻦ ﻮﻳﲔ :ﳏﻤﺪ ﺑﻦ ﺍﳊ ﺐ ﺍﻟﻔﺮﻗﺎﻥ :ﺍﳊﺴﻦ ﻋﻤﺎﺩ ﺍﻟﺪﻳﻦ ﺃﰊ ﺍﻟﻔ ﺍﻟﻘﺎﻫﺮﺓ، ﺍﻻﺳﺘﻘﺎﻣﺔ ﺍﻟﺪﺭﺍﺳﺎﺕ ﺍﻟﻨﺤﻮﻳﺔ ﺍﻟﻘﺎﻫﺮﺓ ، ﺩﺍﺭ ﺍﻟﻔﻜ ﺠﺎﺭ ﺍﻟﻔﺘﻮﺣﻲ ﺍﳊﻨﺒ ﲢﻘﻴﻖ ﺩ، .ﳏﻤﺪ ﻓﺆ ﻪﻘﻔﻟﺍ : ﺍﻟﻔﺨﺮ ﺍﻟﺮﺍﺯ ﻔﻘﻪ ﻋﻠﻰ ﻣﺬﻫﺐ ﻖ ﳏﻤﺪ ﺟﺎﺩ ﺍﳌﻮﱄ ﻖ ﳏﻤﺪ ﳏﻲ ﺍﻟﺪﻳﻦ ،ﺍﳊﻤﻮﻱ، ﺑﲑﻭﺕ ﺁﻥ) :ﻋ ﻣﻘﺪﻣﺔ ﺍﺑﻦ ﺒﺔ ﺍﳋﺎﳒﻲ، ١٩٥٤ ﲑ :ﺍﺑﻦ ﺗﻴﻤﻴﺔ، ﲢﻘﻴﻖ ﻣﻨﻬﺎﺝ ﺍﻟﻮﺻﻮﻝ ﻟﻠ ﺔﻟ : ،ﻲﻌﻓﺎﺸﻟﺍ ﻖﻴﻘﲢ ﰊﺃ ﺩﻭﺍﺩ : ﻥﺎﻤﻴﻠﺳ ﺢ ﻱﺭﺎﺨﺒﻟﺍ ﲝﺎﺷﻴ : ﺢ ﻢﻠﺴﻣ : ،ﺍﻟﻘﺎﻫﺮﺓ ﺕ ﻦﻳﺮﺴﻔﳌﺍ ﴰﺲ : ﺕ ﺍﻟﻨﺤﻮﻳﲔ ﻭﺍﻟﻠﻐﻮ ١٩٧٢ . ﺐ ﻥﺁﺮﻘﻟﺍ ﻭ ﻏﺎﺋﺐﺭ ﻱﱪ . ﻪ) :ﻮﻟﻪﺳ ( ﻞ ﻥﺁﺮﻘﻟﺍ : ﻆﻓﺎﳊﺍ ﺳﺖ : ﺍﺑﻦ ﺍﻟﻨﺪﱘ، ﻥ ﱘﺮﻜﻟﺍ ﻭﺃﺛﺮﻩ ﰲ ﺎﻑ : ،ﻱﺮﺸﳐﺰﻟﺍ ﺍ ﻛﺐ ﲑﻨﳌﺍ : ﺍﺑﻦ ﺍﻟﻨﺠ ١٩٥٧ . . ﻥﺁﺮﻘﻟ : ،ﺃﺑﻮ ﻋﺒﻴﺪﺓ ﻝﻮﰲ ﻋﻠﻢ ﺃﺻﻮﻝ ﰲﺼﺮ ﺃﺻﻮﻝ ﺍﻟﻔ ،ﻭﻓﺴﺖ ١٩٨٠ . ﺮ : ،ﻲﻃﻮﻴﺴﻟﺍ ﻖﻴﻘﲢ ﺓﺩ :ﻝﺁ ﺗﻴﻤﻴﺔ، ﲢﻴﻖ ﻢ ﺀﺎﺑﺩﻷﺍ : ﺍ ﺕﻮﻗﺎﻳ ﺘﺎﻥ ﰲ ﻋﻠﻮﻡ ﺍﻟﻘﺮﺁ ﻱ، ﺍﻟﻘﺎﻫﺮﺓ، ﻣﻜﺘﺒﺔ ﰲﺔ ﺃﺻﻮﻝ ﺍﻟﺘﻔﺴﲑ ﺞ ﻝﻮﻘﻌﻟﺍ ﻣ ﺡﺮﺷ ﺕ . ٢٢- ﻟﺎﺳﺮﻟﺍ ٢٣- ﻦﻨﺳﺃ ٢٤- ﺢﻴﺤﺻ ٢٥- ﺢﻴﺤﺻ ٦٢- ﻃﺒﻘﺎﺕ ٧٢- ﻃﺒﻘﺎﺕ ٢، ١ ٨٢-ﻏﺮﺍﺋﺐ ﱪﻄﻟﺍ ٢٩- ﻞﺋﺎﻀﻓ ٣٠- ﺳﺮﻬﻔﻟﺍ ٣١- ﻥﺁﺮﻘﻟﺍ ٣٢- ﺎﺸﻜﻟﺍ ٣٣- ﻛﻮﻜﻟﺍ ٧، ١ ٣٤- ﺯﺎﳎﺍ ٣٥- ﻮﺼﶈﺍ ٣٦- ﺼﺘﺨﳌﺍ ﻭﻷﺎﺑ ٣٧- ﺮﻫﺰﳌﺍ ٣٨- ﺩﻮﺴﳌﺍ ٣٩- ﻢﺠﻌﻣ ٤٠- ﺘﻣﺪﻘﻣ ﺮﻔﺟ ٤١- ﺔﻣﺪﻘﻣ ٤٢- ﺞﻫﺎﻨﻣ ﺩ.ﺕ  òÌÜÛa @ À @ ÐnÛa pb @ Þìy @ òîvy @a @@@@@@ bèj’
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Changes in Extremely Hot Summers over the Global Land Area under Various Warming Targets
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Changes in Extremely Hot Summers over the Global Land Area under Various Warming Targets Lei Wang, Jianbin Huang*, Yong Luo, Yao Yao, Zongci Zhao Lei Wang, Jianbin Huang*, Yong Luo, Yao Yao, Zongci Zhao Ministry of Education Key Laboratory for Earth System Modeling, Center for Earth System Science, and Joint Center for Global Change Studies (JCGCS), Tsinghua University, Beijing, China Ministry of Education Key Laboratory for Earth System Modeling, Center for Earth System Science, and Joint Center for Global Change Studies (JCGCS), Tsinghua University, Beijing, China * jbh@mail.tsinghua.edu.cn * jbh@mail.tsinghua.edu.cn OPEN ACCESS Citation: Wang L, Huang J, Luo Y, Yao Y, Zhao Z (2015) Changes in Extremely Hot Summers over the Global Land Area under Various Warming Targets. PLoS ONE 10(6): e0130660. doi:10.1371/journal. pone.0130660 Editor: Shang-Ping Xie, University of California San Diego, UNITED STATES Received: February 26, 2015 Accepted: May 22, 2015 Published: June 19, 2015 Copyright: © 2015 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. Editor: Shang-Ping Xie, University of California San Diego, UNITED STATES Editor: Shang-Ping Xie, University of California San Diego, UNITED STATES Received: February 26, 2015 Accepted: May 22, 2015 Published: June 19, 2015 Copyright: © 2015 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. Data Availability Statement: The model data used in this study are available from Earth System Grid Federation at http://cmip-pcmdi.llnl.gov/cmip5/. The GISS data are available from Goddard Institute for Space Studies at http://data.giss.nasa.gov/gistemp/ and CRU data are available from Climate Research Unit at http://www.cru.uea.ac.uk/cru/data/hrg/. Funding: This study was supported by the China Meteorological Administration Special Public Welfare Research Fund (GYHY201306019, http://guoqing. china.com.cn/2012-03/23/content_24968247.htm— received by JH), the Tsinghua University Initiative Scientific Research Program (Grant No. Abstract Summer temperature extremes over the global land area were investigated by comparing 26 models of the fifth phase of the Coupled Model Intercomparison Project (CMIP5) with observations from the Goddard Institute for Space Studies (GISS) and the Climate Research Unit (CRU). Monthly data of the observations and models were averaged for each season, and statistics were calculated for individual models before averaging them to obtain ensemble means. The summers with temperature anomalies (relative to 1951–1980) exceeding 3σ (σ is based on the local internal variability) are defined as “extremely hot”. The models well reproduced the statistical characteristics evolution, and partly captured the spatial distributions of historical summer temperature extremes. If the global mean tempera- ture increases 2°C relative to the pre-industrial level, “extremely hot” summers are projected to occur over nearly 40% of the land area (multi-model ensemble mean projection). Sum- mers that exceed 5σ warming are projected to occur over approximately 10% of the global land area, which were rarely observed during the reference period. Scenarios reaching warming levels of 3°C to 5°C were also analyzed. After exceeding the 5°C warming target, “extremely hot” summers are projected to occur throughout the entire global land area, and summers that exceed 5σ warming would become common over 70% of the land area. In addition, the areas affected by “extremely hot” summers are expected to rapidly expand by more than 25%/°C as the global mean temperature increases by up to 3°C before slowing to less than 16%/°C as the temperature continues to increase by more than 3°C. The area that experiences summers with warming of 5σ or more above the warming target of 2°C is likely to maintain rapid expansion of greater than 17%/°C. To reduce the impacts and dam- age from severely hot summers, the global mean temperature increase should remain low. RESEARCH ARTICLE Hot Summers under Various Warming Targets human health and property, have garnered much attention [5]. For example, the European heat waves in 2003 and 2006 [6–8], the great California heat wave in 2006 [9], the rare high temperatures in Russia in 2010 [10, 11] and the extremely hot Australian summer in 2013, are all well-known for their severity and damage. Additionally, studies have indicated that heat waves will occur with higher intensity and frequency if global warming continues [12–17]. 20131089356, http://kyxxxt.cic.tsinghua.edu.cn/— received by JH), and the National Natural Science Foundation of China (Grant No.41175066, http://www. nsfc.gov.cn/—received by YL). The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript. Heat waves also occur on monthly and seasonal scales as temperature extremes [18–22]. Because monthly data are relatively easy to access, monthly temperatures are widely used to examine changes in past and future climate extremes [23]. The number of warm monthly tem- perature extremes has significantly increased in the past decade [24, 25]. Hansen et al. [18] indicated that the occurrence of extremely hot summers, which rarely occurred in 1951–1980 (less than 1% of the land area), became extensive in the last decade (approximately 10% of the land area). In addition, the latest results from the fifth phase of the Coupled Model Intercom- parison Project (CMIP5) models suggest that extremely hot monthly temperatures will affect approximately 85% of the land area in 2100 under the Representative Concentration Pathway 8.5 (RCP8.5) [19]. Competing Interests: The authors have declared that no competing interests exist. A global mean temperature increase of 2°C relative to pre-industrial level has been accepted by most countries and organizations as a critical target because it seems achievable and would limit climate and ecological impacts [26–30]. Controlling the increase in the global mean tem- perature is as an effective method for avoiding or reducing losses caused by global warming. Consequently, quantifying the responses of climate extremes to a 2°C warming and other warming rates relative to the pre-industrial level will help policymakers, researchers and the public understand the possible consequences of inaction. The seasonal temperatures of the 26 CMIP5 models, which include historical simulations and three RCP scenarios, were analyzed in this study. Compared with the ocean, land areas are more closely associated with human livelihood. Introduction With rapid increases in global mean temperature over the past decade, climate extremes have become stronger and more frequent [1–4]. Particularly, heat waves, which severely threaten PLOS ONE | DOI:10.1371/journal.pone.0130660 June 19, 2015 1 / 11 PLOS ONE | DOI:10.1371/journal.pone.0130660 June 19, 2015 Data and Methods Monthly surface air temperatures from 26 CMIP5 models, which include historical data and three simulated scenarios (RCP2.6, RCP4.5 and RCP8.5) (S1 Table), were used in our research. Historical observations were obtained from the Goddard Institute for Space Studies (GISS) [31] and the Climate Research Unit (CRU) [32]. The monthly GISS data (1880 to the present), which are smoothed to 250 km to include as much regional variability as possible, have a spa- tial resolution of 2°x 2° [31], and the monthly CRU data (1901 to the present) have a high spa- tial resolution of 0.5° x 0.5° [32]. The durations of the 26 CMIP5 historical runs are shown in S1 Table, most of which extend from 1850 to 2005. Most of the 21st century projections, which follow the three emission scenarios, extend from 2006 to 2100. Monthly data are averaged for each season at the beginning, and the summer means of JJA data from the North Hemisphere and DJF data from the South Hemisphere are concurrently analyzed in the following. In our research, the period of 1951–1980 was chosen as a reference period because of its advantages. As mentioned by Hansen et al. [18], the temperature during this period was rela- tively stable and almost without rapid increase, which quite a number of older people have experienced. The definition of temperature extremes used in this study is similar to that proposed by Hansen et al. [18], except that a value of +5σ is included in our research. In a normal distribu- tion, the values between ±0.43σ are defined as “typical” summers; the values greater (less) than +0.43σ (-0.43σ) are defined as “hot” (“cold”) summers; the values greater (less) than +2σ (-2σ) and +3σ (-3σ) are defined as “very hot” (“very cold”) and “extremely hot” (“extremely cold”) summers, respectively. As global warming continues, “extremely hot” events will become more common. Additionally, changes in the occurrence of more severe summers have been quanti- fied: the summers with temperature anomalies greater than +5σ in a normal distribution are referred to as “exceeding 5σ hot” summers. To calculate the local σ, we first fit the summer data from 1901 (the first year of CRU data) to 2005 using a least squares polynomial method and determined the long-term non-linear trend. Next, the summer time series were detrended by deducting the long-term trend. Accordingly, we focused on changes in temperature extremes over global land areas in response to various warming targets. Summer is the focus of our research because humans are more sensitive to hot extremes during the summer than during other seasons. Hansen et al. [18] and Coumou and Robinson [19] also research on the extreme tempera- tures. However, Hansen et al. [18] only investigated the observations by using GISS data, while Coumou and Robinson [19] mainly predicted the area percentage with monthly heat extremes during boreal summer in 21st century and their spatial patterns at the end 20 years of 21st cen- tury under RCP2.6 and RCP8.5 scenarios. In contrast, based on 26 CMIP5 models, a systemic evaluation of the long-term global extreme summer temperatures was performed to quantify the capability of models in simulating extreme summer temperatures in our study, including not only the spatial distributions and total area percentages but also the probability of occur- rence. The global extreme summer temperatures include June-July-August (JJA) in the North- ern Hemisphere and December-January-February (DJF) in the Southern Hemisphere, while boreal summer of JJA was analyzed in both hemispheres in Coumou and Robinson [19]. In addition, we mainly focus on the response of extremely hot summers to the different warming magnitudes relative to the pre-industrial level. Therefore, all simulations from RCP2.6, RCP4.5 and RCP8.5 scenarios (actually 26 simulations in each RCP scenario) are together used in our research to quantify the various warming magnitudes regardless of different emission scenar- ios. So our analysis emphasizes the response of summer climate extremes to different warming targets rather than different emission scenarios as Coumou and Robinson [19]. Furthermore, another big difference between this work and Coumou and Robinson [19] is that the seasonal data are used in this study instead of monthly data in the research of Coumou and Robinson [19]. Since the standard deviation of monthly data is larger than that of seasonal data, the pro- jected future increase of the hot extreme temperatures analyzed by using seasonal data is gener- ally larger than those analyzed by using monthly data. 2 / 11 PLOS ONE | DOI:10.1371/journal.pone.0130660 June 19, 2015 Hot Summers under Various Warming Targets This paper is structured as follows. Section 1 introduces the data and methods used in this research. Next, 26 CMIP5 models are evaluated for simulating historical temperature extremes in section 2. In section 3, the responses of the temperature extremes to various warming targets are analyzed. Finally, a summary and discussion of our finding is presented in section 4. PLOS ONE | DOI:10.1371/journal.pone.0130660 June 19, 2015 Evolution of historical summer temperature extremes Comparing the results from the multi-model ensemble mean and two observations of GISS and CRU reveals that the CMIP5 models perform extremely well when reproducing the evolu- tion of the statistical distribution of summer temperature extremes between 1946 and 2005 (Fig 1). Because the historical CMIP5 runs end in 2005, the temperature extremes are counted every ten years from 1946 to 2005 for the model results and two observational datasets. The curves of the summer temperatures are smoother in the CRU dataset than in the GISS dataset because of the additional observational grids in the CRU data. The distributions of the multi- model ensemble mean are highly consistent with those of the observations. The curves shift to the right and become broader over time, particularly following the 1980s. The probability of an “extremely hot” summer is indicated by a gradually increasing area of the right tail. The curve shifts rightward as the mean temperature increases, and the broader trend may result from the heterogeneous warming rates in different regions rather than the increase of variability [33]. Fei Ji et al. [34] indicated that the most dramatic and rapid warming has occurred in arid or semiarid regions in the Northern Hemisphere at mid-latitudes, and that warming since 1900 has varied spatially and unevenly. However, the global temperature variability has remained stable [35]. As shown in Fig 1, rapid warming has occurred since the 1970s, which resulted in significant rightward curve shifts. In addition, the spatial combined statistics from regions with different warming rates contributes to the broadening of the curves. The occurrence probability of an “extremely hot” summer during each period is shown in Table 1, and the occurrence probability is somewhat underestimated by the multi-model ensemble mean. The multi-model ensemble mean performs well in terms of the total area affected by tem- perature extremes (Fig 2). The “extremely hot” area has rapidly increased since the late 1970s, and the “cold” area has generally decreased, with the exception of a relatively stable period from the early 1940s to the mid-1970s. The trends in the hot and cold areas are highly consis- tent with those of the observations. The averaging process results in smoother multi-model ensemble mean curves than observed for the other two observations. The spatial distributions of the “extremely hot” summers during 1986–2005 are shown in Fig 3. Data and Methods The first, second and third graphs show the GISS, CRU and multi- model ensemble mean results, respectively. To better compare the results from observations and multi- model ensemble mean, the frequencies during the periods of 1951–1980 (reference period), 1976–1985, 1986–1995 and 1996–2005 are shown in the fourth graph. doi:10.1371/journal.pone.0130660.g001 Data and Methods Then, we estimated σ by using this detrended 105 data, which include a sufficient number of data points to estimate the local year-to-year variability. Considering the starting time of the historical simulations of the 26 models (S1 Table), the average temperature from 1861 to 1880 is used to represent the pre-industrial level. When the 11-year running mean global mean temperature exceeds 2°C, the global mean temperature reaches the 2°C target. Additionally, the 20-year climate centered on the year reaching 2°C tar- get is determined. The same methods are used to examine the climate when the global mean temperature reaches various warming targets relative to the pre-industrial level, such as 3°C, 4°C and 5°C. In this analysis, the statistical characteristics of the ensemble mean of the 26 mod- els are obtained after calculating each model independently by considering the weight of each grid area. That is to say, the statistics were first calculated for individual models, and then the results from all models were averaged to present the multi-model ensemble mean. Addition- ally, JJA in the Northern Hemisphere and DJF in the Southern Hemisphere are considered jointly when presenting the summer characteristics. 3 / 11 PLOS ONE | DOI:10.1371/journal.pone.0130660 June 19, 2015 Hot Summers under Various Warming Targets Fig 1. Frequency of summer local temperature anomalies. The temperature anomalies (relative to the 1951–1980 mean) are divided by the local standard deviation of 1901–2005 and binned into intervals of 0.05. The local standard deviation is calculated from the long-term non-linearly detrended time series (details in data and method section). For the multi-model ensemble mean, this process was conducted for each model, and the final frequencies were averaged. The first, second and third graphs show the GISS, CRU and multi- model ensemble mean results, respectively. To better compare the results from observations and multi- model ensemble mean, the frequencies during the periods of 1951–1980 (reference period), 1976–1985, 1986–1995 and 1996–2005 are shown in the fourth graph. Fig 1. Frequency of summer local temperature anomalies. The temperature anomalies (relative to the 1951–1980 mean) are divided by the local standard deviation of 1901–2005 and binned into intervals of 0.05. The local standard deviation is calculated from the long-term non-linearly detrended time series (details in data and method section). For the multi-model ensemble mean, this process was conducted for each model, and the final frequencies were averaged. PLOS ONE | DOI:10.1371/journal.pone.0130660 June 19, 2015 Evolution of historical summer temperature extremes When disregarding the missing data in GISS, the locations with “extremely hot” summers are similar between the two observations, except for the west of South America and the middle of Africa. The present spa- tial distribution and areal coverage of “extremely hot” summer that are simulated using the multi-model ensemble mean is more similar to the GISS results than the CRU results, in which PLOS ONE | DOI:10.1371/journal.pone.0130660 June 19, 2015 4 / 11 Hot Summers under Various Warming Targets Table 1. Occurrence probability of an “extremely hot” summer over the global land area. Period GISS (%) CRU (%) Multi-model ensemble mean Reference period 0.1 0.1 0.1 1946–1955 0.3 0.5 0.3 1956–1965 0.1 0.1 0.1 1966–1975 0.1 0.2 0.2 1976–1985 0.6 0.7 0.4 1986–1995 1.7 2.0 1.4 1996–2005 4.8 5.1 4.3 The second, third and fourth columns in the table present results from the two observational datasets and multi-model ensemble mean. doi:10 1371/journal pone 0130660 t001 Table 1. Occurrence probability of an “extremely hot” summer over the global land area. The second, third and fourth columns in the table present results from the two observational datasets and multi-model ensemble mean. doi:10.1371/journal.pone.0130660.t001 important locations, such as West Europe, South America and North Africa, are all significant, but their exact frequency of “extremely hot” summers is underestimated. important locations, such as West Europe, South America and North Africa, are all significant, but their exact frequency of “extremely hot” summers is underestimated. Response of hot summer extremes to various warming targets doi:10.1371/journal.pone.0130660.g003 Fig 3. Spatial distribution of the “extremely hot” summers frequency from 1986 to 2005. The left, middle and right maps represent the GISS, CRU and averages of the individual models results, respectively. doi:10.1371/journal.pone.0130660.g003 doi:10.1371/journal.pone.0130660.g003 different regions [33, 35]. The details regarding changes in probability are shown in Table 2. Notably the occurrence probability of an “extremely hot” summer would increase to 40.3% as the global temperature reaches the 2°C warming target and 93.3% as the global temperature reaches the 5°C warming target. The probability was only 0.1% during the reference period. When the increase of the global mean temperature exceeds 2°C or 5°C relative to the pre- industrial level, the occurrence probabilities of having an “exceeding 5σ hot” summer may increase to 12.9% and 79.6%, respectively. This probability was zero during the reference period. different regions [33, 35]. The details regarding changes in probability are shown in Table 2. Notably the occurrence probability of an “extremely hot” summer would increase to 40.3% as the global temperature reaches the 2°C warming target and 93.3% as the global temperature reaches the 5°C warming target. The probability was only 0.1% during the reference period. When the increase of the global mean temperature exceeds 2°C or 5°C relative to the pre- industrial level, the occurrence probabilities of having an “exceeding 5σ hot” summer may increase to 12.9% and 79.6%, respectively. This probability was zero during the reference period. Which regions will be vulnerable to hot summer extremes in the future when warming tar- gets are reached? The frequencies of “extremely hot” and “exceeding 5σ hot” summers in response to different warming rates (2°C to 5°C) relative to the pre-industrial level are shown in Fig 5. When the 2°C target is reached, an “extremely hot” summer is projected to occur twice every ten years at most low-mid latitudes. Central and western Asia, central and northern Africa and northern South America are relatively more vulnerable than other areas. Further- more, many areas seem to experience “extremely hot” summers 7 out of 10 years and may even suffer an “exceeding 5σ hot” summer. When the 3°C warming target is reached, this situation Fig 4. Frequency of local summer temperature anomalies. These results are based on different magnitudes of warming relative to the pre-industrial level. Response of hot summer extremes to various warming targets The multi-model ensemble mean well reproduces the statistical characteristics of the probabil- ity density distribution and covering area percentage of the present temperature extremes over the global land area, and partially captures the locations of “extremely hot” summers. This systematic evaluation of the performance of the models on hot summer extremes increases the confidence in the projected future changes in temperature extremes. Under the RCP2.6 sce- nario, the global mean temperatures of 8 models (8/26) reach the 2°C target, even though this scenario is designed to limit the global mean temperature increase to within 2°C relative to the pre-industrial level. A 3°C warming target was reached by 8 models in the RCP4.5 scenario and by all 26 models in the RCP8.5 scenario. Regarding the 4°C and 5°C warming targets, 19 and 9 models, respectively, from the RCP8.5 scenario reached these targets. In this research, we only focused on the response of the temperature extremes to different magnitudes of warming rela- tive to the pre-industrial level. Thus, the emission scenarios (RCP2.6, RCP4.5 and RCP8.5) are not distinguished in the analysis. As a result, 78 simulations are used, from 26 CMIP5 models, each simulating the three scenarios. As shown in Fig 4, the curves of the probability density widen and shift right as the global mean temperature increases. Thus, “extremely hot” summers will occur more readily in the future. The rightward shift is more severe than that during the 1990s due to the gradual aggra- vation of global warming, and significant broadening may result from various warming rates in Fig 2. Percentages of global land areas hit by summer temperature extremes. The time series spans from 1901 to 2005. The thick lines are the results of 26 individual models averages. The shading in the plots denotes the 1σ uncertainty range of the 26 CMIP5 models. Fig 2. Percentages of global land areas hit by summer temperature extremes. The time series spans from 1901 to 2005. The thick lines are the results of 26 individual models averages. The shading in the plots denotes the 1σ uncertainty range of the 26 CMIP5 models. doi:10.1371/journal.pone.0130660.g002 doi:10.1371/journal.pone.0130660.g002 5 / 11 PLOS ONE | DOI:10.1371/journal.pone.0130660 June 19, 2015 Hot Summers under Various Warming Targets Fig 3. Spatial distribution of the “extremely hot” summers frequency from 1986 to 2005. The left, middle and right maps represent the GISS, CRU and averages of the individual models results, respectively. doi:10.1371/journal.pone.0130660.g004 Response of hot summer extremes to various warming targets The temperature anomalies (relative to 1951–1980) are divided by the local standard deviation of 1901–2005 and binned into intervals of 0.05 for individual models before averaging the statistical results. The local standard deviation is calculated from the long-term non-linearly detrended time series (details in data and method section). The total number of simulations that reach the warming targets is shown in brackets. The shading represents the 1σ range of uncertainty. doi:10.1371/journal.pone.0130660.g004 Fig 4. Frequency of local summer temperature anomalies. These results are based on different magnitudes of warming relative to the pre-industrial level. The temperature anomalies (relative to 1951–1980) are divided by the local standard deviation of 1901–2005 and binned into intervals of 0.05 for individual models before averaging the statistical results. The local standard deviation is calculated from the long-term non-linearly detrended time series (details in data and method section). The total number of simulations that reach the warming targets is shown in brackets. The shading represents the 1σ range of uncertainty. doi:10.1371/journal.pone.0130660.g004 6 / 11 PLOS ONE | DOI:10.1371/journal.pone.0130660 June 19, 2015 Hot Summers under Various Warming Targets Table 2. Probability of “extremely hot” (>3σ) and “exceeding 5σ hot” (>5σ) summers in response to different magnitudes of warming relative to the pre-industrial level. Warming targets >3σ (%) >5σ (%) Reference period 0.1 0 1°C 11.0 1.3 2°C 40.3 12.9 3°C 70.4 39.1 4°C 87.3 64.0 5°C 93.3 79.6 doi:10.1371/journal.pone.0130660.t002 Table 2. Probability of “extremely hot” (>3σ) and “exceeding 5σ hot” (>5σ) summers in response to different magnitudes of warming relative to the pre-industrial level. becomes worse. Both “extremely hot” and “exceeding 5σ hot” summers are projected to occur more frequently at a 3°C than at a 2°C warming. Nearly all land areas would experience “exceeding 5σ hot” summers during a 10-year period, excluding Australia. When the global mean temperature reaches the 4°C and 5°C warming targets, essentially no land areas would be excluded from “extremely hot” summer every year. Warming by 5°C relative to pre-industrial level would also make “exceeding 5σ hot” summers extremely common in most land areas. Notably, the impacts on Australia, India and Antarctica are relatively small compared with other locations. Fig 5. Spatial distribution of the frequency of “extremely hot” (>3σ) and “exceeding 5σ hot” summers. The maps show the frequency of “extremely hot” and “exceeding 5σ hot” when the temperature increases by from 2°C to 5°C relative to pre-industrial level. Response of hot summer extremes to various warming targets The σ is calculated from the long-term non-linearly detrended time series (details in data and method section). The left and right columns present the “extremely hot” and “exceeding 5σ hot” summers at different warming targets (2°C, 3°C, 4°C and 5°C in rows 1, 2, 3, and 4, respectively). The upper and lower halves of each plot describe the boreal summer (JJA) and austral summer (DJF), respectively. The color bar is different from that shown in Fig 3. Units: times/10 yrs. doi:10 1371/journal pone 0130660 g005 Fig 5. Spatial distribution of the frequency of “extremely hot” (>3σ) and “exceeding 5σ hot” summers. The maps show the frequency of “extremely hot” and “exceeding 5σ hot” when the temperature increases by from 2°C to 5°C relative to pre-industrial level. The σ is calculated from the long-term non-linearly detrended time series (details in data and method section). The left and right columns present the “extremely hot” and “exceeding 5σ hot” summers at different warming targets (2°C, 3°C, 4°C and 5°C in rows 1, 2, 3, and 4, respectively). The upper and lower halves of each plot describe the boreal summer (JJA) and austral summer (DJF), respectively. The color bar is different from that shown in Fig 3. Units: times/10 yrs. doi:10.1371/journal.pone.0130660.g005 doi:10.1371/journal.pone.0130660.g005 7 / 11 PLOS ONE | DOI:10.1371/journal.pone.0130660 June 19, 2015 Hot Summers under Various Warming Targets The responses of the areas affected by “extremely hot” and “exceeding 5σ hot” summers to different warming targets are specifically examined based on 78 CMIP5 simulations (Fig 6). All 78 simulations reached the 1°C warming target, and 55, 34, 19 and 9 of the simulations reached the 2°C, 3°C, 4°C, 5°C warming targets, respectively. When the 1°C warming target is reached, 9.7% of the global land is projected to experience an “extremely hot” summer, and an “exceed- ing 5σ hot” summer would rarely occur over land. After exceeding the 3°C warming target, more than half of the global land area (67.0%) could experience an “extremely hot” summer. In addition, almost all of the land areas (91.4%) would experience “extremely hot” summers when the 5°C warming target is reached. “Exceeding 5σ hot” summers are projected to occur over more than half of the land area when the 4°C warming target is exceeded. Response of hot summer extremes to various warming targets Notably, the area affected by “extremely hot” summers expands rapidly between warming targets of a 1°C and 3°C, with an area increase of more than 25% per degree. Above the 3°C warming target, the area increases by less than 16% per degree. However, the areas that experience “exceeding 5σ hot” summers continues to rapidly increase by more than 17% per degree after the 2°C warm- ing target is reached. Summary and Discussions Global warming may result in various impacts on the ecology and environment. Our research focused on the changes in summer temperature extremes over land areas around the world in response to different magnitude of warming, showing one aspect of impacts from global warm- ing. To use the models’ future prediction for quantifying the temperature extremes, their simu- lating ability need to be evaluated first. A systematic evaluation of the performance of the models on predicting hot summer extremes showed the multi-model ensemble mean of the 26 CMIP5 models well reproduced the statistical characteristics of historical summer tempera- tures and captured the main locations of “extremely hot” summers. The performance of the multi-model ensemble mean is important for projecting future changes in extreme summer temperatures. The frequency, areal extent and intensity of summer temperature extremes are all projected to increase as global warming continues and higher warming results in more severe hot summers. Mid-low latitude areas are projected to experience more summer temper- ature extremes than high latitudes. Exploring the response of summer temperature extremes to various warming targets will improve our understanding in the importance of controlling the magnitude of warming. To reduce the impacts and damage from severely hot summers, global warming should be limited as much as possible. The observations indicate that the global mean Fig 6. Percentages of the areas with “extremely hot” (left) and “exceeding 5σ hot” (right) summers. The maps show the responses to different magnitudes of warming relative to pre-industrial level. The σ is calculated from the long-term non-linearly detrended time series (details in data and method section). The colors indicate the number of simulations. The line in the middle of each box represents the ensemble mean of the individual model results, and the upper and bottom edges of the box show the range of the 1σ uncertainty. The two bars outside the box represent the maximum and minimum simulation values. doi:10.1371/journal.pone.0130660.g006 Fig 6. Percentages of the areas with “extremely hot” (left) and “exceeding 5σ hot” (right) summers Fig 6. Percentages of the areas with “extremely hot” (left) and “exceeding 5σ hot” (right) summers. The maps show the responses to different magnitudes of warming relative to pre-industrial level. The σ is calculated from the long-term non-linearly detrended time series (details in data and method section). The colors indicate the number of simulations. Supporting Information S1 Table. Affiliations and atmospheric model resolutions of the 26 CMIP5 models. The durations of the historical and RCP runs are also provided. (DOCX) Acknowledgments We acknowledge the World Climate Research Programme’s Working Group on Coupled Modeling, which is responsible for CMIP. We also thank the climate groups (S1 Table) for pro- viding the model output. Our work is completed on the “Explorer 100” cluster system of Tsin- ghua National Laboratory for Information Science and Technology. We also would like to thank the anonymous reviewers for their comments and suggestions that much improved the original manuscript. Author Contributions Conceived and designed the experiments: LW JH YL. Performed the experiments: LW JH YL ZZ. Analyzed the data: LW YY. Contributed reagents/materials/analysis tools: LW JH YL ZZ. Wrote the paper: LW JH. Wrote the paper: LW JH. Hot Summers under Various Warming Targets temperature has currently increased by 0.8°C since the industrial revolution [36]. Thus, stricter greenhouse gas emission policies should be enacted to mitigate global warming as soon as possible. The quantification of temperature extremes relative to local climate mean and variability offers of useful way to analyze the extreme temperatures. Furthermore, distinct from heat waves defined by daily temperatures, this kind of definition emphasizes the overall climate change. The heat waves are of concern mainly because of their significant impacts on the human health, while hot extremes on seasonal scales may have a long-term impact on the eco- logical aspects, such as crop growth and animal breeding. However, one limitation of this defi- nition method is to neglect the absolute temperature. For example, the standard deviations for most high latitudes in the Northern Hemisphere are relatively greater than those in the tropics, and the mean temperature is lower at the high latitude. When the 2°C target is reached, the temperatures exceeding 3σ in most of the high latitude areas remain less than 30°C, while the temperatures less than 3σ in some areas of tropics are higher than 30°C. Thus, this definition of temperature extremes cannot reflect the local absolute temperatures. In addition, only the changes in the extremely hot summer temperatures are analyzed in this study, while the reasons for changes are not discussed. Some researchers have indicated that temperature extremes may be linked to weakening summer circulations, anomalous pat- terns of mid-latitudinal planetary waves, soil and atmospheric moisture, the interactions with precipitation extremes and other factors [7, 37–41]. The temperatures in different regions are affected by various circulation systems and other local characteristics. The mechanisms that cause the changes in extremely hot summers under various warming targets at different loca- tions should be studied in the future. 2. Pezza AB, van Rensch P, Cai W (2012) Severe Heat Waves in Southern Australia: Synoptic Climatol- ogy and Large Scale Connections. Climate Dynamics 38: 209–224. 1. Peterson TC, Heim RR Jr, Hirsch R, Kaiser DP, Brooks H, Diffenbaugh NS, et al. (2013) Monitoring and Understanding Changes in Heat Waves, Cold Waves, Floods, and Droughts in the United States: State of Knowledge. Bulletin of the American Meteorological Society 94: 821–834. Summary and Discussions The line in the middle of each box represents the ensemble mean of the individual model results, and the upper and bottom edges of the box show the range of the 1σ uncertainty. The two bars outside the box represent the maximum and minimum simulation values. Fig 6. Percentages of the areas with “extremely hot” (left) and “exceeding 5σ hot” (right) summers. The maps show the responses to different magnitudes of warming relative to pre-industrial level. The σ is calculated from the long-term non-linearly detrended time series (details in data and method section). The colors indicate the number of simulations. 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Comparison of Clinical Features Between Patients with Positive and Negative Appendectomy Guner Cakmak  (  guner_cakmak@yahoo.com ) Sakarya Üniversitesi Eğitim Ve Araştırma Hastanesi: Sakarya Universitesi Egitim ve Arastirma Hastanesi Baris Mantoglu  Sakarya Üniversitesi Eğitim Ve Araştırma Hastanesi: Sakarya Universitesi Egitim ve Arastirma Hastanesi https://orcid.org/0000-0002-2161-3629 Emre Gonullu  Sakarya Üniversitesi Eğitim Ve Araştırma Hastanesi: Sakarya Universitesi Egitim ve Arastirma Hastanesi Kayhan Ozdemir  Sakarya Üniversitesi Eğitim Ve Araştırma Hastanesi: Sakarya Universitesi Egitim ve Arastirma Hastanesi Burak Kamburoglu  Sakarya Üniversitesi Eğitim Ve Araştırma Hastanesi: Sakarya Universitesi Egitim ve Arastirma Hastanesi Research Keywords: acute appendicitis, mean platelet volume, total bilirubin, negative appendectomy Posted Date: December 3rd, 2020 DOI: https://doi.org/10.21203/rs.3.rs-117309/v1 Guner Cakmak  (  guner_cakmak@yahoo.com ) Comparison of Clinical Features Between Patients with Positive and Negative Appendectomy Guner Cakmak  (  guner_cakmak@yahoo.com ) Sakarya Üniversitesi Eğitim Ve Araştırma Hastanesi: Sakarya Universitesi Egitim ve Arastirma Hastanesi Baris Mantoglu  Sakarya Üniversitesi Eğitim Ve Araştırma Hastanesi: Sakarya Universitesi Egitim ve Arastirma Hastanesi https://orcid.org/0000-0002-2161-3629 Emre Gonullu  Sakarya Üniversitesi Eğitim Ve Araştırma Hastanesi: Sakarya Universitesi Egitim ve Arastirma Hastanesi Kayhan Ozdemir  Sakarya Üniversitesi Eğitim Ve Araştırma Hastanesi: Sakarya Universitesi Egitim ve Arastirma Hastanesi Burak Kamburoglu  Sakarya Üniversitesi Eğitim Ve Araştırma Hastanesi: Sakarya Universitesi Egitim ve Arastirma Hastanesi Research Keywords: acute appendicitis, mean platelet volume, total bilirubin, negative appendectomy Posted Date: December 3rd, 2020 DOI: https://doi.org/10.21203/rs.3.rs-117309/v1 License:   This work is licensed under a Creative Commons Attribution 4.0 International License.   Read Full License Comparison of Clinical Features Between Patients with Positive and Negative Appendectomy Guner Cakmak  (  guner_cakmak@yahoo.com ) Sakarya Üniversitesi Eğitim Ve Araştırma Hastanesi: Sakarya Universitesi Egitim ve Arastirma Hastanesi Baris Mantoglu  Sakarya Üniversitesi Eğitim Ve Araştırma Hastanesi: Sakarya Universitesi Egitim ve Arastirma Hastanesi https://orcid.org/0000-0002-2161-3629 Emre Gonullu  Sakarya Üniversitesi Eğitim Ve Araştırma Hastanesi: Sakarya Universitesi Egitim ve Arastirma Hastanesi Kayhan Ozdemir  Sakarya Üniversitesi Eğitim Ve Araştırma Hastanesi: Sakarya Universitesi Egitim ve Arastirma Hastanesi Burak Kamburoglu  Sakarya Üniversitesi Eğitim Ve Araştırma Hastanesi: Sakarya Universitesi Egitim ve Arastirma Hastanesi Research Keywords: acute appendicitis, mean platelet volume, total bilirubin, negative appendectomy Posted Date: December 3rd, 2020 DOI: https://doi.org/10.21203/rs.3.rs-117309/v1 License:   This work is licensed under a Creative Commons Attribution 4.0 International License. Read Full License Comparison of Clinical Features Between Patients with Positive and Negative Appendectomy Guner Cakmak  (  guner_cakmak@yahoo.com ) Sakarya Üniversitesi Eğitim Ve Araştırma Hastanesi: Sakarya Universitesi Egitim ve Arastirma Hastanesi Baris Mantoglu  Sakarya Üniversitesi Eğitim Ve Araştırma Hastanesi: Sakarya Universitesi Egitim ve Arastirma Hastanesi https://orcid.org/0000-0002-2161-3629 Emre Gonullu  Sakarya Üniversitesi Eğitim Ve Araştırma Hastanesi: Sakarya Universitesi Egitim ve Arastirma Hastanesi Kayhan Ozdemir  Sakarya Üniversitesi Eğitim Ve Araştırma Hastanesi: Sakarya Universitesi Egitim ve Arastirma Hastanesi Burak Kamburoglu  Sakarya Üniversitesi Eğitim Ve Araştırma Hastanesi: Sakarya Universitesi Egitim ve Arastirma Hastanesi Research Keywords: acute appendicitis, mean platelet volume, total bilirubin, negative appendectomy Posted Date: December 3rd, 2020 DOI: https://doi.org/10.21203/rs.3.rs-117309/v1 License:   This work is licensed under a Creative Commons Attribution 4.0 International License. Read Full License Research License:   This work is licensed under a Creative Commons Attribution 4.0 International License. Page 1/14 Page 1/14 Page 1/14 Abstract Background: The objective of this study was to retrospectively compare clinical features and prognostic values between the patients who were referred to the general surgery clinic of our hospital with the presumed diagnosis of acute appendicitis and underwent positive or negative appendectomy. Methods: Patients were divided into two groups as positive (PA) (n:362) and negative appendectomy (NA) (n:284) and the data obtained were compared between these two groups. Laboratory investigations were performed in all patients, and white blood cell (WBC), mean platelet volume (MPV), neutrophils count (NEU), neutrophils (%) (NEU%), C-reactive protein (CRP) and total bilirubin (TBIL) values were studied. Results: The mean MPV value was found as 7.88 fl in PA groups and 8.09 fl in NA group, and the mean MPV value was not statistically significantly difference in PA group, compared to NA groups (p=0.012). Laboratory parameters were also compared between genders. Accordingly, the mean MPV value was statistically significantly higher in female patients compared to male patients in PA group (p = 0.04). The mean TBIL value was 0.97 mg/dl in PA group and 0.69 mg/dl in NA group, and the mean TBIL value was statistically significantly higher in PA group (p< 0.001). Finally, TBIL value was statistically significantly lower in female patients compared to male patients in NA and PA group (p < 0.05). Conclusions: According to the results of our study, MPV and T. BIL values differ in PA and NA groups depending on gender. Therefore, these values may not be used as specific biomarkers in predicting positive acute appendicitis. We believe that these results will contribute to the literature and will be guiding for future studies. Background Appendicitis is the most common emergency surgery, with a lifetime risk for appendicitis estimated as 8.6% in men and 6.7% in women (1). In Europe, 112/100000 persons present to emergency departments each year due to acute appendicitis (2). The rate of morbidity from acute appendicitis has been reported between 7%-16% in the general population (3). Appendicitis usually presents with anorexia and tenderness in the right lower abdominal quadrant (4). However, pain in the right lower quadrant can be associated with numerous pathologies. Because of the wide spectrum of differential diagnosis and lack of specific markers for appendicitis, preoperative diagnosis of this disease is quite challenging (5). Today, the incidence of appendicitis is increasing in developing countries (6). Since serious complications may occur in the case of delayed diagnosis, prompt action is necessary. The management of appendicitis focuses on the prevention with a timely intervention before an uncomplicated appendicitis progresses to a complicated one. Appendectomy, which is sometimes performed on a normal appendicitis is known as “negative appendectomy” (NA) (7). Page 2/14 Page 2/14 Complications of acute appendicitis include perforation, peritonitis and sepsis (8). This can increase the rate of negative appendectomy operations. Negative appendectomy rate (NAR) is defined as the incidence of removing appendices that are pathologically normal (9). Negative appendectomy leads to prolonged hospitalization, morbidity and increased costs. In addition, negative appendectomy may be associated with severe postoperative complications. Therefore, an accurate preoperative diagnosis is essential in the cases of suspected acute appendicitis. In the United States of America (USA) over 250000 appendectomy operations are performed yearly, and the rate of negative is approximately 15% in these operations (10). However, owing to better imaging modalities, a constant decrease is seen in the rate of NAs (11). Gynecological pathologies in female patients in childbearing period mimic acute appendicitis, which can cause an increase in the rate of negative NAs (12). According to the current guidelines, the diagnosis of acute appendicitis is established according to clinical examination and confirmed with imaging investigations and some biomarkers including WBC count and CRP (13). Computed tomography (CT) and ultrasonography are used as imaging modalities in order to set a more accurate diagnosis. The objective of this study was to retrospectively compare clinical features and prognostic values between the patients who were referred to the general surgery clinic of our hospital with the presumed diagnosis of acute appendicitis and underwent positive or negative appendectomy. Methods Data of a total of 2168 patients who were referred to our general surgery clinic with the presumed diagnosis of appendicitis and underwent appendectomy between 2010 and 2019 were retrospectively evaluated. Among these patients, 1522 were excluded from the study because of missing data, and those who underwent interval appendectomy, and incidental appendectomy, and the patients for whom appendectomy operation was delayed due to conservative treatment. Finally, data of 646 patients were obtained and evaluated. Patients aged between 16–90 years. Patients were divided into two groups as positive (PA) (n:362) and negative appendectomy (NA) (n:284) and the data obtained were compared between these two groups. Patients’ demographic data such as age and gender, date and type of operations, investigations, clinical presentation, macroscopic and microscopic findings, imaging and pathological findings were recorded and analyzed. In addition, laboratory investigations were performed in all patients, and white blood cell (WBC), mean platelet volume (MPV), neutrophils count (NEU), neutrophils (%) (NEU%), C-reactive protein (CRP) and total bilirubin (TBIL) values were studied. Page 3/14 Inclusion criteria included all patients operated during the nine years period. Considering clinical conditions of the patients and in the cases of uncertain diagnosis or where the clinical picture did not meet physical findings (age, pregnancy, having gynecological examination, comorbidities etc.) abdominopelvic computed tomography and ultrasonography examinations were performed in some patients. The diagnosis of appendicitis was established based on macroscopic findings. Histological Page 3/14 diagnosis of appendicitis was set according to the infiltration of muscularis propria with neutrophils granulocytes. Appendectomies were performed by conventional or laparoscopic methods. diagnosis of appendicitis was set according to the infiltration of muscularis propria with neutrophils granulocytes. Appendectomies were performed by conventional or laparoscopic methods. Lack of the appendicitis in the pathology reports was considered negative appendectomy. In order to identify appendectomy cases with negative samples, pathology reports were interpreted by an author blinded to whether preoperative imaging was performed. Negative appendectomy was defined as a normal appendix following the resection due to suspected appendicitis or medically unnecessary appendectomy. Medically unnecessary appendectomy was defined as an appendectomy operation performed in the case of typically contraindicated surgery. Before the beginning of the study, necessary ethics approval was received from the local ethics committee of our hospital. Since the study had a retrospective design, receiving informed consent from the patients was waived. The study was conducted in accordance with the ethical principles of the Declaration of Helsinki. Statistical Analysis Data obtained in the study were analyzed utilizing SPSS version 23.0 (Statistical Package for Social Sciences, SPSS, IBM Incusing Chicago, IL, USA) statistical package software. Normality of the variables was analyzed with the Kolmogorov-Smirnov test. In the comparison of the variables between the groups, Independent Student t test among was used for the normally distributed variables, and Mann-Whitney U test for non-normally distributed variables. Continuous variables were expressed with mean ± standard deviation, while categorical variables were given as frequency and percentage. p < 0.05 values were considered statistically significant. Results A total of 646 patients were included in the study with 362 (56%) being in PA group and 284 (44%) in NA group. Of all patients included in the study, 360 (55.7%) were male and 286 (44.3%) were female. A total of 226 (62.4%) patients were male and 136 (37.6%) were female in PA group. Whereas 134 (47.2%) were male and 150 (52.8%) were female in the NA group (Table 1). No statistically significant difference was seen between both groups in terms of gender (p > 0.05). Page 4/14 Table 1 Frequency distribution of patients' gender and age by type of appendectomy   PA. (n = 362) NA. (n = 284)   n % n % Male 226 62.4 134 47.2 Female 136 37.6 150 52,8 Sum 362 100 284 100 Mean Age 28.2 ± 16.65 31.9 ± 17.4 PA: positive appendicitis NA: negative appendicitis Table 1 The mean age of all patients was 30.1 ± 16.9 years, and the mean age was found as 28.2 ± 16.65 years in PA group and 31.9 ± 17.4 years in NA group (Table 1). There was no statistically significant difference between PA and NA groups in terms of age (p > 0.05). The mean age of all patients was 30.1 ± 16.9 years, and the mean age was found as 28.2 ± 16.65 years in PA group and 31.9 ± 17.4 years in NA group (Table 1). There was no statistically significant difference between PA and NA groups in terms of age (p > 0.05). Appendectomies were performed with laparoscopic method in 199 (54.9%) of the patients in PA group and in 207 (72.8%) of the patients in NA group. The remaining patients underwent open surgery. Appendectomies were performed with laparoscopic method in 199 (54.9%) of the patients in PA group and in 207 (72.8%) of the patients in NA group. The remaining patients underwent open surgery. When status of having gynecological examination was examined among the female patients; 105 (77.3%) female patients in PA group and 125 (83.5%) female patients in NA group underwent gynecological examination. When status of having gynecological examination was examined among the female patients; 105 (77.3%) female patients in PA group and 125 (83.5%) female patients in NA group underwent gynecological examination. Laboratory outcomes were examined and compared between the two groups. Results Accordingly, the mean WBC value was found as 9.97 K/mm3 in PA group and 9.51 K/mm3 in NA group, and there was no statistically significant difference between both groups (p = 0.07). The mean MPV value was found as 7.88 fl in PA groups and 8.09 fl in NA group, and there was no statistically significant difference between both groups (p = 0.12). NEU (%) value was found as 76.01 in PA group and 75.69 in NA group. No statistically significant difference was observed between the two groups in terms of the mean NEU (%) value (p =  0.75). The mean neutrophil count was found as 7.33 K/mm3 in the PA group and 7.50 K/mm3 in NA group, and no statistically significant difference was found between the two groups (p = 0.48). The mean CRP value was found as 57.68 mg/dl in PA group and 41.43 mg/dl in NA group. There was a statistically significant difference between both groups in terms of the mean CRP value (p < 0.001). When total bilirubin values were examined; the mean TBIL value was 0.97 mg/dl in PA group and 0.69 mg/dl in NA group, and the mean TBIL value was statistically significantly higher in PA group (p < 0.001) (Table 2). Page 5/14 Page 5/14 Table 2 Comparison results of WBC, MPV, NEU (%), NEU, CRP and TBIL parameters of patients according to the type of appendectomy   Appendectomy Type n x̄ ss t p WBC Positive 362 9.97 3.44 1.81 .07 Negative 284 9.51 2.96 MPV Positive 362 7.88 1.65 -1.55 .12 Negative 284 8.09 1.79 NEU% Positive 362 76.01 12.94 .31 .75 Negative 284 75.69 12.23 NEU Positive 362 7.33 2.72 − .69 .48 Negative 284 7.50 3.64 CRP Positive 362 57.68 58.92 3.54 .00 Negative 284 41.43 56.36 TBIL Positive 362 .97 .57 7.39 .00 Negative 284 .69 .39 In our study, the examined laboratory parameters were also compared between genders. Accordingly, the mean MPV value was statistically significantly higher in female patients compared to male patients in PA group (p = 0.04). NEU (%) value was statistically significant between genders both in PA and NA groups. Accordingly, NEU (%) value was statistically significantly lower in male patients compared to female patients in both PA and NA groups (p < 0.05, p < 0.05; respectively). Neutrophil count was statistically significantly lower in male patients in NA group (p < 0.05). Results Finally, TBIL value was statistically significantly lower in female patients compared to male patients in NA and PA group (p < 0.05) (Table 3– 4). 4). Page 6/14 Table 3 Comparison results of WBC, MPV, NEU (%), NEU, CRP and TBIL parameters according to the gender of the patients' in positive appendectomy group. Type Variables Gender n x̄ ss t p Positive Appendectomy WBC Male 226 10.08 3.58 .76 .45 Female 136 9.80 3.21 MPV Male 226 7.74 1.58 -2.09 .04 Female 136 8.12 1.73 NEU% Male 226 74.98 13.24 -1.96 .04 Female 136 77.73 12.29 NEU Male 226 7.20 2.78 -1.17 .24 Female 136 7.54 2.61 CRP Male 226 57.79 57.72 .05 .96 Female 136 57.49 61.07 TBIL Male 226 1.06 .60 3.98 .00 Female 136 .82 .50 Page 7/14 Table 4 Comparison results of WBC, MPV, NEU (%), NEU, CRP and TBIL parameters according to the gender of the patients' in negative appendectomy group. Type Variables Gender n x̄ ss t p Negative Appendectomy WBC Male 134 9.46 2.59 -.28 .78 Female 150 9.55 3.27 MPV Male 134 7.88 1.59 -1.92 .06 Female 150 8.29 1.95 NEU% Male 134 73.02 12.72 -3.55 .00 Female 150 78.08 11.29 NEU Male 134 7.09 1.99 -1.82 .07 Female 150 7.87 4.62 CRP Male 134 40.99 52.98 − .12 .90 Female 150 41.83 59.39 TBIL Male 134 .75 .37 -2.57 .01 Female 150 .63 .39 When diameters of appendicitis were evaluated; the mean appendicitis diameter was found as 1.09 cm in PA group and 0.88 cm in NA group, and the mean appendicitis diameter was statistically significantly higher in PA group compared to NA group (p < 0.001) (Table 5). Table 5 Comparison results of appendectomy material diameters according to appendectomy type   Appendectomy Type n x̄ ss t p Diame Positive 362 1.09 .63 4.17 .00 Negative 284 .88 .63 Table 5 Comparison results of appendectomy material diameters according to appendectomy type Discussion Surgical intervention of a normal appendicitis exposes patients to unnecessary anesthesia and surgical complications, and this may be resulted from improper clinical evaluation, and lack of diagnostic methods. Despite the high incidence of acute appendicitis and use of laboratory markers and imaging Page 8/14 Page 8/14 modalities, the accurate diagnosis remains challenging. Research of the diagnostic process used for acute appendicitis is highly dynamic, within this context information such as novel inflammatory biomarkers is constantly reported in the literature (14). As in our country, high rates of appendicitis cases have been reported in some other countries (15). Therefore, more effort should be made in order to reduc the incidence of NAR and its complications on patients and hospitals (16). Negative appendectomies lead to both postoperative complications, increased morbidity and mortality rates and costs. Thus, investigation of the factors leading to negative appendectomies is of paramount importance. Among these factors, laboratory parameters take an important place. However, to our knowledge there is no consensus in the literature on this subject. In our study, we first evaluated laboratory values between positive and negative appendectomy cases. We performed preoperative computed tomography by excluding elderly patients, pediatric patients and those with comorbidities. We observed that the rate of negative appendectomy was lower in patients who underwent CT (24.3%). In a study by Wagner et al., the rate of performing preoperative CT raised to 95% from 32% within 10 years (17). In our study, 84.6% of female patients were found to have a gynecological examination. In a study by Joshi et al. 57.1% of female patients were reported to have gynecological examination (16). We believe that higher gynecological examination rate in our study resulted from the necessity of transvaginal ultrasound in addition to abdominal ultrasound in cases of suspected gynecological diseases in young women in order to reduce the incidence of negative appendectomy. Recently, although diagnostic value of laboratory parameters such as MPV, RDW and NAR has been evaluated in patients with suspected appendicitis, results of these studies are highly controversial (18). MPV is a measurement of thrombocyte size that is obtained as a part of routine complete blood count and is usually overlooked by clinicians (19). Changes in platelet counts can lead to changes in MPV. The size and activity of platelets can be influenced by cytokines such as interleukin IL-3 and IL-6. Discussion Elevated MPV levels have been reported in several diseases including chronic obstructive pulmonary disease (COPD), myocardial infarction, diabetes mellitus and high altitude (20). Increases in MPV levels are associated with chronic diseases, while decreases are related to acute diseases (21). In our study, the mean MPV value was not statistically significantly lower in both the PA group and NA group. Although there are a few studies about the role of MPV in acute appendicitis, the results of these studies are variable(20, 22). In a study comparing the healthy control group with patients having acute appendicitis, MPV level was found to be significantly lower in the acute appendicitis group. In the same study, it was emphasized that MPV level should not be overlooked in suspected acute appendicitis cases (20). In another study, a significant reduction was found in MPV level of patients with appendicitis (23). In a study by Uyanik et al., no statistical significance was observed in MPV levels of patients with acute appendicitis (22). In another study, higher MPV levels were found in patients with acute appendicitis compared to the control group (18). In a meta-analysis of five studies including 2101 patients with acute appendicitis, it was reported that MPV can be used as a biomarker for the diagnosis of positive Page 9/14 Page 9/14 appendicitis and is a rapid and inexpensive indicator (24). In our study, in the gender-based evaluation, MPV values were statistically significantly lowered only in male patients in the PA group. Based on our findings, we believe that MPV values under the lower normal range may be affected by gender and requires further evaluation to be used as a biomarker for positive acute appendicitis. Recent studies have investigated the relationship between hyperbilirubinemia and vermiform of inflammation, and some of these studies have reported that bilirubin can be used as a specific marker of appendiceal perforation (25, 26). In our study, TBIL levels were significantly higher in the PA group compared to the NA group (Table 2). However, in gender-based evaluation, TBIL levels were significantly higher in male patients both in PA and NA groups. In a study by Akbulut et al., a TBIL cut-off ≥ 0.67 is an independent factor predicting acute appendicitis (27). Therefore, our study supports the latest literature in line with our findings. Conclusion According to the results of our study, MPV and T. BIL values differ in PA and NA groups depending on gender. Therefore, these values may not be used as specific biomarkers in predicting positive acute appendicitis. We believe that these results will contribute to the literature and will be guiding for future studies. However, further studies are needed to determine the laboratory parameters that can be used as diagnostic biomarkers. Discussion In addition, although the high CRP value in acute appendicitis has been reported in various studies,(13, 28) our outcome of high CRP values in the PA group supports the literature. Although there are several studies about the use of various laboratory markers in the diagnosis of acute appendicitis that were mentioned above, there is still no scientific evidence on the use of blood parameters in predicting acute appendicitis. At this point, it seems possible that NA ratios can be reduced by correlating more than one laboratory data. In imaging examination, one of the most important findings for the diagnosis of acute appendicitis is appendicitis diameter. In the present study, appendicitis diameter was statistically significantly higher in PA group than in NA group (Table 4). Similarly, in a study by Katipoglu et al., the mean appendicitis diameter was significantly higher in the positive appendectomy cases (29). The main limitations of this study are its retrospective design and being conducted in a single center. However, the number of our patients is relatively higher than the other studies in the literature. In addition, unlike the other studies in the literature higher MPV (female group) and TBIL values in the positive appendectomy cases will bring a new projection to the literature. Availability of data and materials There is no additional data available to share with the readers. The datasets used and/or analyzed during the current study are available from the corresponding author on reasonable request. WBC: White blood count MPV: Mean platelet volume NEU: neutrophils count NEU%: neutrophils count% CRP: C-Reactive protein TBIL: Total bilirubin NAR: Negative appendectomy rate CT: Computed Tomography WBC: White blood count MPV: Mean platelet volume NEU: neutrophils count NEU%: neutrophils count% CRP: C-Reactive protein TBIL: Total bilirubin NAR: Negative appendectomy rate CT: Computed Tomography Consent for publication Written informed consent was not received due to the retrospective nature of the study. Written informed consent was not received due to the retrospective nature of the study. Ethics approval and consent to participate Sakarya University Ethics Committee has approved this research project. 71522473/050.01.04/424 Consent for publication Sakarya University Ethics Committee has approved this research project. 71522473/050.01.04/424 Consent for publication Sakarya University Ethics Committee has approved this research project. 71522473/050.01.04/424 Conflict of Interest The authors declared no conflict of interest. The authors declared no conflict of interest. The authors declared no conflict of interest. The authors declared no conflict of interest. PA: Positive appendicitis PA: Positive appendicitis NA: Negative Appendicitis Page 10/14 References 1. Huang TH, Huang YC, Tu CW. Acute appendicitis or not: Facts and suggestions to reduce valueless surgery. J Acute Med. 2013; 3(4):142–7. 2. Ferris M, Quan S, Kaplan BS, Molodecky N, Ball CG, Chernoff GW, et al. The Global Incidence of Appendicitis: A Systematic Review of Population-based Studies. Ann Surg. 2017;266(2):237–41. 3. Tsai MC, Kao LT, Lin HC, Chung SD, Lee CZ. Acute appendicitis is associated with peptic ulcers: A population-based study. Sci Rep. 2015;8;5:18044. 3. Tsai MC, Kao LT, Lin HC, Chung SD, Lee CZ. Acute appendicitis is associated with peptic ulcers: A population-based study. Sci Rep. 2015;8;5:18044. 4. World Health Organization. Surgical care at the district hospital. 2003; 4. World Health Organization. Surgical care at the district hospital. 2003; 5. Poletti PA, Becker M, Becker CD, Halfon Poletti A, Rutschmann OT, Zaidi H, et al. Emergency assessment of patients with acute abdominal pain using low-dose CT with iterative reconstruction: a comparative study. Eur Radiol. 2017; 27(8):3300–9. 5. Poletti PA, Becker M, Becker CD, Halfon Poletti A, Rutschmann OT, Zaidi H, et al. Emergency assessment of patients with acute abdominal pain using low-dose CT with iterative reconstruction: a comparative study. Eur Radiol. 2017; 27(8):3300–9. 6. Sartelli M, Baiocchi GL, Di Saverio S, Ferrara F, Labricciosa FM, Ansaloni L, et al. Prospective Observational Study on acute Appendicitis Worldwide (POSAW). World J Emerg Surg. 2018;13(1):19. 6. Sartelli M, Baiocchi GL, Di Saverio S, Ferrara F, Labricciosa FM, Ansaloni L, et al. Prospective Observational Study on acute Appendicitis Worldwide (POSAW). World J Emerg Surg. 2018;13(1):19. 7. Ruffolo C, Fiorot A, Pagura G, Antoniutti M, Massani M, Caratozzolo E, et al. Acute appendicitis: What is the gold standard of treatment? World Journal of Gastroenterology. 2013;19(47):8799–807. 7. Ruffolo C, Fiorot A, Pagura G, Antoniutti M, Massani M, Caratozzolo E, et al. Acute appendicitis: What is the gold standard of treatment? World Journal of Gastroenterology. 2013;19(47):8799–807. 8. Webb EM, Nguyen A, Wang ZJ, Stengel JW, Westphalen AC, Coakley F V. The negative appendectomy rate: Who benefits from preoperative CT? Am J Roentgenol. 2011;197(4):861–6. 8. Webb EM, Nguyen A, Wang ZJ, Stengel JW, Westphalen AC, Coakley F V. The negative appendectomy rate: Who benefits from preoperative CT? Am J Roentgenol. 2011;197(4):861–6. 9. Raja AS, Wright C, Sodickson AD, Zane RD, Schiff GD, Hanson R, et al. Negative appendectomy rate in the era of CT: An 18-year perspective. Radiology. 2010; 256(2):460–5. 9. Authors’ Contribution GC, BM: Conceived the study design, involved in data collection, performed the statistical analysis, interpreted data and prepared the manuscript draft. Page 11/14 Page 11/14 Page 11/14 EG: Concept and design of study, analysis and draft formation. EG: Concept and design of study, analysis and draft formation. EG: Concept and design of study, analysis and draft formation. EG: Concept and design of study, analysis and draft formation. KO: Concept and design of study, data collection and analysis. KO: Concept and design of study, data collection and analysis. KO: Concept and design of study, data collection and analysis. KO: Concept and design of study, data collection and analysis. BK: Data collection, analysis and results compilation, Analysis, review and final approval of draft. BK: Data collection, analysis and results compilation, Analysis, review and final approval of draft. All the authors critically reviewed the final version of the manuscript and approved the final version Acknowledgement All the authors critically reviewed the final version of the manuscript and approved the final version Acknowledgement References Raja AS, Wright C, Sodickson AD, Zane RD, Schiff GD, Hanson R, et al. Negative appendectomy rate in the era of CT: An 18-year perspective. Radiology. 2010; 256(2):460–5. 10. Florence M, Flum DR, Jurkovich GJ, Lin P, Steele SR, Symons RG, et al. Negative appendectomy and imaging accuracy in the washington state surgical care and outcomes assessment program. Ann Surg. 2008;248(4):557–63. 11. Seetahal SA, Bolorunduro OB, Sookdeo TC, Oyetunji TA, Greene WR, Frederick W, et al. Negative appendectomy: A 10-year review of a nationally representative sample. Am J Surg. 2011;201(4):433–37. 12. Hatipoglu S, Hatipoglu F, Abdullayev R. Acute right lower abdominal pain in women of reproductive age: Clinical clues. World J Gastroenterol. 2014;14:4043–9. Page 12/14 Page 12/14 13. Yu CW, Juan LI, Wu MH, Shen CJ, Wu JY, Lee CC. Systematic review and meta-analysis of the diagnostic accuracy of procalcitonin, C-reactive protein and white blood cell count for suspected acute appendicitis. . Br J Surg. 2013;100(3):322-9. 14. Sack U, Biereder B, Elouahidi T, Bauer K, Keller T, Tröbs RB. Diagnostic value of blood inflammatory markers for detection of acute appendicitis in children. BMC Surg. 2006;6(1):15–15. 15. Kirkil C, Karabulut K, Aygen E, Ilhan YS, Yur M, Binnetoǧlu K, et al. Appendicitis scores may be useful in reducing the costs of treatment for right lower quadrant pain. Ulus Travma ve Acil Cerrahi Derg. 2013;19:13–9. 16. Joshi MK, Joshi R, Alam SE, Agarwal S, Kumar S. Negative Appendectomy: an Audit of Resident- Performed Surgery. How Can Its Incidence Be Minimized? Indian J Surg. 2015;1(3):913–7. 17. Wagner PL, Eachempati SR, Soe K, Pieracci FM, Shou J, Barie PS. Defining the current negative appendectomy rate: For whom is preoperative computed tomography making an impact? Surgery. 2008;144:276–82. 18. Narci H, Turk E, Karagulle E, Togan T, Karabulut K. The role of red cell distribution width in the diagnosis of acute appendicitis: A retrospective case-controlled study. World J Emerg Surg. 2013;8:46. 19. Beyazit Y, Sayilir A, Torun S, Suvak B, Yesil Y, Purnak T, et al. Mean platelet volume as an indicator of disease severity in patients with acute pancreatitis. Clin Res Hepatol Gastroenterol. 2012;36:162-8. 20. Albayrak Y, Albayrak A, Albayrak F, Yildirim R, Aylu B, Uyanik A, et al. Mean platelet volume: A new predictor in confirming acute appendicitis diagnosis. Clin Appl Thromb. 2011;17:362–6. 21. Erdem H, Aktimur R, Cetinkunar S, Reyhan E, Gokler C, Irkorucu O, et al. 29. Katipoglu B, Aygun A CH. The effect of appendix diameter on perforation in acute appendicitis cases. CMJ. 2019;41:392–7. References Evaluation of mean platelet volume as a diagnostic biomarker in acute appendicitis. Int J Clin Exp Med. 2015;8(1):1291–5. 22. Uyanik B, Kavalci C, Arslan ED, Yilmaz F, Aslan O, Dede S, et al. Role of Mean Platelet Volume in Diagnosis of Childhood Acute Appendicitis. Emerg Med Int. 2012; 2012:823095. 22. Uyanik B, Kavalci C, Arslan ED, Yilmaz F, Aslan O, Dede S, et al. Role of Mean Platelet Volume in Diagnosis of Childhood Acute Appendicitis. Emerg Med Int. 2012; 2012:823095. 23. Bilici S, Sekmenli T, Göksu M, Melek M, Avci V. Mean platelet volume in diagnosis of acute appendicitis in children. Afr Health Sci. 2011;11:427–32. 23. Bilici S, Sekmenli T, Göksu M, Melek M, Avci V. Mean platelet volume in diagnosis of acute appendicitis in children. Afr Health Sci. 2011;11:427–32. 24. Fan Z, Zhang Y, Pan J, Wang S. Acute appendicitis and mean platelet volume: A systemic review and meta-analysis. Ann Clin Lab Sci. 2017;47(6):768–72. 24. Fan Z, Zhang Y, Pan J, Wang S. Acute appendicitis and mean platelet volume: A systemic review and meta-analysis. Ann Clin Lab Sci. 2017;47(6):768–72. 25. D’Souza N, Karim D, Sunthareswaran R. Bilirubin; a diagnostic marker for appendicitis. Int J Surg. 2013;11:1114–7. 25. D’Souza N, Karim D, Sunthareswaran R. Bilirubin; a diagnostic marker for appendicitis. Int J Surg. 2013;11:1114–7. 26. Muller S, Falch C, Axt S, Wilhelm P, Hein D, Königsrainer A, et al. Diagnostic accuracy of hyperbilirubinaemia in anticipating appendicitis and its severity. Emerg Med J. 2015; 0:1–5. 26. Muller S, Falch C, Axt S, Wilhelm P, Hein D, Königsrainer A, et al. Diagnostic accuracy of hyperbilirubinaemia in anticipating appendicitis and its severity. Emerg Med J. 2015; 0:1–5. 27. Akbulut S, Koc C, Sahin TT et al. Akut Apandisit ve Perfore Apandisiti öngören faktörlerin belirlenmesi. Ulus Travma Acil Cerrahi Derg. Ahead of Print: UTD-60344. doi:10.14744/tjtes.2020.60344. 28. Mantoğlu B, Karip B, Mestan M, Işcan Y, Ağca B, Altun H, et al. Should appendectomy be performed laparoscopically? Clinical prospective randomized trial. Turkish J Surg. 2015;31(4):224-8. 28. Mantoğlu B, Karip B, Mestan M, Işcan Y, Ağca B, Altun H, et al. Should appendectomy be performed laparoscopically? Clinical prospective randomized trial. Turkish J Surg. 2015;31(4):224-8. 28. Mantoğlu B, Karip B, Mestan M, Işcan Y, Ağca B, Altun H, et al. Should appendectomy be performed laparoscopically? Clinical prospective randomized trial. Turkish J Surg. 2015;31(4):224-8. Page 13/14 Page 13/14 Page 14/14
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Mediernes repræsentationer af ungdommen gennem 50 år
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38 38 // JOURNALISTICA · NR. 1 · 2010 AF CHARLOTTE WIEN OG KLAUS LEVINSEN* Moderne massemedier spiller en stor rolle for, hvordan vi opfatter og forstår ungdommen, og tidligere internationale undersøgelser har vist, at medierne overvejende formidler et negativt billede af de unge. På baggrund af kvantitative indholdsanalyser belyser artiklen hvordan de unge er blevet fremstillet i et udvalg af danske aviser over de seneste 50 år. Vores undersøgelse viser, at kriminalitet, kul- tur og sport er de emner, der hyppigst er i spil, men undersøgelsen kan ikke bekræfte, at der er en dominerende negativ bias i medier- nes beskrivelser af de unge. Dette kunne hverken bekræftes i forhold til undersøgelsen af nutidens mediedækning (2003) eller i undersø- gelsen af langtidsperspektivet (1953-2003). Tvært i mod afslører vo- res data en udvikling i retning af flere neutrale journalistiske beskri- velser af unge, og en tendens til, at de unge i dag citeres direkte i avisartiklerne. Indledning At „ungdommen ikke længere er, hvad den har været“ er et gam- melt ordsprog og – i logisk henseende – et sandt udsagn: Den gruppe, der i fortiden var unge rummer i sagens natur ikke de samme individer som nu, og de vilkår, der gives ungdommen i dag er ikke de samme som i går. Udsagnet er interessant i vores sammenhæng af andre grunde: JOURNALISTICA · NR. 1 · 2010 // 39 Den første er, at der i ligger en antagelse om, at man i en enkelt sætning kan sige noget generelt om alle de individer, der tilhører den sociale gruppe, som vi kalder „ungdommen“. Den anden er, forestillingen om, at de unges adfærd og holdninger ændrer sig over tid. Den første er, at der i ligger en antagelse om, at man i en enkelt sætning kan sige noget generelt om alle de individer, der tilhører den sociale gruppe, som vi kalder „ungdommen“. Den anden er, forestillingen om, at de unges adfærd og holdninger ændrer sig over tid. Ordsproget kan både henvise til „ungdommen“ sådan som den i virkeligheden er, og til vore forestillinger om ungdommen. Grundantagelsen i denne artikel er, at massemedierne spiller en stor rolle for begge dele, og formålet er at undersøge, hvordan „de unge“ og „ungdommen“ i tidens løb er blevet fremstillet i et udvalg af danske aviser Der findes allerede en række internationale studier af, hvordan medierne fremstiller ungdommen, men ingen danske. I flere af disse undersøgelser konkluderes det, at medierne beskriver de unge negativt, og at dette bl.a. skyldes en tiltagende tabloidise- ring og kommercialisering af medierne. De mediemæssige be- tingelser og den journalistiske praksis skaber derved nogle skæv- vredne billeder af de unge som problematiske samfundsborgere, der er mere voldelige, ekstravagante og utilpassede end andre grupper i befolkningen. De fleste af disse studier er imidlertid be- grænsede af deres relativt korte undersøgelsesperioder, og har derved vanskeligt ved at sige noget om forandringerne i medier- nes fremstillinger af de unge. Tidligere studier af hvordan medi- erne gennem tiden har behandlet „de ældre“, viser, at journali- stikken betjener sig af stereotype fremstillinger af sociale grup- per, men også at disse fremstillinger forandrer sig i takt med tiden (Wien 2005). Med dette udgangspunkt undersøger vi her i artiklen hvordan danske medier har repræsenteret ungdommen gennem 50 år, og vil svare på fire spørgsmål, nemlig: • I hvilket omfang indeholder nyhedshistorier om unge hhv. Indledning negative og positive repræsentationer af unge? • Hvilke emner er de foretrukne i nyhedsmediernes dækning af historier om unge? • Er der forskelle i mediernes dækning af hhv. unge mænd og unge kvinder? • Hvilken rolle spiller de unge selv i nyhederne? / JOURNALISTICA · NR. 1 · 2010 40 I de følgende afsnit præsenteres først artiklens teoretiske ud- gangspunkt samt en gennemgang af den tidligere forskning på området. Herefter følger overvejelser vedrørende data og metode samt de empiriske analyser. Medierepræsentationer Talrige studier har vist, at medierne påvirker samfundet: Lige fra den førte politik, vores adfærd og menings- og holdningsdan- nelser (fx Gamson and Modigliani 1989; Donovan and Leivers 1993; Hurtz and Durkin 2004; Wyn 2005; Paterson 2006; Slater 2007; Bleakney, Hennessy et al. 2008) til vores sprog (Gorham 2006). Og netop derfor kan longitudinale studier af mediernes repræsentationer af særlige grupper i samfundet tilvejebringe vi- den om betydningen af strukturelle ændringer i samfundsorde- nen samt om samfundets udvikling. Begrebet medierepræsentation refererer altså til den måde hvorpå forskellige grupper baseret på fx alder, køn eller race be- skrives i medierne og er dermed nært beslægtet med både stu- diet af nyhedsmediernes fortællemæssige rammer (frames) og studiet af stereotyper. Reelt mener vi ikke, at der kan lægges et klart definitorisk snit mellem de tre koncepter: Frames blev op- rindeligt defineret som “schemata of interpretation” (Goffman 1974:21). Begrebet “schemata” er blevet brugt af socialpsykologer til at beskrive de kognitive kategorier, som vi bruger, når vi skal organisere vores hukommelse og kategorisere informationer (Stangor 2000:6; Tajfel and Forgas 2000:50). Og tilsvarende defi- nerede Gitlin “media frames” som “persistent patterns of cogni- tion, by which symbol-handlers routinely organize discourse, whether verbal or textual” (Gitlin 1980:7).1 At Gitlin anskuede mediernes framing som vedvarende (per- sistent) kognitive mønstre gør slægtskabet med social psykolo- gernes stereotyper tydeligt: Ifølge Stangor (Stangor 2000:13) be- tragtes stereotyper generelt som uforanderlige forestillinger. Men forskningen heri har vist, at de rent faktisk ændrer sig over tid (fx (Donovan and Leivers 1993; Devine and Elliott 2000; Wien 2005; Covert and Dixon 2008)). Med andre ord: Stereotyper er ikke ufor- anderlige mønstre. Samme konklusion er nået af forskere, der har JOURNALISTICA · NR. 1 · 2010 // 41 41 arbejdet med longitudinale studier af mediernes framing af sær- lige emner (fx. (Semetko and Valkenburg 2000; Schulz and Zeh 2005; Scheufele and Tewksbury 2007)).2 På samme måde inkluderer studier af medierepræsentationer ofte analyser af stereotyper (fx (Porteous and Colston 1980; Au- brun and Grady 2000; Devlin 2000; Heintz-Knowles 2000; Maz- zarella 2003; Dixon and Maddox 2005; Wyn 2005; Covert and Dixon 2008).3 I sådanne studier inkluderes også typisk analyser af kildebrug, mediernes prioritering af et emne, genrer og et antal kvantitative mål som længde, illustrationer og personlige karak- teristika (fx køn) af personer omtalt i de enkelte nyhedshistorier (Porteous and Colston 1980; Falchikov 1986; Devlin 2000; McMa- nus 2002; Mazzarella 2003; Wyn 2005). Medierepræsentationer Sammenfattende mener vi, at studiet af framing, studiet af stereotyper og studiet af medierepræsentationer er så nært be- slægtede, at vi kan hente inspiration til vores analyse fra litteratur fra alle tre forskningsområder. Men, før vi når så vidt vil vi op- summere, hvad den tidligere forskning i mediernes repræsenta- tioner, stereotyper og framing af ungdommen har vist. Tidligere forskning i mediernes repræsentationer af unge Der foreligger som nævnt allerede et antal undersøgelser, der be- lyser hvordan medierne repræsenterer unge mennesker. Aubrun and Gradys (2000) undersøgte forskellene på de lokale og nationale amerikanske nyhedsmediers dækning af unge. De- res undersøgelse viste, at de lokale medier er mere tilbøjelige til at benytte negative og forsimplede stereotyper om unge. Der er dog stor forskel på, hvilken vinkel medierne anlægger: De lokale medier har en tendens til at indtage en slags tilskuerrolle, hvor det er vanskelligt for læsere, lyttere eller seere at involvere sig fø- lelsesmæssigt. Det gør de gennem hurtigt klippede øjenvidne- agtige nyheder, hvor der fokuseres på unge som ofre for eller årsagen til ulykker eller kriminalitet. De nationale nyhedsmedier er derimod mere tilbøjelige til at præsentere nyheder om unge inden for en tematisk ramme, der rummer såvel problemernes kontekst og proportioner, samt forslag til løsninger (Aubrun and Grady 2000:2-5). / JOURNALISTICA · NR. 1 · 2010 42 Heintz-Knowles (2000) har undersøgt hvordan unge blev fremstillet i amerikanske prime-time TV-serier, og resultaterne viste, at man som seer oftere blev præsenteret for unge kvinder end for unge mænd, og at der oftere var tale om hvide end om nogen anden race. Emnemæssigt var fokus på kærlighed, ven- skab og familie og på emner som narkotika, mens alkohol og kriminalitet kun meget sjældent indgik. Alligevel blev de unge of- test beskrevet som årsagen til et eller andet problem, som „de voksne“ skulle ordne (Heintz-Knowles 2000:6). Hendes resultater adskiller sig dog markant fra de undersøgelser der er foretaget af nyhedsmediernes dækning af de unge. Nichols and Good (2004) undersøger, hvordan mediernes dækning af vold, sex, tobak, alkohol, narkotika osv. har ændret sig over tid. I den forbindelse diskuterer de også mediernes re- præsentationer af unge i sådanne historier. De mener, at ame- rikanernes holdning til de unge bliver mere og mere negativ (Nichols & Good 2004:5), og forklarer årsagerne hertil med en kombination af ændringer i familiemønstre og øget medieindfly- delse. I deres perspektiv er det især underholdnings- og nyheds- medierne, der bærer skylden, fordi der er en skævvredet dæk- ning med alt for stort fokus på negative historier om børn og unge. Ifølge dem er det den hastige vækst på medieområdet gennem de seneste 50 år, der er årsagen til, at medierne leverer en nyhedsdækning, hvor fokus er på historier, der involverer unge som ofre for ulykker eller kriminalitet eller som årsagen der- til. Tidligere forskning i mediernes repræsentationer af unge Ifølge dem er det det, der får mediepublikummet til at “[see] youth in problematic terms”(Nichols & Good 2004:9). Deres arbejde er interessant i vores sammenhæng fordi de når frem til et antal hypoteser, hvoraf vi gerne vil teste to: Den første er, at nyhedshistorier om unge overvejende er negative, og den anden er, at denne tendens forstærkes over tid. Amundson, Lichter et al. fokuserer ligesom Aubrun and Grady (Aubrun and Grady 2000) på forskelle mellem nationale og lokale nyhedsmedier. De konkluderer, at “local news stories about youth tend to treat adolescence as a source of personal problems and so- cial disruptions that need to be addressed by adults”. De hævder videre, at “the operant principle among the journalists seemed to be “if it bleeds it leads”[…].”, når nyheder handler om unge JOURNALISTICA · NR. 1 · 2010 // 43 (Amundson, Lichter et al. 2005:25). Den eneste formildende om- stændighed i nyhedsmediernes dækning af unge er ifølge dem, at nationale nyhedsmedier prioriterer emner relateret til uddan- nelse højt, men at de så til gengæld har en langt mindre intensiv dækning af de unge end af andre grupper i samfundet. Sammenfattende kan man sige, at mediernes repræsentatio- ner af unge mennesker varierer afhængigt af genre, medie og mediets dækning (lokal eller national). Da vi her ønsker at dække en 50-årig periode i Danmark, vælger vi at fokusere på aviserne, fordi disse som de eneste medier er systematisk registreret og ar- kiveret over en tilstrækkelig lang periode. Dermed er det også re- levant at se nærmere på den del af forskningen, som hovedsa- gelig har baseret sig på avisernes fremstilling af de unge. Stensaas (1961) analyserer en række amerikanske aviser og konkluderer, at nyhedshistorier om unge mennesker generelt er negative og sætter de unge i et dårligt lys. Eneste undtagelse fra dette mønster er ifølge Stensaas, at positive historier om unge mennesker til gengæld oftest at finde på forsiderne. Tilsvarende analyserer Porteous og Colston (1980) en række britiske aviser og finder, at de foretrukne emner for nyhedshisto- rier om unge er sport eller kriminalitet og at de unge sædvanlig- vis bliver fremstillet som ofre for – eller årsagen til – ulykker og ulovligheder. Tidligere forskning i mediernes repræsentationer af unge Porteous og Colstons undersøgelse er ikke i sig selv et longitudinalt studium, men da Falchikov (1986) efterfølgende i 1985 foretog en tilsvarende undersøgelse viste det sig, at der i de mellemliggende fem år var sket en række ændringer i mediernes beskrivelser af de unge. I 1985 var de fire foretrukne emner om unge i den skrevne presse: De unge som ofre, kriminalitet, sport eller arbejdsløshed. Falchikovs undersøgelse viste dermed, at det er muligt at spore mindre ændringer i mediedækningen af en social gruppe – fx de unge – over blot en femårig periode. Siden årtusindskiftet er der foretaget yderligere tre undersø- gelser af mediedækningen af de unge: En irsk undersøgelse viser, at hovedparten af de irske nyhedshistorier portrætterer unge mennesker som enten “being a problem or as having problems”: Næsten 85 % af nyhedshistorierne om unge handler om de unge som årsag til eller ofre for kriminalitet og vold, eller beskriver unge som en gruppe, der er særligt udsat for skadelige påvirk- // JOURNALISTICA · NR. 1 · 2010 44 ninger (Devlin 2000:47). Ifølge denne undersøgelse stemmer me- diedækningen godt overnes med en generel samfundsmæssig tendens til at præsentere unge mennesker som “[…] either ac- tively “deviant” or passively “at risk”, and sometimes both simul- taneously” (Devlin 2000, citing Griffin 1997:17-18). McManus (2002) foretager en indholdsanalyse af en række californiske aviser og deres dækning af unge og vold. Han kon- kluderer, at der er signifikante forskelle i hvordan medierne beskriver og repræsenterer de unge i rutinejournalistikken og i efterspillet efter signifikante begivenheder og deraf følgende fo- kusjournalistik (Lund 2002:9). McManus illustrerer denne pointe ved hjælp af en analyse af mediedækningen efter Columbine High-massakren. Han viser, at journalisterne i forbindelse med fokusjournalistik i højere grad benytter en tematisk framing end en episodisk, og at de viser større villighed til at diskutere proble- mets årsag, oprindelse og mulige løsninger på det end i forbin- delse med rutinejournalistikken. Tilsvarende viser hans analyse af rutinejournalistikken, at den fokuserer langt mindre på årsager og løsninger end på de problemer, der beskrives. Han konklude- rer derfor, at der er meget stor kvalitetsforskel på fokusjournali- stikkens og rutinejournalistikkens nyhedsdækning af de unge. Mediedækningen af de unge er senest blevet undersøgt af Wyn (2005). Hun sammenligner mediernes repræsentationer af unge i USA, Australien, New Zealand og Storbritannien baseret på en række udvalgte aviser og TV-stationers nyhedsdækning. Tidligere forskning i mediernes repræsentationer af unge Hun konkluderer, at mediernes repræsentationer af de unge sy- nes at afspejle to konkurrerende diskurser: På den ene side be- skrives unge som en trussel mod samfundsordenen, og på den anden side beskrives de unge som en kilde til håb for fremtiden (Wyn 2005:31-32). Sammenfattende viser den tidligere forskning, at mediernes repræsentationer af unge mennesker varierer afhængigt af genre, medie og mediets dækning (lokal eller national). Men den viser imidlertid også, at det er de negative emner og repræsentationer af ungdommen som dominerer. JOURNALISTICA · NR. 1 · 2010 // 45 Metode At undersøge udviklingen i mediebilledet af de unge over 50 år i alle danske mediers samlede dækning er en ganske uoverkom- melig opgave. Det er derfor nødvendigt at udvælge enkelte ny- hedsmedier. Eftersom der ikke var etableret regionale eller lokale elektro- niske medier i Danmark for 50 år siden, og eftersom der ikke fin- des fuldstændige arkiver over de elektroniske mediers nyheds- dækning gennem de seneste 50 år, må undersøgelsen – som nævnt – basere sig på de skrevne nyhedsmedier. Aviserne er placeret før de elektroniske medier i den journali- stiske fødekæde: TV har med andre ord tendens til at bringe de samme nyheder, som har været bragt i aviserne (Lund 2000:145). Resultaterne i denne undersøgelse kan derfor i et vist omfang forventes også at gøre sig gældende for fremstillingen af unge i f.eks. nyhederne på TV. Gennem de seneste 50 år har der eksisteret et utal af dagblade på regionalt, lokalt og nationalt niveau. Det er derfor nødvendigt at afgrænse empirivalget yderligere. For det første er det nødven- digt, at de analyserede dagblade har eksisteret i hele undersøgel- sesperioden. For andet har vi ønsket at sikre, at vores materiale omfattede både større dagblade, tabloidaviser, lokalaviser, regio- nalaviser og landsdækkende aviser. Følgende aviser er valgt for at give så stor spredning som muligt: • Politiken • Kristeligt Dagblad • B.T. • Fyens Stiftstidende • Næstved Tidende Men selv med blot fem aviser er det en uoverkommelig opgave at analysere alle redaktionelle enheder gennem 50 år. Derfor er der foretaget en række punktnedslag, som begrænser datamængden. En del af den danske og internationale forskning i journalistik baserer sig på den såkaldte nyhedsuge (Lund 2000; Petersen 2003): Uge 46. Denne uge er valgt, fordi den i nyhedsmæssig sam- menhæng skønnes at være en gennemsnitlig uge.4 Men selv med blot fem aviser er det en uoverkommelig opgave at analysere alle redaktionelle enheder gennem 50 år. Derfor er der foretaget en række punktnedslag, som begrænser datamængden. En del af den danske og internationale forskning i journalistik baserer sig på den såkaldte nyhedsuge (Lund 2000; Petersen 2003): Uge 46. Denne uge er valgt, fordi den i nyhedsmæssig sam- menhæng skønnes at være en gennemsnitlig uge.4 JOURNALISTICA · NR. 1 · 2010 46 Da analysen sigter på at identificere udviklingen i mediernes repræsentationer af de unge over tid, er analysen samtidig be- grænset til seks punktnedslagsår: 1953, 1963, 1973, 1983, 1993 og 2003. Metode Hver enkelt avis blev gennemlæst af en forskningsassistent, der udvalgte alle artikler, som handlede om unge. Der blev an- vendt en kombination af to udvalgskriterier: Alle artikler, der en- ten omtalte mennesker mellem 14-24 år5 eller rummede ord som unge, ungdom, teenager eller andre synonymer herfor, blev ud- valgt. I alt blev 1174 artikler udvalgt.6 Vores kodeark omfatter dels en række objektive baggrundsva- riable som dato, placering, længde og kildebrug. Desuden har vi kodet for emne, ud fra polinfos emneordsliste.7 Endelig omfatter kodearket en variabel, som er udviklet specielt til denne under- søgelse: En gennemlæsning af materialet afslørede, at det var re- lativt let at identificere om artiklernes overordnede fortællemæs- sige ramme var positiv eller negativ overfor de unge. Vi beslut- tede derfor at kode artikler, der beskrev unge som en ressource, som socialt engagerede, sympatiske, smukke, pligtopfyldende, samvittighedsfulde e.l. som „positive“, mens artikler, der beskrev de unge som utilpassede, passive, kriminelle, aggressive, fjendt- lige eller som en trussel mod samfundsordenen e.l. som „nega- tive“.8 Tre assistenter kodede materialet og tastede alle data ind i en SPSS datafil.9 En beregning af Krippendorf’s alpha for samtlige variable viste et resultat, der svingede mellem 0.82 til 1.0 og er så- ledes inden for et acceptabelt reliabilitetsniveau. Tabel 1 1953 1963 1973 1983 1993 2003 Alle år Politiken 35 32 52 38 50 58 265 B.T. 33 36 58 38 40 59 264 Næstved Tidende 30 36 56 37 31 28 218 Kr. Dagblad 19 22 40 15 13 25 134 Fyns Stiftstidende 31 28 64 39 57 74 293 N 148 154 270 167 191 244 1174 I vores materiale findes det største antal artikler om unge i 1973. Dette tal mener vi at kunne forklare som et resultat af ung- domsoprøret og dets efterspil: Medierne synes ganske enkelt at have rettet et stigende fokus mod de unge og at have givet dem en stigende prioritering i deres nyhedsdækning. Samtlige aviser har nogenlunde lige mange artikler om unge over hele perioden (218-293) eneste undtagelse herfra er Kr. Dag- blad med 134 artikler. Dette resultat er ikke overraskende efter- som avisen henvender sig til et ældre publikum, og eftersom den rummer færre og længere artikler end de øvrige. Resultater Det samlede antal artikler i de udvalgte aviser er gengivet i tabel 1. Over årene 1953-1973 fandt vi en bemærkelsesværdig stigning på 80 % i antallet af artikler om unge. Vi skal dog i den forbindelse bemærke, at der generelt er sket en stigning i antallet af artikler i aviserne og at stigningen i antallet af artikler om unge derfor ikke nødvendigvis afspejler, at den samlede andel af artikler om ung- dommen er vokset. JOURNALISTICA · NR. 1 · 2010 // 47 Stigende negativ bias? Vores data kan ikke bekræfte en stigende tendens i retning af et stadigt mere negativt mediebillede af de unge, sådan som den tidligere forskning på området har tydet på. Som det fremgår af tabel 2, er der snarere tale om, at medierne i stigende omfang balancerer mellem positive, negative og neutrale repræsentatio- ner af ungdommen. Faktisk er antallet af „neutrale“ repræsen- tationer af de unge signifikant højere fra 1983-2003 end i perioden før. Der er formentlig flere årsager hertil: Dels medførte partipres- sens sammenbrud i slutningen af 1960erne at aviserne i højere og højere grad har stræbt mod en neutral dækning af nyhederne, / JOURNALISTICA · NR. 1 · 2010 48 Tabel 2 Negative og positive beskrivelser af de unge 1953 1963 1973 1983 1993 2003 Alle år Positive 83 86 127 29 55 78 458 56,1% 55,8% 47,0% 17,4% 28,8% 32,0% 39,0% Negative 38 37 90 69 66 78 378 25,7% 24,0% 33,3% 41,3% 34,6% 32,0% 32,2% Neutrale 27 31 52 69 70 88 337 18,2% 20,1% 19,3% 41,3% 36,6% 36,1% 28,7% N 148 154 270 167 191 244 1174 Tabel 2 Negative og positive beskrivelser af de unge dels er journalister og journalistikken blevet stadig mere og mere professionaliseret gennem perioden. Ifølge hypotesen om den øgede kommercialisering af medi- erne ville vi desuden have forventet, at tabloidavisen havde den højeste andel af negative bias i artikler om unge. Men en nær- mere analyse af tallene afslører, at det rent faktisk er den regio- nale avis, der er mest negativ. Dette resultat er interessant af to årsager: Den første er, at dette resultat dermed bekræfter de tidli- gere undersøgelser, der viste, at lokale medier er mere tilbøjelige til at beskrive unge mennesker negativt (Aubrun and Grady 2000; Amundson, Lichter et al. 2005). Den anden er, at undersøgelsen af ældrebilledet i medierne viste, at når det drejede sig om be- skrivelserne af ældre mennesker var regionalavisen langt mere positiv end de landsdækkende aviser (Wien, 2005). Tidligere forskning i mediestereotyper har påvist signifikante forskelle i den måde unge mænd og unge kvinder blev beskrevet i medierne og i hyppigheden af hvor ofte de to køn var repræsen- teret. (Falchikov 1986; Devlin 2000; Mazzarella 2003). Vores data bekræfter også disse forskelle: Mere end en tredjedel af artiklerne handlede udelukkende om unge mænd og det gælder for alle år jfr. tabel 4. Tabel 3 1953 1963 1973 1983 1993 2003 Alle år Kriminalitet (retsvæsen) 23 19 22 30 28 16 22 Kultur 24 34 9 9 13 16 16 Sport 8 10 14 11 7 19 12 Uddannelse 8 9 14 14 12 8 11 Landspolitik 3 2 9 7 17 5 8 Socialforhold 4 6 9 3 5 9 6 Etik, filosofi og religion 5 3 4 4 2 2 3 Ulykker 8 4 2 1 3 2 3 Arbejds- marked 8 4 2 1 3 2 3 Samliv 2 3 1 7 1 3 3 Udlandet 1 2 4 1 3 3 3 Sundhed 2 1 3 4 2 3 3 Andet 7 5 6 7 5 11 7 N 148 154 270 167 191 244 1174 sitive og negative historier om unge fordelt på køn. Materia- let afslører, at der er en klar tendens til, at de negative historier handler om unge mænd, mens de positive historier er domineret af kvinder. Vi finder en tilsvarende kønsskævhed når vi krydstabulerer køn med citater (tabellen er ikke medtaget): Unge mænd citeres meget hyppigere end unge kvinder. Stigende negativ bias? Vi ser dog tegn i retning af en ændring i dette mønster: I 1953-1993 var kvinder kun repræsenteret i 12-15 % af artiklerne, i 2003 gjaldt det for 22 % af artiklerne. Vores data bekræfter også, at der er forskel på hvilken måde medierne omtaler de to køn: Tabel 5 viser fordelingen af hhv. po- JOURNALISTICA · NR. 1 · 2010 // 49 49 Tabel 3 Klassifikation af artikler om unge efter emne pct. Hvilke emner? Som allerede omtalt viste Porteous og Colston (1980) og Falchikov (1986), at de dominerende emner i medierne vedrørende unge var kriminalitet, sport, arbejdsløshed og unge som ofre. Vores materiale viser et lidt anderledes billede, sådan som det ses i ta- // JOURNALISTICA · NR. 1 · 2010 50 Tabel 4 1953 1963 1973 1983 1993 2003 Alle år Mand/mænd 66 52 103 65 83 82 451 44,6% 33,8% 38,1% 38,9% 43,5% 33,6% 38,4% Kvinde/kvinder 21 23 33 26 29 52 184 14,2% 14,9% 12,2% 15,6% 15,2% 21,3% 15,7% Begge køn 12 14 40 13 12 24 115 8,1% 9,1% 14,8% 7,8% 6,3% 9,8% 9,8% Ikke omtalt 49 65 94 63 67 86 424 33,1% 42,2% 34,8% 37,7% 35,1% 35,2% 36,1% N 148 154 270 167 191 244 1174 bel 3. Her har vi klassificeret de enkelte artikler i et antal katego- rier inddelt efter emne. Den hyppigst forekommende kategori i vores materiale er kri- minalitet (22 % af alle artikler). Men både i 1953, 1963 og 2003 er der en ligeså stor eller større andel af artikler, der handler om kultur. Blandt de øvrige fremtrædende kategorier er „kultur“ (16 %), „sport“ (12 %) og „uddannelse“ (11 %). Og lægger man ande- lene af artikler, der handler om kultur, sport og uddannelse sam- men udgør artikler om disse emner en langt større andel end andelen af artikler om kriminalitet. Vores undersøgelse kan heller ikke bekræfte, at flere og flere hi- storier om unge handler om ulykker: Her er kun tale om en lille andel af artikler, som over perioden viser en faldende tendens. I forhold til den tidligere forskning på området adskiller vores resultater sig således markant: Vi finder ikke, at historier om unge i medierne hovedsageligt handler om kriminalitet og ulyk- ker. Tvært i mod: Mediernes foretrukne emner, når det handler om de unge er kultur, sport og uddannelse. Hvilken rolle spiller de unge? Forskningslitteraturen om mediernes stereotypificering lægger vægt på en tendens til at beskrive sociale grupper og gruppead- færd snarere end at beskrive individer. Dette gælder særligt, når JOURNALISTICA · NR. 1 · 2010 // 51 51 Tabel 5 Mand/mænd Kvinde/kvinder Begge køn NA Total Positiv 170 107 57 124 458 37,7% 58,2% 49,6% 29,2% 39,0% Negativ 223 32 26 97 378 49,4% 17,4% 22,6% 22,9% 32,2% Neutral 58 45 32 202 337 12,9% 24,5% 27,8% 47,6% 28,7% N 451 184 115 424 1174 Mand/mænd Kvinde/kvinder Begge køn NA Total man taler om „ungdommen“ som en særlig gruppe med særlige sociale og kulturelle karakteristika: Da man begyndte at forske i børn og unge i begyndelsen af det 20ende århundrede og da moralsk panik over ungdomskulturen blev et centralt emne for både den offentlige og den politiske debat blev „ungdommen“ en særlig gruppe. I medierne blev unge mennesker – og især unge mænd – beskrevet som tilhørende grupper, der forstyrrede den offentlige orden fx som medlemmer af (kriminelle) bander, eller som nogen, der introducerede særlige nye kulturer båret fx af musik (Thompson 1998; Welch 2002). For at undersøge i hvilket omfang unge mennesker beskrives som tilhørende en generel gruppe („ungdommen“) eller en som tilhørende særlige ungdomsgrupper („læderjakker“, „punkere“) eller som individer, har vi kodet materialet i tre forskellige kate- gorier efter hvordan de unge omtales. Tabel 6 viser, at artikler, hvor unge mennesker portrætteres som gruppemedlemmer, var hyppigst i 1953 (n=53), 1963 (n=62) og 1973 (n=112). Den næsten modsatte tendens gælder avisernes beskrivelser af de unge med en samlebetegnelse som fx „ung- dommen“. En fjerdedel af artiklerne fra 1983, 1993 og 2003 inde- holdt en samlebetegnelse for de unge. Når man krydstabulerer dette med de enkelte artiklers emne viser det sig, at 25-30 % af disse artikler havde de dominerende emner: sport, kriminalitet og kultur. Det var altså disse artikler, som havde gruppeperspek- tivet og ikke de negative historier om kriminalitet og ulykker. // JOURNALISTICA · NR. 1 · 2010 52 Tabel 6 Unge som individer eller som gruppemedlemmer? 1953 1963 1973 1983 1993 2003 Alle år Individ (fx navngiven person) 71 60 104 95 111 128 569 48,0% 39,0% 38,5% 56,9% 58,1% 52,5% 48,5% Gruppe (fx „læder- jakker“) 53 62 112 8 25 34 294 35,8% 40,3% 41,5% 4,8% 13,1% 13,9% 25,0% Samle- betegnelse (fx „ungdommen“) 23 27 35 45 43 60 233 15,5% 17,5% 13,0% 26,9% 22,5% 24,6% 19,8% Ved ikke 1 5 19 19 12 22 78 0,7% 3,2% 7,0% 11,4% 6,3% 9,0% 6,6% N 148 154 270 167 191 244 1174 Tabel 6 Unge som individer eller som gruppemedlemmer? Vores data viser altså, at ca. Tabel 5 halvdelen af de artikler, der beskri- ver unge mennesker, beskriver dem som individer. Derfor mener vi ikke det er rimeligt at hævde, at unge hovedsageligt fremstil- les i grove og generaliserende gruppebetegnelser. I Grossbergs analyse af politisk ungdomskultur kalder han ungdommen for “the most silenced generation” (Grossberg 1994, citet hos Mazzarella 2003: 236). Når unge mennesker beskrives i medierne er det – ifølge ham – oftere ved hjælp af citater fra eks- perter end de unge selv. Problemet ved det er iflg. Grossberg, at når sådanne eksperter som psykologer, sociologier og læger får ordet har de en tendens til at generalisere enkeltstående proble- matiske episoder til generelle sociale problemer. Når de unge ikke får lov til at tale for sig selv, bliver de “also subjected to me- dia’s attempts to categorize them” (Mazzarella 2003:236). Vores data giver os mulighed for at undersøge, om dette pessimistiske postulat faktisk stemmer overens med virkeligheden. Vores analyse af hvor ofte de unge selv udtaler sig viser, som det fremgår af tabel 7, at kun en lille del af artiklerne indeholder di- rekte citater af unge mennesker. Men når vi ser på udviklingen JOURNALISTICA · NR. 1 · 2010 // 53 53 Tabel 7 1953 1963 1973 1983 1993 2003 Alle år Citeret 8 22 39 38 49 57 213 5,4% 14,3% 14,4% 22,8% 25,7% 23,4% 18,1% Ikke citeret 140 131 230 129 141 187 958 94,6% 85,1% 85,2% 77,2% 73,8% 76,6% 81,6% Ved ikke 0 1 1 0 1 0 3 ,0% ,6% ,4% ,0% ,5% ,0% ,3% N 148 154 270 167 191 244 1174 over tid, så viser det sig, at der faktisk bliver flere – både i andele og i procent – hvor unge citeres direkte. I 1953 var det således kun i 5 % af artiklerne. I 1963 og 1973 var det 14 % og i 1983, 1993 og 2003 var dette steget til 23-26 %. Med an- dre ord tyder vores undersøgelse ikke på, at der er en stigende tendens til at gøre ungdommen til en “silenced generation” i me- dierne. Vi er dog ikke gået dybere ned i analysen af på hvilken måde og til hvad journalisterne bruger citaterne af de unge og om denne stigning i direkte citater er større end for andre grup- per i samfundet. En forklaring herpå kunne jo netop være at jour- nalistisk praksis har ændret sig. Vores analyse omfatter også brugen af eksperter i artiklerne. Analysen viser, at kun i 5 % af artiklerne fra 1953 optrådte der en ekspert. I resten af årene fra 1963-2003 varierer dette mellem 12- 17 %. En mulig forklaring på dette er den stigende professionali- sering af de forskellige sektorer i velfærdsstaten, som har børn og unge som deres ansvarsområde. Så dermed ser vi i lighed med Grossberg at eksperter inddrages i stigende omfang, men vi er dog knapt så pessimistiske som han, da vi parallelt hermed ser, at de unge i stigende omfang også selv kommer til orde. // JOURNALISTICA · NR. 1 · 2010 54 Konkluderende bemærkninger Sammenfattende kan vi sige, at vores undersøgelse ikke kan be- kræfte, at der er en dominerende negativ bias i mediernes beskri- velser af de unge. Dette kunne hverken bekræftes i forhold til undersøgelsen af nutidens mediedækning (2003) eller i under- søgelsen af langtidsperspektivet herpå (1953-2003) Tvært i mod afslører vores data en udvikling i retning af flere og flere neutrale beskrivelser af unge. Vores undersøgelse viser, at „kriminalitet“, „kultur“ og „sport“ er de emner, der hyppigst er i spil, i artikler om unge. Vores ma- teriale tyder således også her på en afvigelse i forhold til den tid- ligere forskning på området. Når vi ser nærmere på, hvilken rolle de unge spiller i nyheds- dækningen finder vi en tendens til, at medierne citerer de unge direkte. Denne tendens synes at være stærkest i årene efter ung- domsoprøret (1973 og frem). Desuden er der nogle interessante forskelle i avisernes repræsentationer af unge mænd og kvinder: Vores undersøgelse tyder på, at unge kvinder spiller en stigende rolle i medierne. Men krydstabuleringer med øvrige variable i materialet tyder dog fortsat på, at beskrivelserne af de unge mænd er langt mere varierede, selvom unge mænd dog har den tvivlsomme ære af overvejende at blive beskrevet i negative hi- storier. På dette tidspunkt kan vi ikke tilbyde udtømmende forklarin- ger på disse observationer eller på forskellene mellem vores data og den tidligere forskning. Vi vil dog tillade os at fremsætte en række mulige forklaringer til diskussion. En mulig forklaring på afvigelserne er forskelle i dataindsam- lingsteknikkerne: Vores data er indsamlet ved hjælp af stratifice- rede tilfældigt udvalgte stikprøver over en 50-årig periode. Hvert enkelt punktnedslag dækker dog en forholdsvis kort periode på kun en uge. Den tidligere forskning på området er baseret på stikprøver, der er udtaget over længere perioder (to til fire uger). Vi må derfor erkende, at faren for at de udsving vi ser, er tilfældige er relativt stor. Vi imødegår den fare efter bedste evne ved ude- lukkende at tolke meget tydelige tendenser, som strækker sig over flere punktnedslag. JOURNALISTICA · NR. 1 · 2010 // 5 55 Yderligere indsamlede Porteous og Colston (1980) og Falchikov (1986) deres materiale over hhv. en måned i Juni (1979) og to uger i oktober (1984). Ingen af dem præsenterer nogen forklaring på hvorfor netop disse perioder er valgt. Konkluderende bemærkninger Der kan argumenteres for, at det er yderst problematisk at indsamle datamateriale til analy- ser af mediedækningen i sommerperioden, hvis man ønsker at generalisere resultaterne til resten af året, sådan som Porteous og Colston gør. Og der kan yderligere argumenteres for, at det er pro- blematisk at sammenligne data indsamlet over sommerperioden med data indsamlet i efteråret, sådan som Falchikov gør. Vores data er indsamlet på samme tid af året i alle seks punktnedslag. Det er også muligt, at forskelle i mediestystemerne spiller en rolle: Både Porteous og Colston (1980) og Falchikov (1986) ind- samlede deres materiale i en britisk kontekst. Det vil ifølge Hal- lin og Mancini (2004) sige, at data er indsamlet fra nyhedsmedier under „den liberale model“. Vores data er indsamlet fra danske aviser, som kan siges at tilhøre „den demokratiske korporativi- stiske model“, hvor såvel det politiske system som markedsme- kanismerne er forskellige. Yderligere kan forskelle i graden af ta- bloidisering mellem danske og britiske medier forklare en del af forskellen (Esser 1999). N O T E R * Vi ønsker at især at takke studentermedhjælperne Therese Dyrby, Julie Nørrelund, Bo Jessen, Kristian D. Knudsen og Saba Khalaj for assistance i firbindelse med dataindsamling og kodning, samt vore kolleger Christian Elmelund-Præstekær og David Nicolas Hopmann for hjælp og nyttige kommentarer til tidligere versioner af artiklen. 1. Herved bliver det nære slægtskab mellem medieforskningen i fra- ming og social psykologernes arbejde med stereotyper klart: Eksem- pelvis hævder Stangor og Schaller (Stangor and Schaller 2000:64), at stereotyper er det materiale som samfundet er vævet af: Stereoty- perne deles af individerne inden for en given kulturkreds. De udgør en slags fælles informationsbase som alle i kulturkredsen refererer til (Stangor and Schaller 2000:68). Tilsvarende hævder Hinton, at ste- // JOURNALISTICA · NR. 1 · 2010 56 reotyper er en slags kognitive mønstre, som gør tænkning “quick and easy” (Hinton 2000:22-23). 2. Vi finder yderligere støtte for at studiet af framing og af stereotyper er nært beslægtede hos Entmann, der i hans hyppigt citerede artikel fra 1993 oplister fem faktorer, som han mener, bør indgå i analyser af me- diernes fortællemæssige rammer. En af disse er stereotyper (Entman 1993:53). Entman definerer stereotype som ‘mentally stored clusters of ideas that guide the individuals’ processing of information’ (Ent- man 1993:53). De fire øvrige kategorier er “certain keywords, stock phrases, stereotyped images, sources of information and sentences that provide thematically reinforcing clusters of facts or judgements” (Entman 1993:52) 3. I nogle af undersøgelserne bruges begrebet ‘image’ som synonym for stereotype (e.g. Porteous and Colston 1980; Falchikov 1986; Wyn 2005) 3. I nogle af undersøgelserne bruges begrebet ‘image’ som synonym for stereotype (e.g. Porteous and Colston 1980; Falchikov 1986; Wyn 2005) 4. Det skyldes en antagelse om, at der ikke er særlige årligt tilbageven- dende begivenheder i den uge, som kan påvirke nyhedsstrømmen. Valget af nyhedsugen er imidlertid ikke uproblematisk. For det før- ste er antagelsen om gennemsnitlighed stort set ubegrundet. For det andet indeholder brugen af nyhedsugen den ubegrundede og ofte usagte antagelse, at man ved analyse af én uge kan udtale sig gene- relt om alle uger i det pågældende år. Alligevel anvendes „nyheds- ugen“ i dette projekt i mangel af bedre. Det skal dog bemærkes, at også andre perioder er blevet anvendt i beslægtede undersøgelser. Pedersen & Horst (2000) anvender perioden fra den 1.-7. februar i tre punktnedslagsår for at dække udviklingen i den politiske journalistik fra 1958 til 1998. Albæk et al. N O T E R (2002) anvender 15. januar til 14. februar i tiårsintervaller i en analyse af dagbladendes brug af ekspertkilder. 4. Det skyldes en antagelse om, at der ikke er særlige årligt tilbageven- dende begivenheder i den uge, som kan påvirke nyhedsstrømmen. Valget af nyhedsugen er imidlertid ikke uproblematisk. For det før- ste er antagelsen om gennemsnitlighed stort set ubegrundet. For det andet indeholder brugen af nyhedsugen den ubegrundede og ofte usagte antagelse, at man ved analyse af én uge kan udtale sig gene- relt om alle uger i det pågældende år. Alligevel anvendes „nyheds- ugen“ i dette projekt i mangel af bedre. Det skal dog bemærkes, at også andre perioder er blevet anvendt i beslægtede undersøgelser. Pedersen & Horst (2000) anvender perioden fra den 1.-7. februar i tre punktnedslagsår for at dække udviklingen i den politiske journalistik fra 1958 til 1998. Albæk et al. (2002) anvender 15. januar til 14. februar i tiårsintervaller i en analyse af dagbladendes brug af ekspertkilder. 5. Svarende til FNs definition af unge. http://www.un.org/esa/soc- dev/unyin/qanda.htm#1, 24. september, 2007 6. McManus’s (2002) konkluderer, at ca. 14 % af nyhedsartiklerne om- handler unge. Vi har ikke optalt samtlige artikler, men vi mener, at tallet for de danske aviser næppe adskiller sig signifikant herfra. 7. Se http://www.db.dk/binaries/polinfoclassic_2001.doc. Vi har dog kun benyttet emneordslistens øverste niveau og kun indekseret med en enkelt term pr. artikel 8. Kodearket kan ses på https://e-learn.sdu.dk/bbcswebdav/orgs/bbc omm477/Tekster/Diverse_faelles_dok JOURNALISTICA · NR. 1 · 2010 // 57 9. Datafilen kan downloades fra https://e-learn.sdu.dk/bbcswebdav/ orgs/bbcomm477/SPSS_filer A N V E N D T L I T T E R AT U R Amundson, D. R., L. S. Lichter, et al. (2005). What’s The Matter With Kids Today – Television Coverage of Adolescents in America. Washington, Frameworks Institute. Aubrun, A. and J. Grady (2000). Aliens in the Living Room: How TV Sha- pes Our Understanding of “Teens”. Washington, Frameworks Insti- tute. Bleakney, A., M. Hennessy, et al. (2008). “It works both Ways: Teh Rela- tionship between Exposure to Sexual Content in the Media and Ado- lescent Sexual Behaviour.” Media Psychology 11(1): 443-461. Casas, M. C. and T. L. Dixon (2003). The impact of stereotypical and Counter-stereotypical News on Viewer Perception of Blacks and Latinos. An Explorative Study. A companion to media studies. A. N. Valdivia. Oxford, Blackwell: 480-492. Covert, J. J. and T. L. Dixon (2008). “A changing view – Representation and effects of the portrayal of women of color in mainstream wo- men’s magazines.” Communication Research 35(2): 232-256. Devine, P. G. and A. J. Elliott (2000). Are Racial Stereotypes Really Fading? The Princeton Trilogy Revisited. Stereotypes and Prejudice. C. Stan- gor. Philadelphia, Psychology Press: 86-100. 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A. and A. Modigliani (1989). “Media Discourse and Public Opinion on Nuclear Power: A Constructionists Approach.” The Ame- rican Journal of Sociology 95(1): 1-37. Gitlin, T. (1980). The Whole World Is Watching – mass media in the making & unmaking of the new left. Los Angeles, University of California Press. Goffman, E. (1974). Frame Analysis – An Essay on the Organization of Ex- periente. New York, Harper Colophon Books. Gorham, B. W. (2006). Petersen, J. H. (2003). “Lippmann Revisited.” Journalism 4(2): 249-259. A N V E N D T L I T T E R AT U R “News media’s relationship with stereotyping: The linguistic intergroup bias in response to crime news.” Journal of Communication 56(2): 289-308. Hallin, D. C. and P. Mancini (2004). Comparing Media Systems: Three Mo- dels of Media and Politics. Cambridge, Cambridge University Press. Heintz-Knowles, K. E. (2000). Images of Youth: A content Analysis of Adolescents In Prime-Time Entertainment Programming. Washing- ton, Frameworks Institute. Hinton, P. R. (2000). Stereotypes, cognition and culture. Hove, Psychology Press. Hurtz, W. and K. Durkin (2004). “The effects of gender-stereotyped radio commercials.” Journal of Applied Social Psychology 34(9): 1974-1992. Kite, M. E. and L. S. Wagner (2002). Attitudes toward Older Adults. Ageism. T. D. Nelson. Cambridge, MIT Press: 129-161. Lund, A. B., Ed. (2000). Først med det sidste – en nyhedsuge i Danmark. Århus, Ajour. Lund, A. B. (2002). Den redigerende magt. Århus, Århus Universitetsfor- lag. Mazzarella, S. R. (2003). Constructing Youth. A companion to media stu- dies. A. N. Valdivia. Oxford, Blackwell: 227-246. McManus, J. (2002). “Youth Violence Stories Focus On Events, Not Cau- ses.” Newspaper Research Journal 23(4): 6-19. Nichols, S. L. T. L. G. (2004). America’s Teenagers:Myths and Realities:Me- dia Images, Schooling, and the Social Costs of Careless Indifference. Mahwah, NJ, Lawrence Erlbaum Associates, Inc.,. Paterson, B. (2006). “Newspaper representations of mental illness and the impact of the reporting of ‘events’ on social policy: the ‘framing’ of Isabel Schwarz and Jonathan Zito.” Journal of Psychiatric and Mental Health Nursing 13(3): 294-300. Petersen, J. H. (2003). “Lippmann Revisited.” Journalism 4(2): 249-259. JOURNALISTICA · NR. 1 · 2010 // 59 59 Porteous, M. A. and N. J. Colston (1980). “How adolessents are reported in the Brittish Press.” Journal of Adolescence 3(3): 197-207. Ramasubramanian, S. (2007). “Media-based strategies to reduce racial stereotypes activated by news stories.” Journalism & Mass Commu- nication Quarterly 84(2): 249-264. Scheufele, D. A. and D. Tewksbury (2007). “Framing, Agenda Setting, and Priming: The Evolution of Three Media Effects Models.” Journal of Communication 57: 9-20. Schulz, W. and R. Zeh (2005). “The Changing Election Coverage of Ger- man Television. A Content Analysis: 1990-2002.” Communications 30: 385-407. Semetko, H. A. and P. M. Valkenburg (2000). “Framing European Politics: A Content Analysis of Press and Television News.” Journal of Com- munication 50(2): 93-109. Slater, M. D. (2007). “Reinforcing Spirals: The Mutual Influence of Media Selectivity on Individual Behavious and Social Identity.” Communi- cation Theory 17: 281-303. Stangor, C., Ed. (2000). Stereotypes and Prejudice. Philadelphia, Psycho- logy Press. A N V E N D T L I T T E R AT U R Stangor, C. and M. Schaller (2000). Stereotypes as individual and Collec- tive Representations. Stereotypes and Prejudice. C. Stangor. Philadel- phia, Psychology Press. Stensaas, H. S. (1961). “The front page teenager: How 111 dailies treat him.” Journalism Quarterly 38: 373-375. Tajfel, H. and J. P. Forgas (2000). Social Categorization: Cognitions, Va- lues and Groups. Stereotypes and Prejudice. C. Stangor. Maryland, Psychology press. Thompson, K. (1998). Moral Panic. London, Routledge. Welch, M., Eric A. Price, Nana Yankey (2002). “Moral Panic Over Youth Violence: Wilding and the Manufacture of Menace in the Media.” Youth & Society 34(1): 3-30. Wien, C. (2005). Ældrebilledet i medierne gennem 50 år. En undersøgelse af ældrestereotyper i dagbladene fra 1953 til 2003. Odense, Syddansk Universitetsforlag. Wien, C. (2005). Ældrebilledet i medierne gennem 50 år. En undersøgelse af ældrestereotyper i dagbladene fra 1953 til 2003. Odense, Syddansk Universitetsforlag. Wyn, J. (2005). “Youth in the Media: Adult stereotypes of young people”, in A. Williams and C. Thurlow, (Eds). Communication in Adolescence: Perspectives on Language and Social Interaction in the Teenage Years, Peter Lang: New York. Wyn, J. (2005). “Youth in the Media: Adult stereotypes of young people”, in A. Williams and C. Thurlow, (Eds). Communication in Adolescence: Perspectives on Language and Social Interaction in the Teenage Years, Peter Lang: New York. y , J ( 5) yp y g p p , in A. Williams and C. Thurlow, (Eds). Communication in Adolescence: Perspectives on Language and Social Interaction in the Teenage Years, Peter Lang: New York. JOURNALISTICA · NR. 1 · 2010 // 135 135 Media representations of youth over 50 years: A content analysis of Danish newspapers from 1953 to 2003 CHARLOTTE WIEN AND KLAUS LEVINSEN The modern mass media affects how we perceive and under- stand the youth; and previous international studies have shown that the media mostly conveys a negative image of young people. Based on a quantitative content analysis, this article examines how young people are presented in at selection of Danish new- spapers over the past 50 years. Our study shows that crime, cul- ture and sports are the most frequent topics in the newspapers relating to young people, but the study could not confirm the ex- istence of a dominant negative bias in the newspaper stories. This could neither be confirmed in relation to the study of con- temporary media coverage (2003) or in the examination of the long-term perspective (1953-2003). Quite the contrary, our data reveals a trend towards more and more neutral journalistic des- criptions of young people, and a tendency to quote young people in newspaper articles.
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Clustering of Tir during enteropathogenic E. coli infection triggers calcium influx–dependent pyroptosis in intestinal epithelial cells
PLoS biology
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PLOS BIOLOGY PLOS BIOLOGY PLOS BIOLOGY RESEARCH ARTICLE OPEN ACCESS Citation: Zhong Q, Roumeliotis TI, Kozik Z, Cepeda-Molero M, Ferna´ndez LA´, Shenoy AR, et al. (2020) Clustering of Tir during enteropathogenic E. coli infection triggers calcium influx–dependent pyroptosis in intestinal epithelial cells. PLoS Biol 18(12): e3000986. https://doi.org/10.1371/journal. pbio.3000986 Academic Editor: Hans-Uwe Simon, University of Bern, SWITZERLAND Received: June 3, 2020 Accepted: November 24, 2020 Published: December 30, 2020 Copyright: © 2020 Zhong 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. Received: June 3, 2020 Accepted: November 24, 2020 Published: December 30, 2020 Copyright: © 2020 Zhong 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. Received: June 3, 2020 Accepted: November 24, 2020 Published: December 30, 2020 Copyright: © 2020 Zhong et al. This is an open access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited. Data Availability Statement: Data are available from the Pride Database, the accession number is PXD018763. Funding: T.I.R and J.S.C are supported by the Cancer Research UK Centre grant C309/A25144. GF is supported a Wellcome Investigator Award 107057/Z/15/Z. QZ is supported by Imperial College President’s PhD Scholarship. GF is supported by a Wellcome Investigator Award (107057/Z/15/Z). T.I.R and J.S.C were funded by Abstract Clustering of the enteropathogenic Escherichia coli (EPEC) type III secretion system (T3SS) effector translocated intimin receptor (Tir) by intimin leads to actin polymerisation and pyroptotic cell death in macrophages. The effect of Tir clustering on the viability of EPEC-infected intestinal epithelial cells (IECs) is unknown. We show that EPEC induces pyroptosis in IECs in a Tir-dependent but actin polymerisation-independent manner, which was enhanced by priming with interferon gamma (IFNγ). Mechanistically, Tir clustering triggers rapid Ca2+ influx, which induces lipopolysaccharide (LPS) internalisation, followed by activation of caspase-4 and pyroptosis. Knockdown of caspase-4 or gasdermin D (GSDMD), translocation of NleF, which blocks caspase-4 or chelation of extracellular Ca2+, inhibited EPEC-induced cell death. IEC lines with low endogenous abundance of GSDMD were resistant to Tir-induced cell death. Conversely, ATP-induced extracellular Ca2+ influx enhanced cell death, which confirmed the key regulatory role of Ca2+ in EPEC-induced pyr- optosis. We reveal a novel mechanism through which infection with an extracellular patho- gen leads to pyroptosis in IECs. Academic Editor: Hans-Uwe Simon, University of Bern, SWITZERLAND RESEARCH ARTICLE Clustering of Tir during enteropathogenic E. coli infection triggers calcium influx– dependent pyroptosis in intestinal epithelial cells Qiyun ZhongID1, Theodoros I. RoumeliotisID2, Zuza Kozik2, Massiel Cepeda-Molero1, Luis A´ ngel Ferna´ndez3, Avinash R. Shenoy4, Chris BakalID5, Gad FrankelID1*, Jyoti S. ChoudharyID2* a1111111111 a1111111111 a1111111111 a1111111111 a1111111111 a1111111111 a1111111111 a1111111111 a1111111111 a1111111111 1 Centre for Molecular Bacteriology and Infection, Department of Life Sciences, Imperial College, London, United Kingdom, 2 Functional Proteomics Group, Chester Beatty Laboratories, The Institute of Cancer Research, London, United Kingdom, 3 Department of Microbial Biotechnology, Centro Nacional de Biotecnologı´a, Consejo Superior de Investigaciones Cientı´ficas (CNB-CSIC), Campus UAM-Cantoblanco, Madrid, Spain, 4 Centre for Molecular Bacteriology & Infection, Department of Infectious Disease, Imperial College, London, United Kingdom, 5 Dynamical Cell Systems, Chester Beatty Laboratories, The Institute of Cancer Research, London, United Kingdom * g.frankel@imperial.ac.uk (GF); jyoti.choudhary@icr.ac.uk (JSC) * g.frankel@imperial.ac.uk (GF); jyoti.choudhary@icr.ac.uk (JSC) PLOS BIOLOGY PLOS BIOLOGY EPEC Tir triggers calcium influx-dependent pyroptosis they hijack multiple cell signalling pathways, including those regulating cytoskeletal dynamics, inflammation, vesicle trafficking, and cell survival [4]. Multiple T3SS effectors trigger or antag- onise host immune responses, including those mediated by nuclear factor kappa B (NF-κB) (e.g., translocated intimin receptor (Tir), NleF, NleC, NleD and NleE) [5–8], caspase-1 (NleA) [9], and caspase-4 (NleF) [10,11]. T3SS effector translocation is a highly regulated process which involves the transmembrane gatekeeper effector EspZ [12]; uncontrolled effector trans- location is cytotoxic [12]. the CRUK Centre grant (C309/A25144). "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". PLOS BIOLOGY Abbreviations: A/E, attaching and effacing; AGC, automatic gain control; ANOVA, analysis of variance; APOB, apolipoprotein B; APOE, apolipoprotein E; ASC, apoptosis-associated speck-like protein containing a CARD; BSA, bovine serum albumin; CANX, calnexin; CASP4, caspase- 4; CFU, colony forming unit; CID, collision-induced dissociation; CREB1, cAMP responsive element binding protein 1; DAMP, damage-associated molecular pattern; DMEM, Dulbecco’s Modified Eagle Medium; DMTU, N,N0-dimethylthiourea; EPEC, enteropathogenic Escherichia coli; ER, endoplasmic reticulum; FBS, foetal bovine serum; FDR, false discovery rate; GAPDH, glyceraldehyde 3-phosphate dehydrogenase; GBP2, guanylate- binding protein 2; GEO, Gene Expression Omnibus; GOBP, Gene Ontology Biological Process; GSDMD, gasdermin D; GSDME, gasdermin E; HCD, higher- energy collisional dissociation; HEPES, N-2- hydroxyethylpiperazine-N-2-ethane sulphonic acid; HPLC, high-performance liquid chromatograph; IAA, iodoacetamide; IEC, intestinal epithelial cell; IFNGR, interferon gamma receptor; IFNγ, interferon gamma; IL, interleukin; IPTG, isopropyl β-d-1- thiogalactopyranoside; IRF, interferon regulatory factor; IT, injection time; ITPR2, inositol 1,4,5- trisphosphate receptor type 2; JAK, Janus kinase; JAK-STAT, Janus kinase/signal transducers and activators of transcription; LBP, LPS-binding protein; LC–MS, liquid chromatography–mass spectrometry; LPS, lipopolysaccharide; MOI, multiplicity-of-infection; Nec1, necrostatin-1; NF- κB, nuclear factor kappa B; NLR, NOD leucine-rich repeat protein; NLRP3, NLR family pyrin domain containing 3; NOD, nucleotide-binding oligomerisation domain; NSA, necrosulfonamide; OCR, oxygen consumption rate; OMV, outer membrane vesicle; PAMP, pathogen-associated molecular pattern; PARP1, poly [ADP-ribose] polymerase 1; PBS, phosphate buffered saline; PBST, phosphate buffered saline, 0.05% Tween 20; PCA, principal component analysis; PI, propidium iodide; PI3K, phosphatidylinositol 3-kinase; PMA, phorbol 12-myristate 13-acetate; PRIDE, PRoteomics IDEntifications database; PVDF, polyvinylidene difluoride; qRT-PCR, real-time quantitative PCR; RIPA, radioimmunoprecipitation; RIPK, receptor-interacting serine/threonine-protein kinase; ROS, reactive oxygen species; RP, reversed-phase; RPE, retinal pigment epithelium; EPEC colonise intestinal epithelial cells (IECs) via a mechanism known as attaching and effacing (A/E) lesions, which are characterised by intimate bacterial attachment and effacing of the brush border microvilli [13]. Intimate EPEC attachment is mediated by avid interactions between the bacterial outer membrane adhesin intimin (encoded by the eae gene) and the effector Tir [14,15]. Binding to intimin leads to Tir clustering, phosphorylation of Tir tyrosine residue Y474 by host non-receptor tyrosine kinases [16,17], and recruitment of the host adap- tor protein Nck, which, in turn, recruits N-WASP that activates Arp2/3 [18–20]. In addition, Tir Y454 forms a complex with phosphatidylinositol 3-kinase (PI3K) [21] and binds the host adaptors IRTKS/IRSp53, scaffold proteins that regulate actin organisation [22,23]. PLOS BIOLOGY While the signalling downstream of Y474 cascade leads to robust actin polymerisation and formation of actin-rich pedestal-like structures at the site of bacterial attachment [18–20], pathway down- stream of Y454 Triggers weak actin polymerisation activity [17,24]. Mutating both tyrosine residues to alanine or phenylalanine abolishes Tir phosphorylation and recruitment of host proteins involved in actin polymerisation [17,25]. Recently, in order to determine whether intimin–Tir interaction is necessary and sufficient for colonisation of the gut mucosa, we systematically deleted effector genes from the prototype EPEC strain E2348/69, generating EPEC-0 missing all the effector genes, EPEC-1 expressing only Tir and EPEC-2 that expresses only Tir and EspZ [26]. EPEC-0, 1, and 2 express intimin and a functional T3SS, while only EPEC-1 and EPEC-2 can trigger actin polymerisation in infected cells [26]. We have recently shown that intimin-induced Tir clustering, phosphorylation, and actin polymerisation trigger inflammasome-mediated pyroptotic cell death in EPEC-infected mac- rophages [25]. The inflammasome is a multi-protein complex which canonically involves cas- pase-1, the adaptor protein apoptosis-associated speck-like protein containing a CARD (ASC) and the nucleotide-binding oligomerisation domain (NOD) leucine-rich repeat proteins (NLRs), which act as sensors for pathogen/damage-associated molecular patterns (PAMPs/ DAMPs) such as extracellular ATP, reactive oxygen species (ROS), bacterial toxins, and secre- tion system/flagella subunits [27–29]. The caspase-1 inflammasome cleaves and activates gas- dermin D (GSDMD), a pore-forming protein that executes pyroptotic cell death [30,31] and secretion of interleukin (IL)-1β and IL-18 [25]. The noncanonical inflammasome comprises caspase-4, which is activated by direct binding to bacterial lipopolysaccharide (LPS) [30–32], known to enter the cytosol during bacterial invasion [33], via outer membrane vesicles (OMVs) [34,35] or via specific cellular receptors [36]. Recent studies have shown that the receptor-interacting serine/threonine-protein kinase 1 (RIPK1)/caspase-8 axis plays an impor- tant role in regulating apoptotic, necroptotic, and pyroptotic cell death pathways in IECs [37]. Our current understanding on the mechanisms of EPEC-induced cell death is mainly based on studies performed in macrophages. Nonetheless, pyroptosis can also be triggered in non- phagocytic IECs during bacterial infection [38]. Deep proteomic analysis of a panel of 50 intes- tinal cancer cell lines shows that caspase-4 and GSDMD are constitutively expressed, while NLR family pyrin domain containing 3 (NLRP3), gasdermin E (GSDME), and caspase-5 are undetectable under standard growth conditions [39]. Introduction The extracellular pathogen enteropathogenic Escherichia coli (EPEC) causes persistent infan- tile diarrhoea [1] and has been found to preferentially colonise colorectal cancer tissue in adult patients [2]. EPEC infection of the intestinal mucosa is mediated by a type III secretion system (T3SS) [3], a molecular syringe that injects 21 effectors directly into the host cell cytosol where PLOS Biology | https://doi.org/10.1371/journal.pbio.3000986 December 30, 2020 1 / 31 The interaction of EPEC with the colorectal epithelial cell line SNU-C5 We aimed to investigate whether EPEC could trigger cell death in IECs and the potential role of IFNγ in this process. Most studies of EPEC infection have used either HeLa or Caco-2 cell lines; however, the latter contains several mutations in genes involved in IFNγ signalling path- way, including interferon gamma receptor 2 (IFNGR2) and Janus kinase 1 (JAK1). For this reason, we first sought to identify a suitable intestinal cell model for this study. Using publicly available proteomics data for a panel of 50 colorectal cancer cell lines [39], we analysed the abundance levels of proteins mapping in a characteristic immune response module, selected based on a weighted correlation network analysis (WGCNA) [42]. We ranked all 50 cell lines by the median scaled abundance of the immune response module [39] (Fig 1A) and selected the SNU-C5 cell line as a representative of moderate abundance of the proteins in this module. In addition, SNU-C5 cell line has no mutations in the genes involved in IFNγ signalling and noncanonical inflammasome: IFNGR1, IFNGR2, JAK1, JAK2, signal transducer and activator of transcription 1 (STAT1), interferon regulatory factor 1 (IRF1), IRF2, caspase-4 (CASP4), and GSDMD, according to the Colorectal Cancer Atlas [43]. We did not consider the varia- tions of the canonical inflammasome components due to the undetectable protein expression levels of all known pyroptosis-related NLRs [39]. Additionally, the increased expression of guanylate-binding protein 2 (GBP2), an interferon-induced gene, in SNU-C5 primed with IFNγ for 24 h was validated by real-time quantitative PCR (qRT-PCR) (S1A Fig), confirming that SNU-C5 cells respond to IFNγ. To further evaluate the cell model, we examined whether SNU-C5 cells are susceptible to EPEC infection. We used Tir-induced actin polymerisation and NF-κB activation as indicators of successful infection. SNU-C5 cells were infected with EPEC wild-type (WT), EPEC-1 (Tir only), and EPEC-0 (effectorless). While all 3 strains adhered to >98% of SNU-C5 cells (Fig 1B and 1C), robust actin polymerisation was detected underneath EPEC-WT and EPEC-1, but not EPEC-0 (Fig 1D). Immunofluorescence staining of NF-κB p65 subunit showed that EPEC-WT did not trigger NF-κB activation up to 4 h postinfection (Fig 1E and 1F), consistent with the notion that it expresses multiple effectors that inhibit the NF-κB pathway [6–8]. PLOS BIOLOGY The expression level of some of these pyroptotic proteins may be elevated via NF-κB or Janus kinase/signal transducers and PLOS Biology | https://doi.org/10.1371/journal.pbio.3000986 December 30, 2020 2 / 31 PLOS BIOLOGY EPEC Tir triggers calcium influx-dependent pyroptosis rpm, revolutions per minute; RPMI, Roswell Park Memorial Institute; SCARB1, scavenger receptor class B member; SDC, sodium deoxycholate; SDS- PAGE, sodium dodecyl sulphate polyacrylamide gel electrophoresis; SERCA, sarco/endoplasmic reticulum Ca2+; shRNA, short-hairpin RNA; siRNA, small interfering RNA; SPS, synchronous precursor selection; STAT1, signal transducer and activator of transcription 1; STING, stimulator of interferon genes; STS, staurosporine; T3SS, type III secretion system; TBST, tris buffered saline, 0.1% Tween 20; TCEP, tris-2-carboxyethyl phosphine; TEAB, triethylammonium bicarbonate; Th, Thomson unit; Tir, translocated intimin receptor; TMEM173, transmembrane protein 173; TMT, tandem mass tag; TRP, transient receptor potential; WGCNA, weighted correlation network analysis; WT, wild-type; YFP, yellow fluorescent protein. activators of transcription (JAK-STAT) signalling pathways during a process called priming, via pretreatment with LPS or interferon gamma (IFNγ), respectively [28]. Although priming is most extensively used for phagocytic cells, IFNγ induces expression of caspase-1 and caspase-5 in the colonic epithelial cell line HT-29 [40]. Moreover, EPEC infection of the colorectal cancer cell line Caco-2 triggers caspase-4–dependent release of IL-18 [41], suggesting that EPEC may also induce pyroptosis in IECs. The aim of this study was to investigate whether EPEC causes pyroptosis in an IEC line model and to decipher its molecular mechanism. The interaction of EPEC with the colorectal epithelial cell line SNU-C5 In contrast, EPEC-0, lacking these effectors, provoked modest NF-κB activation, which is likely due to the activity of the T3SS apparatus [44], while infection with EPEC-1 triggered signifi- cant NF-κB activation above EPEC-0 (Fig 1E and 1F). These results suggest that in the absence of the anti-inflammatory effectors and the gatekeeper EspZ [12], high-level Tir translocation in EPEC-1 triggers strong pro-inflammatory responses. Together, these data indicate that SNU-C5 responds as expected to EPEC infection and thus provides a robust model to study EPEC infection of IECs. EPEC-induced lytic cell death in SNU-C5 is enhanced by IFNγ priming We next investigated whether EPEC infection induces death in SNU-C5 cells in a manner dependent on its effectors, using propidium iodide (PI) uptake as a reporter for membrane PLOS Biology | https://doi.org/10.1371/journal.pbio.3000986 December 30, 2020 3 / 31 PLOS BIOLOGY EPEC Tir triggers calcium influx-dependent pyroptosis Fig 1. EPEC induces Tir-dependent actin polymerisation and NF-κB activation in SNU-C5 cells. (A) Ranking of 50 colorectal cell lines based on the scaled abundance of proteins in the immune response module according to WGCNA [39]. (B) Adhesion of EPEC WT, EPEC-0, and EPEC-1 to SNU-C5. Means ± SEM from n = 3 independent biological repeats. (C, D) Immunofluorescence labelling of SNU-C5 cells infected with EPEC WT, EPEC-0 and EPEC-1 or 4 h. (C) Percentage of infected cells. (D) Immunofluorescent images. DAPI: blue; EPEC: green; Phalloidin: red. Representative images from n = 3 independent biological repeats. Scale bar: 5 μm. Example bacteria are marked with white arrows. (E) Immunofluorescence staining of p65 in UI and EPEC-infected cells, analysed by high content imaging. Representative images from n = 3 independent biological repeats. Scale bar: 20 μm. (F) Nuclear/cytosolic ratio of the NF-κB p65 immunofluorescence intensity calculated by the Columbus-2 software. Statistical significance was determined using 1-way ANOVA with Tukey posttest. ns, nonsignificant;  p  0.05;  p  0.01;  p  0.001. The underlying data for this figure can be found in S1 Data. ANOVA, analysis of variance; DAPI, 40,6-diamidino- 2-phenylindole; EPEC, enteropathogenic Escherichia coli; NF-κB, nuclear factor kappa B; SEM, standard error of the Fig 1. EPEC induces Tir-dependent actin polymerisation and NF-κB activation in SNU-C5 cells. (A) Ranking of 50 colorectal cell lines based on the scaled abundance of proteins in the immune response module according to WGCNA [39]. (B) Adhesion of EPEC WT, EPEC-0, and EPEC-1 to SNU-C5. Means ± SEM from n = 3 independent biological repeats. (C, D) Immunofluorescence labelling of SNU-C5 cells infected with EPEC WT, EPEC-0 and EPEC-1 or 4 h. (C) Percentage of infected cells. (D) Immunofluorescent images. DAPI: blue; EPEC: green; Phalloidin: red. Representative images from n = 3 independent biological repeats. Scale bar: 5 μm. Example bacteria are marked with white arrows. (E) Immunofluorescence staining of p65 in UI and EPEC-infected cells, analysed by high content imaging. Representative images from n = 3 independent biological repeats. Scale bar: 20 μm. PLOS Biology | https://doi.org/10.1371/journal.pbio.3000986 December 30, 2020 EPEC-induced lytic cell death in SNU-C5 is enhanced by IFNγ priming (F) Nuclear/cytosolic ratio of the NF-κB p65 immunofluorescence intensity calculated by the Columbus-2 software. Statistical significance was determined using 1-way ANOVA with Tukey posttest. ns, nonsignificant;  p  0.05;  p  0.01;  p  0.001. The underlying data for this figure can be found in S1 Data. ANOVA, analysis of variance; DAPI, 40,6-diamidino- 2-phenylindole; EPEC, enteropathogenic Escherichia coli; NF-κB, nuclear factor kappa B; SEM, standard error of the Fig 1. EPEC induces Tir-dependent actin polymerisation and NF-κB activation in SNU-C5 cells. (A) Ranking of 50 colorectal cell lines based on the scaled abundance of proteins in the immune response module according to WGCNA [39]. (B) Adhesion of EPEC WT, EPEC-0, and EPEC-1 to SNU-C5. Means ± SEM from n = 3 independent biological repeats. (C, D) Immunofluorescence labelling of SNU-C5 cells infected with EPEC WT, EPEC-0 and EPEC-1 or 4 h. (C) Percentage of infected cells. (D) Immunofluorescent images. DAPI: blue; EPEC: green; Phalloidin: red. Representative images from n = 3 independent biological repeats. Scale bar: 5 μm. Example bacteria are marked with white arrows. (E) Immunofluorescence staining of p65 in UI and EPEC-infected cells, analysed by high content imaging. Representative images from n = 3 independent biological repeats. Scale bar: 20 μm. (F) Nuclear/cytosolic ratio of the NF-κB p65 immunofluorescence intensity calculated by the Columbus-2 software. Statistical significance was determined using 1-way ANOVA with Tukey posttest. ns, nonsignificant;  p  0.05;  p  0.01;  p  0.001. The underlying data for this figure can be found in S1 Data. ANOVA, analysis of variance; DAPI, 40,6-diamidino- 2-phenylindole; EPEC, enteropathogenic Escherichia coli; NF-κB, nuclear factor kappa B; SEM, standard error of the PLOS Biology | https://doi.org/10.1371/journal.pbio.3000986 December 30, 2020 4 / 31 PLOS BIOLOGY EPEC Tir triggers calcium influx-dependent pyroptosis mean; Tir, translocated intimin receptor; UI, uninfected; WGCNA, weighted correlation network analysis; WT, wild- type. mean; Tir, translocated intimin receptor; UI, uninfected; WGCNA, weighted correlation network analysis; WT, wild- type. https://doi.org/10.1371/journal.pbio.3000986.g001 damage and necrotic cell lysis. This revealed that EPEC-WT induced 19% increase in PI uptake after 8 h of infection compared with uninfected control cells, while EPEC-0 increased PI uptake by 11% (Fig 2A, S1B Fig). Notably, EPEC-1 induced 35% increase in PI uptake, sig- nificantly higher than that of EPEC-WT and EPEC-0 (Fig 2A). damage and necrotic cell lysis. EPEC-induced lytic cell death in SNU-C5 is enhanced by IFNγ priming This revealed that EPEC-WT induced 19% increase in PI uptake after 8 h of infection compared with uninfected control cells, while EPEC-0 increased PI uptake by 11% (Fig 2A, S1B Fig). Notably, EPEC-1 induced 35% increase in PI uptake, sig- nificantly higher than that of EPEC-WT and EPEC-0 (Fig 2A). y g g In order to determine the effect of IFNγ priming on EPEC-induced cell death, we pre- treated the SNU-C5 monolayer with IFNγ 24 h prior to infection. IFNγ priming alone did not induce an increase in PI uptake (S1C Fig). Infecting the primed cells with EPEC-0 for 8 h showed no increase in PI uptake (12%), compared with unprimed cells. In contrast, IFNγ priming increased PI uptake to 27% and 51% following infection with EPEC-WT and EPEC-1, respectively (Fig 2A and 2B). The time course of PI uptake during EPEC infection revealed that cell death first started to increase at around 1.5 h and plateaued at 6 to 8 h postinfection (Fig 2B). To confirm that Tir is responsible for enhanced cell death, a control infection with EPECΔtir revealed markedly reduced cell death compared with EPEC WT in primed cells (Fig 2C). Collectively, these results show that, similar to macrophages [25], Tir triggers cell death in an epithelial cell model and that this activity is amplified by IFNγ. We therefore decided to investigate EPEC-induced cell death mechanisms in IFNγ-primed cells. PLOS Biology | https://doi.org/10.1371/journal.pbio.3000986 December 30, 2020 Tir-induced cell death is dependent on caspase-4 and GSDMD To test the involvement of caspases in Tir-induced cell death in IECs, we pretreated SNU-C5 cells with either a pan-caspase inhibitor z-VAD-fmk (zVAD) or the pyroptotic caspase-1/4/5 inhibitor z-YVAD-fmk (YVAD). Both zVAD and YVAD significantly reduced cell death caused by EPEC-1 at 8 h postinfection, indicating the involvement of pyroptosis, while the low level of Tir-independent cell death in EPEC-0 was unaffected by caspase inhibition (Fig 2D). Furthermore, western blot of infected cells showed no cleavage of the apoptosis marker poly [ADP-ribose] polymerase 1 (PARP1), ruling out apoptosis in Tir-dependent cell death (S2A Fig). In contrast to macrophage [25], Tir-dependent cell death in SNU-C5 was not inhibited by the NLRP3 inhibitor MCC950 (S2B and S2C Fig). As Yersinia, a related Gram-negative pathogen carrying T3SS, has recently been shown to trigger macrophage pyroptosis via RIPK1- and caspase-8-dependent cleavage of GSDMD [45, 46, 47], we infected SNU-C5 cells in the presence of the RIPK1 inhibitor Nec1 (S2D–S2F Fig) or with caspase-8 small interfering RNA (siRNA) silencing (S2G and S2H Fig), both of which revealed no inhibition of cell death. The pan-caspase inhibition did not completely abolish Tir-dependent cell death, with ca. 15% of EPEC-1-infected cells sill exhibiting PI uptake compared with EPEC-0-infected cells (Fig 2D). As necroptosis can be activated during pyroptosis inhibition [48], we investigated if the residual cell death is due to necroptosis. For this, we used necrostatin-1 (Nec1) to inhibit RIPK1, necrosulfonamide (NSA) to inhibit MLKL and siRNA to silence RIPK3. While control experiments have shown that these treatments prevented necroptosis induced by staurospor- ine (STS) and zVAD co-stimulation [49], they did not significantly affect Tir-dependent cell death, either on their own or in combination with zVAD or caspase-4 silencing (S2D–S2K Fig). Taken together, these results implicate the pyroptotic caspases as the main contributor in EPEC-induced cell death, while the mechanism leading to the residual cell death remains elusive. As neither caspase-1 nor caspase-5 is detected in SNU-C5 [39], we next investigated the involvement of caspase-4 which is detected in the proteome. Western blotting of cell lysates and supernatants collected at 8 h postinfection revealed cleavage of the approximately 45 kDa PLOS Biology | https://doi.org/10.1371/journal.pbio.3000986 December 30, 2020 5 / 31 PLOS BIOLOGY EPEC Tir triggers calcium influx-dependent pyroptosis Fig 2. IFNγ enhances Tir- and caspase-4-dependent SNU-C5 cell death. (A) PI uptake into SNU-C5 cells infected with EPEC WT, EPEC-0, and EPEC-1 for 8 h. Tir-induced cell death is dependent on caspase-4 and GSDMD Cells were either unprimed or primed with 10 ng/ml of IFNγ. PI uptake results were normalised to uninfected cells. Means ± SEM from n = 5 independent biological repeats. (B) Time-course PI uptake into primed SNU-C5 cells throughout the 8 h of infection period. Measurements were taken every 10 min. Means ± SEM from n = 5 independent biological repeats are shown. (C) PI uptake into primed SNU-C5 cells infected with EPEC WT and Δtir. Means ± SEM from n = 3 independent biological repeats. (D) PI uptake into primed SNU-C5 cells treated with zVAD and/or YVAD 30 min before infection with EPEC WT and EPEC-1. Means ± SEM from n = 3 (EPEC WT & EPEC-0) and 7 (EPEC-1) independent biological repeats. (E) Caspase-4 western blot of lysates and supernatants from primed cells infected by EPEC WT, EPEC-0, and EPEC-1. Representative blot from n = 3 independent biological repeats. (F) Caspase-4 western blot of lysates from primed cells transfected with caspase-4 siRNA for 2 days. Representative blot from n = 3 independent biological repeats are shown. (G) Control and caspase-4 siRNA transfection into primed SNU-C5 cells 2 days before infection. Shown is PI uptake following infection with Fig 2. IFNγ enhances Tir- and caspase-4-dependent SNU-C5 cell death. (A) PI uptake into SNU-C5 cells infected with EPEC WT, EPEC-0, and EPEC-1 for 8 h. Cells were either unprimed or primed with 10 ng/ml of IFNγ. PI uptake results were normalised to uninfected cells. Means ± SEM from n = 5 independent biological repeats. (B) Time-course PI uptake into primed SNU-C5 cells throughout the 8 h of infection period. Measurements were taken every 10 min. Means ± SEM from n = 5 independent biological repeats are shown. (C) PI uptake into primed SNU-C5 cells infected with EPEC WT and Δtir. Means ± SEM from n = 3 independent biological repeats. (D) PI uptake into primed SNU-C5 cells treated with zVAD and/or YVAD 30 min before infection with EPEC WT and EPEC-1. Means ± SEM from n = 3 (EPEC WT & EPEC-0) and 7 (EPEC-1) independent biological repeats. (E) Caspase-4 western blot of lysates and supernatants from primed cells infected by EPEC WT, EPEC-0, and EPEC-1. Representative blot from n = 3 independent biological repeats. (F) Caspase-4 western blot of lysates from primed cells transfected with caspase-4 siRNA for 2 days. PLOS Biology | https://doi.org/10.1371/journal.pbio.3000986 December 30, 2020 Tir-induced cell death is dependent on caspase-4 and GSDMD Representative blot from n = 3 independent biological repeats are shown. (G) Control and caspase-4 siRNA transfection into primed SNU-C5 cells 2 days before infection. Shown is PI uptake following infection with PLOS Biology | https://doi.org/10.1371/journal.pbio.3000986 December 30, 2020 6 / 31 PLOS BIOLOGY EPEC Tir triggers calcium influx-dependent pyroptosis EPEC WT and EPEC-1 or transfection with LPS. Means ± SEM from n = 4 (LPS & EPEC WT) and 7 (EPEC-1) independent biological repeats. (H) PI uptake into primed SNU-C5 cells infected with EPEC-1 and EPEC-1-NleF. Means ± SEM from n = 3 independent biological repeats. (I) GSDMD western blot of lysates from primed cells infected by EPEC WT, EPEC-0 and EPEC-1. Lysate of THP-1 cells after LPS/Nigericin treatment were used as positive control for GSDMD activation. Representative blot from n = 3 independent biological repeats. (J) GSDMD western blot of lysates from primed cells transfected with GSDMD siRNA for 3 days. Representative blot from n = 3 independent biological repeats are shown. (K) GSDMD western blot of lysates from primed GSDMD stable knockdown cells. Representative blot from n = 3 independent biological repeats are shown. (L) Control and GSDMD siRNA transfection into primed SNU-C5 cells 3 days before infection. Shown is PI uptake following infection with EPEC WT and EPEC-1 or transfection with LPS. Means ± SEM from n = 4 independent biological repeats. (M) Control and GSDMD stable knockdown cells were primed and transfected by LPS, infected by EPEC WT or EPEC-1. Means ± SEM from n = 3 independent biological repeats. (N) Left panel: the scaled endogenous abundance of caspase- 4, GSDMD, caspase-1, 5 and 8 in the 4 cell lines [39]. N/A: undetectable. Right panel: PI uptake into primed SNU-C5, HT-29, SNU-C2B, and COLO-320-HSR cells infected with EPEC-0 and EPEC-1. Means ± SEM from n = 3 independent biological repeats. Statistical significance was determined using 2-way ANOVA with Bonferroni posttest (A, D, G, L, M, N) or 2-tailed t test (C, H).  p  0.05;  p  0.01;  p  0.001. The underlying data for this figure can be found in S1 Data. ANOVA, analysis of variance; EPEC, enteropathogenic Escherichia coli; GSDMD, gasdermin D; IFNγ, interferon gamma; LPS, lipopolysaccharide; PI, propidium iodide; SEM, standard error of the mean; siRNA, small interfering RNA; Tir, translocated intimin receptor; WT, wild-type; YVAD, z-YVAD-fmk; zVAD, z-VAD-fmk. Tir-induced cell death is dependent on caspase-4 and GSDMD https://doi.org/10.1371/journal.pbio.3000986.g002 pro-caspase-4 into the active form approximately 30 kDa caspase-4 large subunit in EPEC- 1-infected cells (Fig 2E). To verify its functional role, we silenced caspase-4 by siRNA, which reduced cell death when LPS transfection was used as a positive control (Fig 2F and 2G). Upon infection with either EPEC WT or EPEC-1, cell death was suppressed by caspase-4 siRNA (Fig 2G), suggesting that Tir-induced cytotoxicity is mediated by caspase-4-driven pyroptosis. Indeed, ectopic expression of NleF, which inhibits caspase-4 [10,11], in EPEC-1 resulted in strong inhibition of Tir-induced cell death (Fig 2H). Moreover, EPEC-1-infected cells exhib- ited higher levels of cleaved N-terminal GSDMD fragment that is responsible for the pyropto- tic pores (Fig 2I). Silencing GSDMD in SNU-C5 by siRNA only mildly reduced EPEC- 1-induced cell death (Fig 2J and 2L). Consequently, we constructed GSDMD stable knock- down SNU-C5 cell line using the microRNA30E short-hairpin RNA (shRNA) strategy [25,50]. GSDMD shRNA strongly reduced cell death by the positive control LPS transfection as well as both EPEC WT and EPEC-1, confirming that GSDMD is required for Tir-dependent cell death (Fig 2K and 2M). g To further verify the dependence of cell death on GSDMD, we selected 3 additional IEC lines, HT-29, SNU-C2B, and COLO-320-HSR, in which the abundance of GSDMD is 1.9, 15.5, and 36.7 times lower compared with SNU-C5, while the abundance ratios for caspase-4 of these cell lines over SNU-C5 are 1.1, 0.7, and 0.1, respectively (Fig 2N, left panel). These cell lines display no mutations in either caspase-4 or GSDMD genes. Caspase-5 was undetectable, and caspase-8 was expressed in all cell lines, while caspase-1 was only detected at a low level in HT-29 and SNU-C2B cells (Fig 2N, left panel). Adhesion of EPEC WT, EPEC-0, and EPEC-1 and Tir translocation was confirmed in all cell lines (S3 Fig). Infections of IFNγ-primed SNU-C5 and HT-29 with EPEC-1 resulted in higher cell death compared with those with EPEC-0 (Fig 2N, right panel). In contrast, EPEC-0 caused similar lower level of cell death upon infection of COLO-320-HSR and SNU-C2B (Fig 2N, right panel). These results reveal a direct and specific correlation between the endogenous level of GSDMD and Tir-dependent cell death. Tir clustering triggers epithelial cell death Binding of intimin to Tir leads to its clustering and activation of a robust actin polymerisation cascade, which is essential for triggering cell death in human macrophages [25]. In order to determine whether Tir-induced actin polymerisation is also necessary for epithelial cell death, PLOS Biology | https://doi.org/10.1371/journal.pbio.3000986 December 30, 2020 7 / 31 PLOS BIOLOGY EPEC Tir triggers calcium influx-dependent pyroptosis we generated a TirY454A/Y474A mutant in EPEC-1 (EPEC-1-TirAA), which should not induce actin polymerisation. As controls, we confirmed that EPEC-1-TirAA adhered to SNU-C5 cells similar to EPEC-1 (Fig 3A and 3B), did not induce detectable actin polymerisation (Fig 3C), and triggered p65 nuclear translocation at a level comparable with EPEC-1 (Fig 3D and 3E). Importantly, contrary to the observation in human macrophages [25], EPEC-1-TirAA still induced pyroptosis following infection of SNU-C5 cells (Fig 3F). Both YVAD and ectopic expression of NleF markedly reduced EPEC-TirAA-induced cell death, suggesting that like WT Tir, TirAA activates caspase-4 in this model (Fig 3G and 3H). To further confirm that actin polymerisation is dispensable for cell death, we pretreated SNU-C5 cells with the actin poly- merisation inhibitor cytochalasin D, which prevented actin pedestal formation but not cell death (Fig 3I and 3J). To assess the role of Tir clustering, we used a deletion mutant lacking intimin from EPEC-1 (EPEC-1Δeae) [26]. The infection of SNU-C5 cells with EPEC-1Δeae resulted in significant reduction of cell death (Fig 3K). This implied that while intimin- induced Tir clustering is essential, the actin polymerisation function of Tir is dispensable for induction of cell death in IECs. EspZ has been shown to limit the level of Tir translocation [12]. In order to determine if the magnitude of cell death is impacted by the level of Tir translocation, we measured cell death induced by EPEC-2 (EPEC-1 expressing EspZ) and EPEC-1-TirAA ectopically expressing EspZ (EPEC-1-TirAA-EspZ). EPEC-2 and EPEC-1-TirAA-EspZ induced significantly less cell death compared with EPEC-1 and EPEC-1-TirAA, respectively (Fig 3F). Notably, the relatively high level of cell death in EPEC-1-TirAA- and EPEC-1-TirAA-EspZ-infected cells compared with EPEC-1- and EPEC-2-infected cells, respectively (Fig 3F), as well as the mild increase in cell death caused by cytochalasin D, suggests that the loss of actin polymerisation ability enhances cell death. Based on these data, we conclude that the dosage of translocated Tir impacts on cell death. Tir clustering triggers epithelial cell death Moreover, it shows a strain-dependent gradient of EPEC-induced cell death in both primed and unprimed cells: EPEC-0 < EPEC-2 < EPEC-1-TirAA-EspZ < EPEC-1 < EPEC- 1-TirAA (Fig 3F, S3C Fig). PLOS Biology | https://doi.org/10.1371/journal.pbio.3000986 December 30, 2020 Tir affects the abundance of Ca2+ transport and response proteins (F–H) PI uptake into primed SNU-C5 cells infected with EPEC-0, EPEC-2, EPEC-1-TirAA-EspZ (F), EPEC-1 (F, I), EPEC-1-TirAA (F–H), and EPEC-1-TirAA-NleF (H). The effector compositions in EPEC-0, EPEC-2, EPEC-1-TirAA-EspZ, EPEC-1, and EPEC- 1-TirAA are listed (F). Primed SNU-C5 cells were treated by YVAD and infected with EPEC-1-TirAA and used for PI uptake assays. Means ± SEM from n = 5 (F) and 3 (G-H) independent biological repeats. (I) Immunofluorescence labelling of SNU-C5 cells infected with EPEC-1 in the presence of absence of cytochalasin D treatment. DAPI: blue; EPEC: green; Phalloidin: red. Representative images from n = 4 independent biological repeats. Scale bar: 5 μm. Example bacteria are marked with white arrows. (J) PI uptake into EPEC-1-infected SNU-C5 cells in the presence of absence of cytochalasin D. Means ± SEM from n = 3 independent biological repeats. (K) PI uptake into SNU-C5 cells infected by EPEC-1 or EPEC-1 Δeae. Means ± SEM from n = 3 independent biological repeats. Statistical significance was determined using 2-tailed t test (A, D, G, H, J, K) or 1-way ANOVA with Tukey posttest (F).  p  0.05;  p  0.01;  p  0.001. The underlying data for this figure can be found in S1 Data. ANOVA, analysis of variance; DAPI, 40,6-diamidino-2-phenylindole; EPEC, enteropathogenic Escherichia coli; NF-κB, nuclear factor kappa B; PI, propidium iodide; SEM, standard error of the mean; Tir, translocated intimin receptor; YVAD, z-YVAD-fmk. Fig 3. Clustering by intimin is essential for Tir-induced cell death. (A) Infection rates of EPEC-1 and EPEC-1-TirAA on SNU-C5 cells. Means ± SEM from n = 3 independent biological repeats. (B, C) Immunofluorescence labelling of SNU-C5 cells infected with EPEC-1 and EPEC-1-TirAA for 2 h. (B) Percentage of infected cells. (C) DAPI: blue; EPEC: green; Phalloidin: red. Representative images from n = 3 independent biological repeats. Scale bar: 5 μm. Example bacteria are marked with white arrows. (D, E) Nuclear/cytosolic ratio of the NF-κB p65 immunofluorescence intensity calculated by the Columbus-2 software (D) and representative images (E) from n = 3 independent biological repeats of unprimed SNU-C5 cells infected with EPEC-1 and EPEC-1-TirAA. Scale bar: 20 μm. (F–H) PI uptake into primed SNU-C5 cells infected with EPEC-0, EPEC-2, EPEC-1-TirAA-EspZ (F), EPEC-1 (F, I), EPEC-1-TirAA (F–H), and EPEC-1-TirAA-NleF (H). The effector compositions in EPEC-0, EPEC-2, EPEC-1-TirAA-EspZ, EPEC-1, and EPEC- 1-TirAA are listed (F). Tir affects the abundance of Ca2+ transport and response proteins In order to gain mechanistic insights of Tir-induced caspase-4 activation, we performed prote- omics analysis of SNU-C5 cells following infection of unprimed and IFNγ-primed cells with EPEC-0, EPEC-2, EPEC-1-TirAA-EspZ, EPEC-1, and EPEC-1-TirAA, using isobaric labelling (S5A Fig). We selected 2 h postinfection for the analysis representing the time point where phenotypic signs of cell death are evident but with limited cell loss (Fig 2B, S1B Fig). We quan- tified a total of 8,888 human proteins, of which 1,752 were found differentially regulated between the different infections (analysis of variance [ANOVA], adjusted P < 0.05). Principal component analysis (PCA) using all proteins showed a strong separation by IFNγ priming as well as by the strain-dependent gradient of cell death, with the largest variation observed between EPEC-0/EPEC-2 and EPEC-1-TirAA for both primed and unprimed cells (S5B Fig). Hierarchical clustering revealed groups of differentially regulated proteins either positively or negatively correlating with the cell death gradient as well as clusters of proteins regulated by IFNγ-priming in an infection-independent manner (Fig 4A). Enrichment analysis identified a diverse range of signalling pathways and biological processes with up- or down-regulation trends across infections (Fig 4B). As expected, proteins involved in antigen presentation and response to IFNγ were highly up-regulated in all primed samples (Fig 4B). A heatmap of the 106 differentially regulated proteins upon IFNγ priming, including caspase-4 and GSDMD, is shown in S5 Fig. Several basic cellular processes including, proteolysis, spliceosome, cell cycle, and cytoskeleton proteins showed a decreased abundance that is correlated with Tir-induced PLOS Biology | https://doi.org/10.1371/journal.pbio.3000986 December 30, 2020 8 / 31 PLOS BIOLOGY EPEC Tir triggers calcium influx-dependent pyroptosis Fig 3. Clustering by intimin is essential for Tir-induced cell death. (A) Infection rates of EPEC-1 and EPEC-1-TirAA on SNU-C5 cells. Means ± SEM from n = 3 independent biological repeats. (B, C) Immunofluorescence labelling of SNU-C5 cells infected with EPEC-1 and EPEC-1-TirAA for 2 h. (B) Percentage of infected cells. (C) DAPI: blue; EPEC: green; Phalloidin: red. Representative images from n = 3 independent biological repeats. Scale bar: 5 μm. Example bacteria are marked with white arrows. (D, E) Nuclear/cytosolic ratio of the NF-κB p65 immunofluorescence intensity calculated by the Columbus-2 software (D) and representative images (E) from n = 3 independent biological repeats of unprimed SNU-C5 cells infected with EPEC-1 and EPEC-1-TirAA. Scale bar: 20 μm. PLOS Biology | https://doi.org/10.1371/journal.pbio.3000986 December 30, 2020 https://doi.org/10.1371/journal.pbio.3000986.g003 Tir affects the abundance of Ca2+ transport and response proteins (F) Hierarchical clustering of proteins selected by creb1_gpl1261_gds3660_down (TF-LOF Expression from GEO) and Calcium (Keywords). The underlying data for this figure can be found in S1 Data. ANOVA, analysis of variance; CREB1, cAMP responsive element binding protein 1; EPEC, enteropathogenic Escherichia coli; FDR, false discovery rate; IFNγ, interferon gamma; GEO, Gene Expression Omnibus; TF-LOF transcription factor loss of function; Tir, translocated intimin receptor. Fig 4. Proteomic analysis of SNU-C5 infected with EPEC variants inducing variable levels of cell death. (A) Hierarchical clustering (Euclidean distance) of differentially regulated proteins (ANOVA, FDR < 0.05) between EPEC-0, EPEC-2, EPEC-1-TirAA-EspZ, EPEC-1, and EPEC-1-TirAA-infected cells with and without INFNγ priming. Data represent log2 ratios versus the uninfected sample and 2 independent biological repeats were performed per infection. Individual repeats are labelled in the heatmap (R1 and R2). (B) Representative enriched pathways shown as a heatmap of the enrichment scores (Benjamini–Hochberg FDR < 0.05). (C) Box plot of changes in Ca2+ transporter proteins selected using Keywords across the different samples. The boxes are color-coded according to the enrichment scores shown in panel B. (D) Transcription factor enrichment was performed using the “TF-LOF Expression from GEO” library. (E) Box plot of protein changes of a gene set with known reduced expression upon CREB1 perturbation (therefore positively regulated by CREB1). These proteins tend to be up-regulated in our data set, suggesting the activation of CREB1. The boxes are color-coded according to the enrichment scores shown in panel D. (F) Hierarchical clustering of proteins selected by creb1_gpl1261_gds3660_down (TF-LOF Expression from GEO) and Calcium (Keywords). The underlying data for this figure can be found in S1 Data. ANOVA, analysis of variance; CREB1, cAMP responsive element binding protein 1; EPEC, enteropathogenic Escherichia coli; FDR, false discovery rate; IFNγ, interferon gamma; GEO, Gene Expression Omnibus; TF-LOF, transcription factor loss of function; Tir, translocated intimin receptor. htt //d i /10 1371/j l bi 3000986 004 Fig 4. Proteomic analysis of SNU-C5 infected with EPEC variants inducing variable levels of cell death. (A) Hierarchical clustering (Euclidean distance) of differentially regulated proteins (ANOVA, FDR < 0.05) between EPEC-0, EPEC-2, EPEC-1-TirAA-EspZ, EPEC-1, and EPEC-1-TirAA-infected cells with and without INFNγ priming. Data represent log2 ratios versus the uninfected sample and 2 independent biological repeats were performed per infection. Individual repeats are labelled in the heatmap (R1 and R2). (B) Representative enriched pathways shown as a heatmap of the enrichment scores (Benjamini–Hochberg FDR < 0.05). Tir affects the abundance of Ca2+ transport and response proteins Primed SNU-C5 cells were treated by YVAD and infected with EPEC-1-TirAA and used for PI uptake assays. Means ± SEM from n = 5 (F) and 3 (G-H) independent biological repeats. (I) Immunofluorescence labelling of SNU-C5 cells infected with EPEC-1 in the presence of absence of cytochalasin D treatment. DAPI: blue; EPEC: green; Phalloidin: red. Representative images from n = 4 independent biological repeats. Scale bar: 5 μm. Example bacteria are marked with white arrows. (J) PI uptake into EPEC-1-infected SNU-C5 cells in the presence of absence of cytochalasin D. Means ± SEM from n = 3 independent biological repeats. (K) PI uptake into SNU-C5 cells infected by EPEC-1 or EPEC-1 Δeae. Means ± SEM from n = 3 independent biological repeats. Statistical significance was determined using 2-tailed t test (A, D, G, H, J, K) or 1-way ANOVA with Tukey posttest (F).  p  0.05;  p  0.01;  p  0.001. The underlying data for this figure can be found in S1 Data. ANOVA, analysis of variance; DAPI, 40,6-diamidino-2-phenylindole; EPEC, enteropathogenic Escherichia coli; NF-κB, nuclear factor kappa B; PI, propidium iodide; SEM, standard error of the mean; Tir, translocated intimin receptor; YVAD, z-YVAD-fmk. https://doi.org/10.1371/journal.pbio.3000986.g003 9 / 31 PLOS Biology | https://doi.org/10.1371/journal.pbio.3000986 December 30, 2020 PLOS BIOLOGY EPEC Tir triggers calcium influx-dependent pyroptosis Fig 4. Proteomic analysis of SNU-C5 infected with EPEC variants inducing variable levels of cell death. (A) Hierarchical clustering (Euclidean distance) of differentially regulated proteins (ANOVA, FDR < 0.05) between EPEC-0, EPEC-2, EPEC-1-TirAA-EspZ, EPEC-1, and EPEC-1-TirAA-infected cells with and without INFNγ priming. Data represent log2 ratios versus the uninfected sample and 2 independent biological repeats were performed per infection. Individual repeats are labelled in the heatmap (R1 and R2). (B) Representative enriched pathways shown as a heatmap of the enrichment scores (Benjamini–Hochberg FDR < 0.05). (C) Box plot of changes in Ca2+ transporter proteins selected using Keywords across the different samples. The boxes are color-coded according to the enrichment scores shown in panel B. (D) Transcription factor enrichment was performed using the “TF-LOF Expression from GEO” library. (E) Box plot of protein changes of a gene set with known reduced expression upon CREB1 perturbation (therefore positively regulated by CREB1). These proteins tend to be up-regulated in our data set, suggesting the activation of CREB1. The boxes are color-coded according to the enrichment scores shown in panel D. https://doi.org/10.1371/journal.pbio.3000986.g004 Tir affects the abundance of Ca2+ transport and response proteins (C) Box plot of changes in Ca2+ transporter proteins selected using Keywords across the different samples. The boxes are color-coded according to the enrichment scores shown in panel B. (D) Transcription factor enrichment was performed using the “TF-LOF Expression from GEO” library. (E) Box plot of protein changes of a gene set with known reduced expression upon CREB1 perturbation (therefore positively regulated by CREB1). These proteins tend to be up-regulated in our data set, suggesting the activation of CREB1. The boxes are color-coded according to the enrichment scores shown in panel D. (F) Hierarchical clustering of proteins selected by creb1_gpl1261_gds3660_down (TF-LOF Expression from GEO) and Calcium (Keywords). The underlying data for this figure can be found in S1 Data. ANOVA, analysis of variance; CREB1, cAMP responsive element binding protein 1; EPEC, enteropathogenic Escherichia coli; FDR, false discovery rate; IFNγ, interferon gamma; GEO, Gene Expression Omnibus; TF-LOF, transcription factor loss of function; Tir, translocated intimin receptor. https://doi.org/10.1371/journal.pbio.3000986.g004 PLOS Biology | https://doi.org/10.1371/journal.pbio.3000986 December 30, 2020 10 / 31 PLOS BIOLOGY EPEC Tir triggers calcium influx-dependent pyroptosis death profile (Fig 4B). Strikingly, Ca2+ transport, protein folding in endoplasmic reticulum (ER), and mitochondrial metabolism were among the most up-regulated pathways by EPEC-1 and EPEC-1-TirAA, and to a lesser extent by EPEC-1-TirAA-EspZ, compared with EPEC-2 and EPEC-0 (Fig 4B). In particular, Ca2+ transport proteins showed a clear trend of up-regulation correlating with the cell death gradient (Fig 4C). To investigate whether any of the observed proteomic changes could be explained by early activation or inhibition of upstream factors, we performed enrichment analysis using expres- sion gene sets from transcription factor perturbations from the Gene Expression Omnibus (GEO) database [51]. This analysis identified cAMP responsive element binding protein (CREB1) as the most significantly enriched transcription factor with enrichment scores posi- tively correlating with the cell death gradient (Fig 4D and 4E). CREB1 is activated by Ca2+ influx and subsequently regulates the downstream expression of Ca2+ transport proteins as a feedback response [52,53]. Proteins related to Ca2+ signalling, including the ER Ca2+ trans- porters ATP2A2 (1 of the sarco/endoplasmic reticulum Ca2+ ATPases (SERCA); the main ER Ca2+ importer) and inositol 1,4,5-trisphosphate receptor type 2 (ITPR2, ER Ca2+ release chan- nel), Ca2+ response protein calnexin (CANX) which senses and regulates Ca2+ oscillations, and several other Ca2+-binding proteins, were also predicted downstream of CREB1 signalling and show changes in expression similar to the cell death gradient (Fig 4F). Tir affects the abundance of Ca2+ transport and response proteins In addition, ER and mitochondria are both major Ca2+ storage sites and modulated by Ca2+ signalling. Many enzymes involved in protein folding in ER and mitochondrial metabolism require Ca2+ binding and are regulated by Ca2+ homeostasis [54,55]. Therefore, it is likely that the up-regulation of ER protein folding and mitochondrial metabolism proteins during EPEC infection resulted from enhanced Ca2+ signalling. Importantly, caspase-4 can be activated by increased intracellular Ca2+ level, for example, ER Ca2+ release mediated by transmembrane protein 173 (TMEM173) (also known as stimulator of interferon genes (STING)) [56] or the SERCA inhibitor thapsigargin [57]. Based on our current findings and published data, we hypothesised that a change in Ca2+ concentration in the cytosol modulates the viability of the infected cells. Ca2+ influx mediates Tir-induced cell death Based on the changes in the abundance of the plasma membrane Ca2+ influx transporters and the ER Ca2+ importer ATP2A2 along the cell death gradient (Fig 4), we hypothesised that EPEC infection leads to Ca2+ influx. To test this hypothesis, we used the Fluo-4 Ca2+ reporter to measure cytosolic-free Ca2+ levels. We found that 40 min postinfection with EPEC-1 and EPEC-1-TirAA, SNU-C5 cells exhibit increased cytosolic Ca2+ levels compared with EPEC- 0-infected and uninfected cells (Fig 5A, S6A Fig). In addition, Tir-dependent Ca2+ influx was detected in unprimed cells infected with either EPEC-1 or EPEC-1-TirAA at similar level to primed cells, suggesting IFNγ treatment has negligible effect on Ca2+ influx (S7B Fig). Although mitochondrial proteins were up-regulated and ROS generated from respiration as a result of Ca2+ influx could lead to cell death [58], we found that the ROS scavenger and the respiration inhibitor oligomycin had no significant effect on Tir-induced cell death (S8 Fig). In order to investigate the source of Ca2+ influx in cell death, we pretreated SNU-C5 cells with extracellular Ca2+ chelating agent EGTA. This completely diminished the increase in cytosolic Ca2+ and resulted in the attenuation of pyroptosis induced by both EPEC-1 and EPEC-1-TirAA (Fig 5C and 5D; S7A and S7B Fig), without affecting EPEC-1-mediated actin polymerisation (Fig 5B). Moreover, cell death induced by EPEC-1 was significantly lower when SNU-C5 cells were grown in Ca2+-free, compared with complete DMEM media (Fig 5E). Enumeration of CFU showed similar bacterial attachment in Ca2+-free medium (Fig 5F). Based on the changes in the abundance of the plasma membrane Ca2+ influx transporters and the ER Ca2+ importer ATP2A2 along the cell death gradient (Fig 4), we hypothesised that EPEC infection leads to Ca2+ influx. To test this hypothesis, we used the Fluo-4 Ca2+ reporter to measure cytosolic-free Ca2+ levels. We found that 40 min postinfection with EPEC-1 and EPEC-1-TirAA, SNU-C5 cells exhibit increased cytosolic Ca2+ levels compared with EPEC- 0-infected and uninfected cells (Fig 5A, S6A Fig). In addition, Tir-dependent Ca2+ influx was detected in unprimed cells infected with either EPEC-1 or EPEC-1-TirAA at similar level to primed cells, suggesting IFNγ treatment has negligible effect on Ca2+ influx (S7B Fig). PLOS Biology | https://doi.org/10.1371/journal.pbio.3000986 December 30, 2020 Ca2+ influx mediates Tir-induced cell death The Fluo-4 emission at 40 min postinfection was plotted. Means ± SEM from n = 3 independent biological repeats. (H) PI uptake into SNU-C5 cells grown in complete or Ca2+-free DMEM transfected with LPS with or without 30 min pretreatment with YVAD. Means ± SEM from n = 3 independent biological repeats. Statistical significance was determined using 2-way ANOVA with Bonferroni posttest (A, G, H) or 2-tailed t test (C-F).  p  0.05;  p  0.01;  p  0.001. The underlying data for this figure can be found in S1 Data. ANOVA, analysis of variance; DAPI, 40,6-diamidino-2-phenylindole; EGTA, ethylene glycol-bis(β-aminoethyl ether)-N,N,N0,N0-tetraacetic acid; EPEC, enteropathogenic Escherichia coli; PI, propidium iodide; SEM, standard error of the mean; Tir, translocated intimin receptor; YVAD, z-YVAD-fmk. Fig 5. Tir-induced extracellular Ca2+ influx promotes cell death. (A) Fluo-4 assay of primed SNU-C5 cells infected with EPEC-0, EPEC-1 and EPEC-1-TirAA with or without 30 min pretreatment with EGTA. The Fluo-4 emission at 40 min postinfection was plotted. Means ± SEM from n = 3 independent biological repeats. (B) Immunofluorescence labelling of SNU-C5 cells infected with EPEC-1 for 2 h with and without EGTA pretreatment. DAPI: blue; EPEC: green; Phalloidin: red. Representative images from 3 independent biological repeats are shown. Error bar: 5 μm. (C, D) PI uptake into SNU-C5 cells infected with EPEC-1 (C) or EPEC-1-TirAA (D) with or without 30 min pretreatment with EGTA. Means ± SEM from n = 3 independent biological repeats. (E) PI uptake into SNU-C5 cells grown in complete DMEM or Ca2+-free DMEM infected with EPEC-1. Means ± SEM from n = 4 independent biological repeats. (F) Adhesion of EPEC-1 to SNU-C5 cells grown in complete or Ca2+-free DMEM. Means ± SEM from n = 3 independent biological repeats. (G) Fluo-4 of SNU-C5 cells infected with EPEC-1 with or without 30 min pretreatment with YVAD. The Fluo-4 emission at 40 min postinfection was plotted. Means ± SEM from n = 3 independent biological repeats. (H) PI uptake into SNU-C5 cells grown in complete or Ca2+-free DMEM transfected with LPS with or without 30 min pretreatment with YVAD. Means ± SEM from n = 3 independent biological repeats. Statistical significance was determined using 2-way ANOVA with Bonferroni posttest (A, G, H) or 2-tailed t test (C-F).  p  0.05;  p  0.01;  p  0.001. The underlying data for this figure can be found in S1 Data. Ca2+ influx mediates Tir-induced cell death Means ± SEM from n = 3 independent biological repeats. (B) Immunofluorescence labelling of SNU-C5 cells infected with EPEC-1 for 2 h with and without EGTA pretreatment. DAPI: blue; EPEC: green; Phalloidin: red. Representative images from 3 independent biological repeats are shown. Error bar: 5 μm. (C, D PI uptake into SNU-C5 cells infected with EPEC-1 (C) or EPEC-1-TirAA (D) with or without 30 min pretreatment with EGTA. Means ± SEM from n = 3 independent biological repeats. (E) PI uptake into SNU-C5 cells grown in complete DMEM or Ca2+-free DMEM infected with EPEC-1. Means ± SEM from n = 4 independent biological repeats. (F) Adhesion of EPEC-1 to SNU-C5 cells grown in complete or Ca2+-free DMEM. Means ± SEM from n = 3 independent biological repeats. (G) Fluo-4 of SNU-C5 cells infected with EPEC-1 with or without 30 min pretreatmen with YVAD. The Fluo-4 emission at 40 min postinfection was plotted. Means ± SEM from n = 3 independent biological repeats. (H) PI uptake into SNU-C5 cells grown in complete or Ca2+-free DMEM transfected with LPS with or without 30 min pretreatment with YVAD. Means ± SEM from n = 3 independent biological repeats. Statistical significance was determined using 2-way ANOVA with Bonferroni posttest (A, G, H) or 2-tailed t test (C-F).  Fig 5. Tir-induced extracellular Ca2+ influx promotes cell death. (A) Fluo-4 assay of primed SNU-C5 cells infected with EPEC-0, EPEC-1 and EPEC-1-TirAA with or without 30 min pretreatment with EGTA. The Fluo-4 emission at 40 min postinfection was plotted. Means ± SEM from n = 3 independent biological repeats. (B) Immunofluorescence labelling of SNU-C5 cells infected with EPEC-1 for 2 h with and without EGTA pretreatment. DAPI: blue; EPEC: green; Phalloidin: red. Representative images from 3 independent biological repeats are shown. Error bar: 5 μm. (C, D) PI uptake into SNU-C5 cells infected with EPEC-1 (C) or EPEC-1-TirAA (D) with or without 30 min pretreatment with EGTA. Means ± SEM from n = 3 independent biological repeats. (E) PI uptake into SNU-C5 cells grown in complete DMEM or Ca2+-free DMEM infected with EPEC-1. Means ± SEM from n = 4 independent biological repeats. (F) Adhesion of EPEC-1 to SNU-C5 cells grown in complete or Ca2+-free DMEM. Means ± SEM from n = 3 independent biological repeats. (G) Fluo-4 of SNU-C5 cells infected with EPEC-1 with or without 30 min pretreatment with YVAD. Ca2+ influx mediates Tir-induced cell death Although mitochondrial proteins were up-regulated and ROS generated from respiration as a result of Ca2+ influx could lead to cell death [58], we found that the ROS scavenger and the respiration inhibitor oligomycin had no significant effect on Tir-induced cell death (S8 Fig). In order to investigate the source of Ca2+ influx in cell death, we pretreated SNU-C5 cells with extracellular Ca2+ chelating agent EGTA. This completely diminished the increase in cytosolic Ca2+ and resulted in the attenuation of pyroptosis induced by both EPEC-1 and EPEC-1-TirAA (Fig 5C and 5D; S7A and S7B Fig), without affecting EPEC-1-mediated actin polymerisation (Fig 5B). Moreover, cell death induced by EPEC-1 was significantly lower when SNU-C5 cells were grown in Ca2+-free, compared with complete DMEM media (Fig 5E). Enumeration of CFU showed similar bacterial attachment in Ca2+-free medium (Fig 5F). PLOS Biology | https://doi.org/10.1371/journal.pbio.3000986 December 30, 2020 11 / 31 PLOS BIOLOGY EPEC Tir triggers calcium influx-dependent pyroptosis Fig 5. Tir-induced extracellular Ca2+ influx promotes cell death. (A) Fluo-4 assay of primed SNU-C5 cells infected with EPEC-0, EPEC-1 and EPEC-1-TirAA with or without 30 min pretreatment with EGTA. The Fluo-4 emission at 40 min postinfection was plotted. Means ± SEM from n = 3 independent biological repeats. (B) Immunofluorescence labelling of SNU-C5 cells infected with EPEC-1 for 2 h with and without EGTA pretreatment. DAPI: blue; EPEC: green; Phalloidin: red. Representative images from 3 independent biological repeats are shown. Error bar: 5 μm. (C, D) PI uptake into SNU-C5 cells infected with EPEC-1 (C) or EPEC-1-TirAA (D) with or without 30 min pretreatment with EGTA. Means ± SEM from n = 3 independent biological repeats. (E) PI uptake into SNU-C5 cells grown in complete DMEM or Ca2+-free DMEM infected with EPEC-1. Means ± SEM from n = 4 independent biological repeats. (F) Adhesion of EPEC-1 to SNU-C5 cells grown in complete or Ca2+-free DMEM. Means ± SEM from n = 3 independent biological repeats. (G) Fluo-4 of SNU-C5 cells infected with EPEC-1 with or without 30 min pretreatment with YVAD. The Fluo-4 emission at 40 min postinfection was plotted. Means ± SEM from n = 3 independent Fig 5. Tir-induced extracellular Ca2+ influx promotes cell death. (A) Fluo-4 assay of primed SNU-C5 cells infected with EPEC-0, EPEC-1 and EPEC-1-TirAA with or without 30 min pretreatment with EGTA. The Fluo-4 emission at 4 min postinfection was plotted. PLOS Biology | https://doi.org/10.1371/journal.pbio.3000986 December 30, 2020 Tir induces Ca2+-dependent pyroptosis on a primary epithelial cell line To determine if Tir-induced cell death is restricted to tumorigenic epithelial cells, we pro- ceeded to use the primary human retinal pigment epithelium (RPE) cell, a non-tumorigenic model cell line that has been previously used in epithelial cell death studies, including caspase- 4-dependent cell death [61]. Infection of RPE cells by EPEC WT and EPEC-1, but not EPEC-0, showed actin pedestal formation, confirming EPEC attachment (Fig 7A). In IFNγ-primed RPE cells, EPEC-1 induced a significantly higher level of cell death compared with both EPEC WT and EPEC-0 (Fig 7B). Similar to SNU-C5, cell death induced by EPEC-1 in RPE cells was inhibited by the pyroptosis inhibitor YVAD as well as by the chelation of extracellular Ca2+ (Fig 7C and 7D). This suggests that the ability of Tir to induce Ca2+-dependent pyroptosis is conserved across the tumorigenic and primary epithelial cell lines. Ca2+ influx mediates Tir-induced cell death ANOVA, analysis of variance; DAPI, 40,6-diamidino-2-phenylindole; EGTA, ethylene glycol-bis(β-aminoethyl ether)-N,N,N0,N0-tetraacetic acid; EPEC, enteropathogenic Escherichia coli; PI, propidium iodide; SEM, standard error of the mean; Tir, translocated intimin receptor; YVAD, z-YVAD-fmk. https://doi.org/10.1371/journal.pbio.3000986.g005 https://doi.org/10.1371/journal.pbio.3000986.g005 12 / 31 PLOS Biology | https://doi.org/10.1371/journal.pbio.3000986 December 30, 2020 PLOS BIOLOGY EPEC Tir triggers calcium influx-dependent pyroptosis While the GSDMD pore has been previously shown to induce Ca2+ influx [59], YVAD did not affect Tir-induced Ca2+ influx, suggesting that this event lies upstream of caspase-4 and GSDMD cleavage (Fig 5G, S7C Fig). Notably, cell death caused by LPS transfection was not reduced in Ca2+-free DMEM (Fig 5H), indicating that extracellular Ca2+ influx is not necessary for activation of caspase-4 by cytosolic LPS. To further substantiate the role of Ca2+ in cell death, we used ATP, an agonist of the Ca2+-permeable purinergic channels [60], to pharmacologically induce Ca2+ influx before EPEC infection. First, to confirm the effect of ATP on Ca2+ influx on SNU-C5 cells, we per- formed Fluo-4 assay on cells treated with 0.5 mM ATP, which revealed increased Ca2+ influx within 2 min after ATP treatment (Fig 6A). We then performed PI uptake assays on EPEC-0 and EPEC-1-infected cells pretreated with ATP for 30 min. ATP treatment significantly enhanced cell death by EPEC-1 but not EPEC-0, while ATP alone did not affect the viability of uninfected cells (Fig 6B). Therefore, although Ca2+ augments Tir-dependent cell death, it can- not substitute Tir. Furthermore, treating cells with EGTA before the addition of ATP reduced the rapid Ca2+ uptake and Tir-induced cell death (Fig 6C and 6D). To study whether ATP- induced Ca2+ influx enhances Tir-dependent cell death via caspase-4, we treated the cells with YVAD which by itself did not inhibit Ca2+ influx (Fig 6C). YVAD prevented ATP-dependent increase in Tir-induced cell death, suggesting that increased Ca2+ influx cannot bypass the need of caspases (Fig 6D). In support of this, we also found that ATP failed to promote Tir- induced cell death in the presence of NleF (Fig 6E). Importantly, ATP pretreatment did not elevate cell death caused by LPS transfection (Fig 6F). Hence, Ca2+ influx specifically promotes Tir-induced cell death. PLOS Biology | https://doi.org/10.1371/journal.pbio.3000986 December 30, 2020 Tir induces Ca2+-dependent LPS internalisation Our data thus far show that Tir-induced cell death is dependent on Ca2+ influx and caspase-4 activation. In order to mechanistically integrate these observations, we investigated if Ca2+ influx could promote LPS internalisation. To this end, E. coli LPS conjugated with Alexa-488 was added as a tracer before infection. Fluorescent LPS was detected as clusters surrounding the perinuclear region in approximately a third of EPEC-1- and EPEC-1-TirAA-infected cells, which was significantly higher than EPEC-0-infected cells (Fig 8A and 8B), suggesting that Tir promotes the internalisation of LPS independent of actin polymerisation. Depletion of extra- cellular Ca2+ reduced LPS perinuclear localisation (Fig 8A), indicating the role of Ca2+ in the LPS internalisation process. The requirement of both Ca2+ and Tir for LPS internalisation mir- rors the Tir-induced cell phenotype. Taken together, these results show a novel cascade leading to EPEC-induced cell death, starting with Tir translocation and clustering and induction of Ca2+ influx, which promote LPS internalisation and activation of caspase-4. PLOS Biology | https://doi.org/10.1371/journal.pbio.3000986 December 30, 2020 13 / 31 PLOS BIOLOGY EPEC Tir triggers calcium influx-dependent pyroptosis Fig 6. Pharmacologically induced Ca2+ influx promotes Tir-dependent cell death upstream of pyroptotic caspase activation. (A) Fluo-4 assay of SNU-C5 cells with or without 2 min pretreatment with ATP. Means ± SEM from n = 3 independent biological repeats. (B) PI uptake into SNU-C5 cells infected with EPEC-0 or EPEC-1, with or without 30 min pretreatment with ATP. Means ± SEM from n = 6 (UI and EPEC-1) and 3 (EPEC-0) independent biological repeats. (C, D) PI uptake into SNU-C5 cells infected with EPEC-1 with 30 min pretreatment with ATP, EGTA, or YVAD or 45 min pretreatment with EGTA or YVAD, followed by ATP for 30 min. The order of treatment in each sample is indicated by the arrow. Fluo-4 assay was performed on the uninfected cells with the same treatment at 2 min after ATP addition. Means ± SEM from n = 5 (EPEC-1 and EPEC-1 + ATP in D) and 3 (C, D) independent biological repeats. (E) PI uptake into SNU-C5 cells infected with EPEC-1 and EPEC-1-NleF with or without 30 min pretreatment with ATP. Means ± SEM from n = 3 independent biological repeats. (F) PI uptake into SNU-C5 cells transfected with LPS with or without pretreatment of ATP for 30 min. Means ± SEM from n = 4 independent biological repeats are shown. PLOS Biology | https://doi.org/10.1371/journal.pbio.3000986 December 30, 2020 Tir induces Ca2+-dependent LPS internalisation https://doi.org/10.1371/journal.pbio.3000986.g006 14 / 31 PLOS Biology | https://doi.org/10.1371/journal.pbio.3000986 December 30, 2020 PLOS BIOLOGY EPEC Tir triggers calcium influx-dependent pyroptosis Fig 7. EPEC induces Tir- and Ca2+-dependent pyroptosis in RPE cells. (A) Immunofluorescence labelling of RPE cells infected with EPEC WT, EPEC-0 and EPEC-1 for 2 h. DAPI: blue; EPEC: green; Phalloidin: red. Representative images from n = 3 independent biological repeats. Scale bar: 5 μm. Example bacteria are marked with white arrows. (B) PI uptake into primed RPE cells infected with EPEC WT, EPEC-0, and EPEC-1 for 8 h. Means ± SEM from n = 3 independent biological repeats. (C) PI uptake into RPE cells in the presence or absence of YVAD and infected with EPEC-1. Means ± SEM from n = 3 independent biological repeats. (D) PI uptake into RPE cells infected with EPEC-1 in complete DMEM or Ca2+-free DMEM. Means ± SEM from n = 3 independent biological repeats. Statistical significance was determined using 1-way ANOVA with Tukey posttest (B) or 2-tailed t test (C, D) ns, nonsignificant;  p  0.05;  p  0.01. The underlying data for this figure can be found in S1 Data. ANOVA, analysis of variance; DMEM, Dulbecco’s Modified Eagle’s medium; EGTA, ethylene glycol-bis(β-aminoethyl ether)-N,N,N0,N0-tetraacetic acid; EPEC, enteropathogenic Escherichia coli; PI, propidium iodide; SEM, standard error of the mean; Tir, translocated intimin receptor; YVAD, z-YVAD-fmk. Fig 7. EPEC induces Tir- and Ca2+-dependent pyroptosis in RPE cells. (A) Immunofluorescence labelling of RPE cells infected with EPEC WT, EPEC-0 and EPEC-1 for 2 h. DAPI: blue; EPEC: green; Phalloidin: red. Representative images from n = 3 independent biological repeats. Scale bar: 5 μm. Example bacteria are marked with white arrows. (B) PI uptake into primed RPE cells infected with EPEC WT, EPEC-0, and EPEC-1 for 8 h. Means ± SEM from n = 3 independent biological repeats. (C) PI uptake into RPE cells in the presence or absence of YVAD and infected with EPEC-1. Means ± SEM from n = 3 independent biological repeats. (D) PI uptake into RPE cells infected with EPEC-1 in complete DMEM or Ca2+-free DMEM. Means ± SEM from n = 3 independent biological repeats. Statistical significance was determined using 1-way ANOVA with Tukey posttest (B) or 2-tailed t test (C, D) ns, nonsignificant;  p  0.05;  p  0.01. The underlying data for this figure can be found in S1 Data. Tir induces Ca2+-dependent LPS internalisation Statistical significance was determined using 2-way ANOVA with Bonferroni posttest (A), 2-tailed t test (B, E, F) or 1-way ANOVA with Tukey posttest (C, D). ns, nonsignificant;  p  0.05;  p  0.01;  p  0.001. The underlying data for this figure can be found in S1 Data. ANOVA, analysis of variance; ATP, adenosine triphosphate; EGTA, ethylene glycol-bis(β-aminoethyl ether)-N,N,N0,N0-tetraacetic acid; EPEC, enteropathogenic Escherichia coli; PI, propidium iodide; SEM, standard error of the mean; Tir, translocated intimin receptor; YVAD, z-YVAD-fmk. https://doi org/10 1371/journal pbio 3000986 g006 Fig 6. Pharmacologically induced Ca2+ influx promotes Tir-dependent cell death upstream of pyroptotic caspase activation. (A) Fluo-4 assay of SNU-C5 cells with or without 2 min pretreatment with ATP. Means ± SEM from n = 3 independent biological repeats. (B) PI uptake into SNU-C5 cells infected with EPEC-0 or EPEC-1, with or without 30 min pretreatment with ATP. Means ± SEM from n = 6 (UI and EPEC-1) and 3 (EPEC-0) independent biological repeats. (C, D) PI uptake into SNU-C5 cells infected with EPEC-1 with 30 min pretreatment with ATP, EGTA, or YVAD or 45 min pretreatment with EGTA or YVAD, followed by ATP for 30 min. The order of treatment in each sample is indicated by the arrow. Fluo-4 assay was performed on the uninfected cells with the same treatment at 2 min after ATP addition. Means ± SEM from n = 5 (EPEC-1 and EPEC-1 + ATP in D) and 3 (C, D) independent biological repeats. (E) PI uptake into SNU-C5 cells infected with EPEC-1 and EPEC-1-NleF with or without 30 min pretreatment with ATP. Means ± SEM from n = 3 independent biological repeats. (F) PI uptake into SNU-C5 cells transfected with LPS with or without pretreatment of ATP for 30 min. Means ± SEM from n = 4 independent biological repeats are shown. Statistical significance was determined using 2-way ANOVA with Bonferroni posttest (A), 2-tailed t test (B, E, F) or 1-way ANOVA with Tukey posttest (C, D). ns, nonsignificant;  p  0.05;  p  0.01;  p  0.001. The underlying data for this figure can be found in S1 Data. ANOVA, analysis of variance; ATP, adenosine triphosphate; EGTA, ethylene glycol-bis(β-aminoethyl ether)-N,N,N0,N0-tetraacetic acid; EPEC, enteropathogenic Escherichia coli; PI, propidium iodide; SEM, standard error of the mean; Tir, translocated intimin receptor; YVAD, z-YVAD-fmk. Tir induces Ca2+-dependent LPS internalisation ANOVA, analysis of variance; DMEM, Dulbecco’s Modified Eagle’s medium; EGTA, ethylene glycol-bis(β-aminoethyl ether)-N,N,N0,N0-tetraacetic acid; EPEC, enteropathogenic Escherichia coli; PI, propidium iodide; SEM, standard error of the mean; Tir, translocated intimin receptor; YVAD, z-YVAD-fmk. PLOS Biology | https://doi.org/10.1371/journal.pbio.3000986 December 30, 2020 Discussion In this study, we investigate if EPEC can trigger cell death in IECs. We show that similar to macrophages (both THP1 and primary human monocyte-derived macrophages) [25], infec- tion of SNU-C5 cells induced PI uptake that was dependent on Tir, with the magnitude of cell death directly correlated with the extent of Tir translocation. Importantly, in contrast to mac- rophages, EPEC-induced cell death in IECs was independent of both actin polymerisation and the canonical inflammasome (i.e., NLRP3 and caspase-1). This study provides the first evi- dence for caspase-4-dependent pyroptosis in the absences of NLRP3 and caspase-1 in IECs upon EPEC infection. The cell death of SNU-C5 was independent of caspase-8 or caspase-5 (which was undetect- able) or necroptosis but, similar to macrophages [25], dependent on caspase-4 and GSDMD. Tir-induced pyroptosis was enhanced by IFNγ, which triggered expression of caspase-4 and GSDMD in SNU-C5, while cell lines endogenously expressing low level of GSDMD were refractory to EPEC-induced cell death. These results demonstrate not only that the genetic background of host cells affects susceptibility to infection, but also that GSDMD is the rate lim- iting factor impacting on EPEC-induced cell death. PLOS Biology | https://doi.org/10.1371/journal.pbio.3000986 December 30, 2020 15 / 31 PLOS BIOLOGY EPEC Tir triggers calcium influx-dependent pyroptosis Fig 8. Tir promotes Ca2+-dependent LPS internalisation. (A) Alexa-488 conjugated LPS (or non-fluorescent LPS as negative control for autofluorescence) and EGTA was added to primed SNU-C5 cells 30 min before infection. Cells were infected by EPEC-0, EPEC-1 and EPEC-1-TirAA for 2 h. DAPI: blue; LPS: green; EPEC: purple. Representative images from n = 4 biological repeats are shown. Scale bar: 5 μm. Enlarged images of the bacteria (autofluorescence) and perinuclear regions of infected cells are shown with blue and red borders, respectively. (B) The percentage of cells with perinuclear LPS clusters. Means ± SEM from n = 4 independent biological repeats are shown. Statistical significance was determined using 1-way ANOVA with Tukey posttest.  p  0.05. The underlying data for this figure can be found in S1 Data. ANOVA, analysis of variance; DAPI, 40,6-diamidino-2-phenylindole; EGTA, ethylene glycol- bis(β-aminoethyl ether)-N,N,N0,N0-tetraacetic acid; EPEC, enteropathogenic Escherichia coli; LPS, lipopolysaccharide; SEM, standard error of the mean; Tir, translocated intimin receptor. https://doi.org/10.1371/journal.pbio.3000986.g008 Fig 8. Tir promotes Ca2+-dependent LPS internalisation. (A) Alexa-488 conjugated LPS (or non-fluorescent LPS as negative control for autofluorescence) and EGTA was added to primed SNU-C5 cells 30 min before infection. PLOS Biology | https://doi.org/10.1371/journal.pbio.3000986 December 30, 2020 Discussion Cells were infected by EPEC-0, EPEC-1 and EPEC-1-TirAA for 2 h. DAPI: blue; LPS: green; EPEC: purple. Representative images from n = 4 biological repeats are shown. Scale bar: 5 μm. Enlarged images of the bacteria (autofluorescence) and perinuclear regions of infected cells are shown with blue and red borders, respectively. (B) The percentage of cells with perinuclear LPS clusters. Means ± SEM from n = 4 independent biological repeats are shown. Statistical significance was determined using 1-way ANOVA with Tukey posttest.  p  0.05. The underlying data for this figure can be found in S1 Data. ANOVA, analysis of variance; DAPI, 40,6-diamidino-2-phenylindole; EGTA, ethylene glycol- bis(β-aminoethyl ether)-N,N,N0,N0-tetraacetic acid; EPEC, enteropathogenic Escherichia coli; LPS, lipopolysaccharide; SEM, standard error of the mean; Tir, translocated intimin receptor. https://doi.org/10.1371/journal.pbio.3000986.g008 EPEC-induced cell death is not limited to tumorigenic cells, as the primary cell line RPE showed similar susceptibility. Induction of pyroptosis during EPEC infection could benefit the host, by means of secretion of pro-inflammatory cytokines (e.g., IL-18) [11] and specific elimi- nation of infected cells, as cell death is contact dependent (i.e., intimin–Tir interactions). Indeed, the EPEC-like murine pathogen Citrobacter rodentium [62,63] lacking EspZ is highly attenuated [64], which is consistent with our findings that uncontrolled Tir translocation leads to removal of infected cells by pyroptosis. On the pathogen side, as intimin–Tir interactions, which are essential for colonisation, lead to unintentional cell death, EPEC injects NleF, an effector expressed in the majority of typical EPEC strains [65], to mitigate the impact by inhib- iting caspase-4 and blocking pyroptosis. NleF was also found to be associated more frequently with diarrhoeagenic cases compared with asymptomatic cases in atypical EPEC isolates recently found in Brazil [66], indicating the importance of cell death inhibition in bacterial vir- ulence. Moreover, our data suggest that Tir-induced actin polymerisation also contributes to the moderation of cell death in IECs. This would benefit the pathogen as it could preserve the colonisation site and sustain the infection. PLOS Biology | https://doi.org/10.1371/journal.pbio.3000986 December 30, 2020 16 / 31 PLOS BIOLOGY EPEC Tir triggers calcium influx-dependent pyroptosis To mechanistically link plasma membrane-associated Tir with cytosolic caspase-4, we per- formed proteomic analysis to identify proteins and pathways correlated with the cell death lev- els induced by the different EPEC variants. This revealed the remodelling of diverse cellular processes upon IFNγ treatment and EPEC infection. Discussion Several pathways and proteins strongly correlated with cell death levels were induced by the different EPEC strains. Others showed a negative correlation with cell death level, including cell cycle, cell division, and protein degra- dation machinery, which have also been demonstrated in previous researches to be released by pyroptotic cells [67]. Notably, Ca2+-related cellular processes, including activation of the tran- scription factor CREB1, clearly correlated with the cell death gradient. While infection resulted in higher abundance of Ca2+ transporters, the precise route of Ca2+ influx remains unclear. The rapid onset of Ca2+ influx independent of priming suggests the involvement of an IFNγ- independent posttranslational mechanism. Intimin-mediated Tir clustering has been shown to cause plasma membrane curvature, resulting in localised membrane disruption that could facilitate the influx of Ca2+. This hypothesis is consistent with the correlation between the level of Tir translocation and the magnitude of pyroptosis. Tir clustering may trigger the activation of Ca2+ channels in the plasma membrane. For example, members of the transient receptor potential (TRP) cation channel family that responds to mechanical changes in the local mem- brane [68,69] could be activated by such stimulus. In addition, E. coli LPS can intercalate into the plasma membrane leading to membrane compression which activates certain members of the TRP family [69–72]. TRPV2 and TRPM7, expressed in SNU-C5 [39], can facilitate Ca2+ influx in response to both membrane stretch [73–75] and LPS exposure [76,77]. The ability of TRPV2 to promote LPS-dependent NF-κB activation [76] also mirrors our finding during EPEC-1 and EPEC-1-TirAA infection. While extracellular Ca2+ is essential for Tir-induced pyroptosis, it is dispensable for cell death by caspase-4 in another scenario, for example, the delivery of LPS by transfection. Indeed, our results indicate that the role of Ca2+ influx is in promoting LPS internalisation. Increased intracellular Ca2+ concentration can activate diverse cell signalling events, while Ca2+ overload in mitochondria can lead to opening of the mitochondrial permeability tran- sition pore, release of cytochrome c, and ROS production [58,78]. However, we have shown that neither ROS nor apoptosis are involved in EPEC-induced cell death in SNU-C5 cells. While inhibiting Ca2+ influx by extracellular Ca2+ chelation and growing SNU-C5 cells in Ca2+-free medium inhibited Tir-induced cell death, inducing a sharp preinfection Ca2+ influx by ATP increased the level of Tir-induced pyroptosis, while not affecting LPS- induced cell death. PLOS Biology | https://doi.org/10.1371/journal.pbio.3000986 December 30, 2020 Discussion Together, these findings strongly suggest that Ca2+ influx plays a key role upstream of cell death. Notably, Ca2+ influx alone did not induce cell death in the absence of Tir, suggesting that Ca2+ synergies but does not replace Tir-dependent down- stream processes. Recent studies have shown that E. coli and Streptococcus pneumoniae induce cytosolic Ca2+ up-regulation in murine macrophages, which was dependent on the cooperation between the mitochondrial proteins TMEM173 and ITPR1 (the main ER Ca2+ release channel) [56]. Moreover, TMEM173-mediated Ca2+ release from intracellular stores promoted cleavage of GSDMD by caspase-1/11 or caspase-8 in response to E. coli or S. pneumoniae, respectively [56]. Conversely, the overexpression of ATP2A2, which was found to be up-regulated by Tir and predicted to be downstream of CREB1 in our study, limited E. coli and S. pneumoniae- induced GSDMD N-terminus fragment formation [56], suggesting the presence of negative feedback during Tir-induced cell death. Intracellular compartmentalisation of LPS was detected in Tir- and Ca2+-dependent man- ners. LPS uptake has been attributed to multiple plasma membrane proteins, including the LPS-binding protein (LBP) and TLR4 [79,80]. Although TLR4-dependent LPS uptake also PLOS Biology | https://doi.org/10.1371/journal.pbio.3000986 December 30, 2020 17 / 31 PLOS BIOLOGY EPEC Tir triggers calcium influx-dependent pyroptosis Fig 9. A model portraying the pathway of EPEC-induced cell death. EPEC infection is initiated with Tir translocation. Binding of intimin leads to intimate attachment, Tir clustering, and local membrane curvature, which could activate Ca2+ transporters and influx. Ca2+ influx leads to LPS entry, which can lead to the activation of caspase-4 and pyroptosis, while NleF can inhibit caspase-4 activation. IFNγ priming induces expression of caspase-4 and GSDMD, enhancing cell death. EPEC, enteropathogenic Escherichia coli; GSDMD, gasdermin D; IFNγ, interferon gamma; LPS, lipopolysaccharide; Tir, translocated intimin receptor. https://doi.org/10.1371/journal.pbio.3000986.g009 Fig 9. A model portraying the pathway of EPEC-induced cell death. EPEC infection is initiated with Tir translocation. Binding of intimin leads to intimate attachment, Tir clustering, and local membrane curvature, which could activate Ca2+ transporters and influx. Ca2+ influx leads to LPS entry, which can lead to the activation of caspase-4 and pyroptosis, while NleF can inhibit caspase-4 activation. IFNγ priming induces expression of caspase-4 and GSDMD, enhancing cell death. EPEC, enteropathogenic Escherichia coli; GSDMD, gasdermin D; IFNγ, interferon gamma; LPS, lipopolysaccharide; Tir, translocated intimin receptor. https://doi.org/10.1371/journal.pbio.3000986.g009 https://doi.org/10.1371/journal.pbio.3000986.g009 https://doi.org/10.1371/journal.pbio.3000986.g009 requires extracellular Ca2+ influx [77], TLR4 is not detected in the SNU-C5 proteome [39]. Discussion Scavenger receptor-mediated LPS uptake and the subsequent perinuclear compartmentalisa- tion have been characterised in HeLa cell overexpressing the scavenger receptor class B mem- ber 1 (SCARB1) [81]. SCARB1 and the LPS-binding apolipoproteins apolipoprotein B (APOB) and apolipoprotein E (APOE) are up-regulated upon EPEC-1 and EPEC-1-TirAA infections correlating with the cell death gradient, suggesting a possible role of these proteins in LPS uptake. Intimin-mediated Tir clustering has been shown to cause plasma membrane curvature, which could facilitate the influx of both Ca2+ and LPS. This hypothesis is consistent with the correlation between the level of Tir translocation and the magnitude of pyroptosis. As Ca2+ has been shown to stabilise LPS at the bacterial outer membrane [82,83], it may also facilitate adhesion of free LPS to the plasma membrane of infected cells, which likely increases its chance of entry via Tir-induced local membrane permeability. Overall, our results converge to a novel pathway, which relies on influx of Ca2+ through the plasma membrane during infec- tion with an extracellular pathogen, in a Tir-dependent manner, leading to LPS internalisation followed by caspase-4-mediated GSDMD cleavage (Fig 9). Materials and methods Bacterial strains and cell lines Infection of IECs Cells were seeded 1 day before infection. EPEC strains were primed by diluting the overnight cultures 50× in non-supplemented DMEM (low glucose) and growing for 3 h static at 37˚C with 5% CO2. Isopropyl β-d-1-thiogalactopyranoside (IPTG) (Sigma-Aldrich) at 1 mM was added to the bacterial culture 30 min before infection when required. Infection was carried out at a multiplicity-of-infection (MOI) of 50:1. Spent medium was replaced with fresh serum-free RPMI (SNU-C5, HT-29, SNU-C2B, COLO-320-HSR and THP-1) or DMEM medium (RPE) 1 h before infection. SNU-C5 and RPE cells for infection in Ca2+-free condition were grown either in Ca2+-free DMEM (high glucose) (Gibco) (supplemented with 1 mM sodium pyruvate to the same level as Ca2+-containing DMEM) or Ca2+-containing DMEM (high glucose) (Sigma-Aldrich) as control from 1 h before infection. Infected cells were centrifuged at 700 g for 10 min and incubated for 2 h static at 37˚C, 5% CO2. For over 2 h of infection, 200 μg/ml gentamicin (Sigma-Aldrich) or 250 μg/ml kanamycin (Sigma-Aldrich) (for EPEC-1-TirAA strain) was added 2 h postinfection. Generating of EPEC-1-TirAA strain Genomic DNA from WT EPEC was extracted using the DNeasy Blood & Tissue Kit (Qiagen, Hilden, Germany). All primers were ordered from Merck (Darmstadt, Germany). Primers 1.1.1 and 1.1.2 were used to amplify tir gene from the genomic DNA. The tir gene and the pSE- VA612S plasmid were digested by BamHI and HindIII and ligated to make pSEVA612S-tir fol- lowed by transformation into E. coli CC118λpir. Primers 1.3.2 and 1.4.2 were used to mutate the Y454 and Y474 in pSEVA612S-tir to alanine. The resulting linear PCR product was digested by DpnI and ligated to create pSEVA612S-tirAA plasmid and then transformed into E. coli CC118λpir. Mutations were confirmed by plasmid sequencing (Eurofins Genomics, Con- stance, Germany) using primers M13-FP, M13-RP, 1.7, and 1.8. pSEVA612S-tirAA plasmid was conjugated from E. coli CC118λpir pSEVA612S-tirAA into EPEC-0 (containing pACBSR plasmid) using the helper strain E. coli 1047 pRK2013. Colonies that grew on agar plates with both gentamicin and streptomycin were selected, and chromo- somal integration of pSEVA612S-tirAA was confirmed by colony PCR using primers 1.5 and 1.6. All plasmid descriptions and primer sequences are listed in S1B and S1C Table, respectively. Bacterial strains and cell lines EPEC E2348/69 strains (S1A Table) were grown in Luria Bertani (LB) (Sigma-Aldrich, St. Louis, Missouri, United States of America) broth or agar. Overnight bacterial cultures were 18 / 31 PLOS Biology | https://doi.org/10.1371/journal.pbio.3000986 December 30, 2020 PLOS BIOLOGY EPEC Tir triggers calcium influx-dependent pyroptosis grown at 37˚C 180 revolutions per minute (rpm) shaking (liquid) or static (agar) and primed in Dulbecco’s Modified Eagle Medium (DMEM) as described below for infections. SNU-C5, HT-29, SNU-C2B, COLO-320-HSR, and THP-1 cell lines were cultured in Ros- well Park Memorial Institute (RPMI) medium (Sigma-Aldrich) with 10% (v/v) foetal bovine serum (FBS) (Gibco, Carlsbad, California, USA), 2 mM Glutamax (Gibco), 1 mM sodium pyruvate (Sigma-Aldrich), 10 mM N-2-hydroxyethylpiperazine-N-2-ethane sulphonic acid (HEPES) (Sigma-Aldrich), and 2,500 μg/ml glucose (Sigma-Aldrich). THP-1 cells were differ- entiated using 100 ng/ml phorbol 12-myristate 13-acetate (PMA) (Sigma-Aldrich) for 48 h and PMA withdrawn for 24 h. RPE cell line was cultured in DMEM/F-12 medium (Sigma- Aldrich) with 10% (v/v) FBS, 2 mM Glutamax, and 0.26% sodium bicarbonate (Gibco). HEK293E cell line was cultured in DMEM (high glucose, 4,500 mg/L) (Sigma-Aldrich) with 10% (v/v) FBS, 1 mM sodium pyruvate, and 10 mM HEPES (pH 7.4). siRNA transfection A total of 1.25 × 104 cells/well were seeded in black clear-bottom 96-well plates 3 days prior to infection. In addition, 50 mM siRNA (0.25 μl) (Dharmacon, Lafayette, Colorado, USA) (S1D Table) mixed with 0.3 μl of TransIT-X2 transfection reagent (Mirus Bio, Madison, Wisconsin, USA) in 9 μl Opti-MEM were added to the cells 2 days before infection (caspase-4 siRNA) or 6 h after seeding, i.e., 3 days before infection (GSDMD, caspase-8 and RIPK3 siRNA). The medium was replaced with IFNγ-containing RPMI 1 day before infection. Immunofluorescence staining A total of 1.5 × 105 cells/well were seeded in 24-well plates on glass coverslips for imaging with Zeiss AxioImager Z1 microscope (Carl Zeiss, Jena, Germany). A total of 1.5 × 104 cells/well were seeded in black clear-bottom 96-well plates for imaging with Opera Cell::Explorer auto- mated spinning-disk confocal microscope (Perkin-Elmer, Waltham, Massachusetts, USA). Infection experiments were carried out as described. Alexa-488-conjugated E. coli O55:B5 LPS (Thermo Fisher Scientific, Waltham, Massachusetts, USA) was added to the cells at 5 μg/ml 30 min before infection when required. Cells were fixed by 4% paraformaldehyde for 15 min, washed by 3× PBS, permeabilised by 0.2% Triton X-100 (Sigma-Aldrich) for 4 min, washed again and blocked with 1% bovine serum albumin (BSA) for 10 min before being incubated with primary antibodies (S1E Table) for 45 min. Cells were re-blocked with 1% BSA for 10 min and incubated with secondary antibodies (S1E Table) for 30 min. For imaging using Z1 microscope using 100× oil lens, coverslips were mounted on glass slides (VWR, Radnor, Penn- sylvania, USA) with Gold-Pro-Long-Anti-fade (Invitrogen). Cytokine and drug treatment A total of 10 ng/ml human recombinant-IFNγ (R&D Systems, Minneapolis, Minnesota, USA) was added to the cells 24 h before infection. Moreover, 50 μM z-VAD-fmk (zVAD) (R&D 19 / 31 PLOS Biology | https://doi.org/10.1371/journal.pbio.3000986 December 30, 2020 PLOS BIOLOGY EPEC Tir triggers calcium influx-dependent pyroptosis Systems), 50 μM z-YVAD-fmk (YVAD) (R&D Systems), 25 μM necrostatin-1 (Nec1) (Santa Cruz Biotechnology, Dallas, Texas, USA), 5 μM necrosulfonamide (NSA) (Tocris), 200 nM cyto- chalasin D (Sigma-Aldrich), 1 mM EGTA (Sigma-Aldrich), 0.5 mM ATP (Sigma-Aldrich), 5 μM MCC950 (Tocris), 200 μM Tiron (Sigma-Aldrich), 50 mM N,N0-dimethylthiourea (DMTU) (Sigma-Aldrich), 10 μM oligomycin (Sigma-Aldrich) or 2 μM staurosporine (STS) (Calbiochem, San Diego, California, USA) were added to the cells 30 min before infection. For co-treatment, ATP was added 30 min before infection, while EGTA or YVAD were added 45 min before infec- tion. Ultrapure E. coli O111:B4 LPS (Invivogen, San Diego, California, USA) transfection was performed using Lipofectamine 2000 (Invitrogen, Waltham, Massachusetts, USA) at 5 μg/ml. GSDMD stable silencing Retroviral plasmid pMX-CMV-YFP-LacZ-miRNA30E and pMX-CMV-YFP-GSDMD-miR- NA30E (S1B and S1D Table) [25, 50, 84] were packaged in HEK293E cells with the packaging plasmid pCMV-MMLV-Gag/Pol and pCMV-VSV-G, in a 5:4:1 ratio, using Lipofectamine 2000. After 48 h of viral transfection, the medium containing the virus was filtered through a 0.45-μm filter and transferred to pre-seeded SNU-C5 cells. Puromycin (5 μg/ml) (Gibco, Carlsbad, California, USA) was added to the SNU-C5 cells 48 h after viral transduction. Cells were sorted by FACS Aria III Flow Sorter in CMBI high throughput single-cell analysis facility (HTSCAF) for yellow fluorescent protein (YFP) expression. Puromycin treatment was contin- ued after sorting to maintain a stable YFP-expressing population. Infection rate measurement A total of 1.5 × 105 cells/well were seeded in 24-well plates. Infection was carried out 1 day post-seeding as described for 2 h (no antibiotic). Infected cells were washed by 3× PBS and treated by 0.5% porcine pancreas trypsin and 0.02% EDTA in PBS (Sigma-Aldrich) for count- ing by haemocytometer (Hecht Glaswarenfabrik, Rho¨n, Germany). Counted cells were lysed with 0.1% Triton X-100, serially diluted and plated. Colony forming units (CFUs) were counted after overnight incubation at 37˚C. Infection rate was calculated by CFU per cell. High-content image acquisition and analysis Imaging was performed using an Opera Cell::Explorer automated spinning-disk confocal microscope using 40× lens (Perkin-Elmer). Twenty-five evenly distributed fields were imaged per well. Columbus-2 System (Perkin-Elmer) was used for image analysis. Nucleus and cyto- solic segmentation was performed using the DAPI and phalloidin staining, respectively. NF-κB activation was represented by the p65 nuclear-to-cytosolic ratio measured and cal- culated by Columbus-2. The nucleus region for p65 intensity measurement was 2 pixels inward from the nuclear–cytosolic boundary defined by DAPI to avoid segmentation errors at the boundary. The cytosolic region was a 2-pixel-ring further 2-pixel-outward from the nuclear–cytosolic boundary to avoid measurement skewing by cell shape [85]. Western blotting Cells were lysed with lysis buffer containing radioimmunoprecipitation (RIPA) buffer (50 mM Tris-HCl, pH 8, 2 mM EDTA, 300 mM NaCl, 2% NP-40, 1% sodium deoxycholate) and 1× PLOS Biology | https://doi.org/10.1371/journal.pbio.3000986 December 30, 2020 20 / 31 PLOS BIOLOGY EPEC Tir triggers calcium influx-dependent pyroptosis protease inhibitor (Pierce, Waltham, Massachusetts, USA) and mixed with 1× Laemmli sample buffer (Bio-Rad, Hercules, California, USA) and 5% β-mercaptoethanol (Sigma-Aldrich). The lysates were run on sodium dodecyl sulphate polyacrylamide gel electrophoresis (SDS-PAGE) gels. Western blotting was performed using a TransBlot Turbo Transfer System (Bio-Rad) to transfer the protein bands to the polyvinylidene difluoride (PVDF) membrane. Membranes were blocked with 5% milk in phosphate buffered saline, 0.05% Tween 20 (PBST) or tris buff- ered saline, 0.1% Tween 20 (TBST) for 1 h at room temperature and probed with primary anti- bodies (S1F Table) overnight at 4˚C. The secondary antibodies (S1F Table) were added for 1 h at room temperature. Membranes were developed using the ECL Western Blotting Reagents (GE, Amersham, UK) and imaged using the ChemiDoc MP imaging system (Bio-Rad). PLOS Biology | https://doi.org/10.1371/journal.pbio.3000986 December 30, 2020 Agilent Seahorse metabolic assays A total 1.5 × 104 cells/well were seeded in XFp Cell Culture Miniplates (Agilent Technologies, Santa Clara, California, USA) 1 day prior to measurement. Cells were incubated at 37˚C in non-CO2 incubator for 45 min in XF RPMI base medium with 2 mM glutamine, 1 mM sodium pyruvate, and 10 mM glucose, with pH 7.4. Measurements of oxygen consumption rate (OCR) were carried out in a Seahorse XFp Analyzer (Agilent Technologies). After mea- surements, OCR was normalised by the number of cells counted by haemocytometer after trypsin digestion. qRT-PCR A total of 5 × 105 cells/well were seeded in 12-well plates 1 day prior to RNA extraction. When required, IFNγ treatments were applied as described previously at the time of seeding. RNA extraction using the RNeasy kit (Qiagen) and DNase digestion using the RQ1 RNase-free DNase (Qiagen) were performed following the manufacturer’s instructions. A total of 0.5 μl each of oligo-dT and random primers (Promega, Madison, Wisconsin, USA) were added to every 1 μg RNA and incubated at 70˚C for 5 min. M-MLV reverse transcriptase (Promega) was used to perform the RT reaction at 42˚C for 60 min following the manufacturer’s instruc- tions. q-PCR reactions were performed on 10 ng cDNA/sample using gene-specific primers and PowerUp SyBr-Green Mastermix in a StepOne Real-Time PCR system (Thermo). Data analysis was performed using StepOne Software v2.3. Glyceraldehyde 3-phosphate dehydroge- nase (GAPDH) cDNA level was used as an internal control. Data used for statistical analysis were then normalised by log 2 transformation of the fold change to untreated control. PI uptake assay A total of 5 × 104 cells/well were seeded in black clear-bottom 96-well plates 1 day prior to infection and IFNγ and inhibitor treatment were applied as described previously. Alterna- tively, 1.25 × 104 cells/well were seeded in 3 days prior to infection for siRNA transfection as described before. Bacteria were primed in phenol-red-free DMEM (low glucose). Cells were incubated in phenol-red-free RPMI or DMEM, for different cell lines as described previously, supplemented with 5 μg/ml PI (Sigma-Aldrich) 1 h prior to infection and throughout the infection. Cell-free medium-only wells were prepared as blank. Positive control wells were pre- pared by cell lysis using RPMI with 5 μg/ml PI and 0.05% Triton X-100 (Sigma-Aldrich) 10 min prior to infection. Infections were carried out as described before. Time course measure- ments were carried out from 10 min to 8 h postinfection with 10-min intervals, measuring 620-nm emission with 520-nm excitation. The percentage of PI uptake of each well was calcu- lated by dividing each blank-normalised reading over blank-normalised positive control read- ing at the same time point. The increase in percentage PI uptake of the infected cells compared with uninfected was then calculated by subtraction of the percentage PI uptake in uninfected samples (with the same drug/siRNA treatments). 21 / 31 PLOS Biology | https://doi.org/10.1371/journal.pbio.3000986 December 30, 2020 PLOS BIOLOGY EPEC Tir triggers calcium influx-dependent pyroptosis Fluo-4 assay A total of 5 × 104 cells/well were seeded in black clear-bottom 96-well plates 1 day prior to infection, and IFNγ was applied as described. Cells were incubated in Fluo-4 Direct reagent (Molecular Probes, Eugene, Oregon, USA) diluted 2-fold in phenol-red-free RPMI for 30 min before the first measurement. Fluorescence readings were performed in Omega microplate reader measuring 520-nm emission with 485-nm excitation. All drugs were added 30 min before infection, unless specified otherwise. ATP-treated samples were followed by a second measurement at 2 min after treatment. Cell-free medium-only wells were prepared as blank. When required, time course measurements were carried out from 20 min before to 1 h after infection with 10-min intervals. Raw measurements of Fluo-4 fluorescence of each well were normalised by subtraction of the fluorescence of the blank well. PLOS Biology | https://doi.org/10.1371/journal.pbio.3000986 December 30, 2020 High-pH reversed-phase peptide fractionation and LC–MS analysis Offline high-pH reversed-phase (RP) peptide fractionation and desalting were performed using the Waters (Milford, Massachusetts, USA) XBridge C18 column (2.1 × 150 mm, 3.5 mm) on a Dionex Ultimate 3000 (Thermo Fisher Scientific) high-performance liquid chro- matograph (HPLC) system. Mobile phase A was 0.1% ammonium hydroxide, and mobile phase B was acetonitrile and 0.1% ammonium hydroxide. The TMT-labelled peptides were reconstituted in 100 μl of mobile phase A and fractionated using a multistep gradient elution method at 200 μl/min as follows: 5 min at 5% phase B, for 35 min up to 35% phase B, up to 80% phase B in 5 min, isocratic for 5 min and re-equilibration to 5% phase B. Fractions were collected in a 96-well plate every 42 s to a total of 65 fractions, then concatenated into 28 frac- tions and vacuum dried. Liquid chromatography–mass spectrometry (LC–MS) analysis was performed on the Dionex Ultimate 3000 system coupled with the Orbitrap Fusion Lumos mass spectrometer (Thermo Fisher Scientific). Each peptide fraction was reconstituted in 50 μl 0.1% formic acid, and 10 μl were loaded to the Acclaim PepMap 100, 100 μm × 2 cm, 5 μm, 100 Å C18 trapping column (Thermo Fisher Scientific) at a 10 μl/min flow rate. The sample was then analysed with the EASY-Spray C18 capillary column (75 μm × 50 cm, 2 μm) (Thermo Fisher Scientific) at 45˚C. Mobile phase A was 0.1% formic acid, and mobile phase B was 80% acetonitrile and 0.1% formic acid. The gradient separation at a flow rate of 300 nl/min was gra- dient for 90 min from 5% to 38% phase B and for 10 min up to 95% phase B, isocratic for 5 min at 95% B, re-equilibrated to 5% phase B in 5 min, and isocratic for 10 min at 5% phase B. Precursors between 375 and 1,500 m/z were selected with mass resolution of 120,000, auto- matic gain control (AGC) of 4 × 105, and injection time (IT) of 50 ms, with the top speed mode in 3 s, and were isolated for collision-induced dissociation (CID) fragmentation with a quadrupole isolation width of 0.7 Th (Thomson unit). Collision energy was set at 35%, with AGC at 1 × 104 and IT at 50 ms. MS3 quantification was obtained with higher-energy colli- sional dissociation (HCD) fragmentation of the top 5 most abundant CID fragments isolated with synchronous precursor selection (SPS). High-pH reversed-phase peptide fractionation and LC–MS analysis Quadrupole isolation width was set at 0.7 Th, col- lision energy was applied at 65%, and the AGC setting was at 1 × 105 with IT at 105 ms. The HCD MS3 spectra were acquired for the mass range of 100 to 500 m/z with a resolution of 50,000. Targeted precursors were dynamically excluded for further isolation and activation for 45 s with 7 ppm mass tolerance. The raw mass spectrometry files have been deposited to PRo- teomics IDEntifications database (PRIDE accession: PXD018763). Sample preparation and TMT labelling Cell pellets were solubilised in lysis buffer (100 mM triethylammonium bicarbonate (TEAB), 1% sodium deoxycholate (SDC), 10% isopropanol, 50 mM NaCl) supplemented with halt pro- tease and phosphatase inhibitor cocktail (Thermo Fisher Scientific, Catalog Number 78442), assisted with pulsed probe sonication. The samples were subsequently boiled for 5 min at 90˚C and then subjected to a second round of sonication. The protein concentration was measured with the Coomasie Plus assay (Thermo Fisher Scientific) according to manufacturer’s instruc- tions. Aliquots containing 100 μg of protein were prepared for trypsin digestion. Samples were reduced with 5 mM tris-2-carboxyethyl phosphine (TCEP) and alkylated with 10 mM iodoace- tamide (IAA). Proteins were then digested by adding trypsin at the 75 ng/μl final concentra- tion and incubating the samples for 18 h at room temperature, shaking at 300 rpm. The resultant peptides were diluted up to 100 μl with 100 mM TEAB buffer and labelled with PLOS Biology | https://doi.org/10.1371/journal.pbio.3000986 December 30, 2020 22 / 31 PLOS BIOLOGY EPEC Tir triggers calcium influx-dependent pyroptosis tandem mass tags (TMTs; TMT11plex) multiplex reagents (Thermo Fisher Scientific) accord- ing to manufacturer’s instructions. The quenching reaction was performed by adding hydrox- ylamine to a final concentration of 0.27% (v/v) and incubating the samples for 15 min at room temperature. Finally, 11 samples per batch were combined in equal amounts to a single tube. Formic acid was added to a final concentration of 2% (v/v), and samples were centrifuged for 5 min at 10,0000 rpm to precipitate SDC. The supernatant containing TMT-labelled peptides was moved to a new tube and dried with a centrifugal vacuum concentrator. Database search and protein quantification The acquired mass spectra were searched for protein identification and quantification in Pro- teome Discoverer 2.3 (Thermo Fisher Scientific) with the SEQUEST-HT search engine. The spectra were searched for fully tryptic peptides with maximum of 2 missed-cleavages, 20 ppm precursor mass tolerance, and 0.5 Da fragment ion mass tolerance. The search considered static carbamidomethylation of Cys residues and TMT6plex modification of peptide N-term and Lys residues, as well as dynamic oxidation of Met and deamidation of Asp and Glu 23 / 31 PLOS Biology | https://doi.org/10.1371/journal.pbio.3000986 December 30, 2020 PLOS BIOLOGY EPEC Tir triggers calcium influx-dependent pyroptosis residues. The Percolator node was used to estimate the confidence of peptide identifications at 1% false discovery rate (FDR) and was based on the q-value and a decoy database search. The spectra were searched against UniProt annotated reference proteomes of Homo sapiens and E. coli. The reporter ion quantifier node included a TMT-11-plex quantification method with an integration window tolerance of 15 ppm and integration method based on the most confident centroid peak at MS3 level. Quantification was performed using unique peptides only, with protein groups considered for peptide uniqueness. Proteomics data used for statistical analysis were scaled and then normalised by log 2 transformation of the fold change to uninfected control. Statistical analysis All experiments except proteomics were independently repeated at least 3 times as indicated in the figure legends. Proteomics were independently repeated twice. For qRT-PCR, high-con- tent imaging analysis and CFU assays, 2 to 3 technical repeats were performed to calculate means for each biological repeat. For manual cell counting in microscopy images, at least 4 images from randomly selected positions in each coverslip containing a total of 50 to 100 cells (over 250 cells from 4 biological repeats) were used. For high-content image analysis, 25 images evenly distributed in predefined positions containing a total of over 100 cells in each well were used. Methods of data transformation are described in their corresponding method sections. Statistical analysis of all biochemical/biological experimental data was performed using GraphPad Prism 5.1. Student t test and 1-way or 2-way ANOVA followed by Tukey posttest or Bonferroni posttest, respectively, were performed on the means as listed in the figure legends. Significant result was defined as having a p-value of <0.05. Statistical analysis, including PCA, ANOVA, t test, and pathway enrichment analysis of the proteomic data, was performed in Perseus 1.6.2.2 (Max Planck Institute of Biochemistry, Ger- many) [86]. Pathway enrichment was performed using Uniprot Keywords, Gene Ontology Biological Process (GOBP) and KEGG pathways [87]. For transcription factor analysis, the “TF-LOF Expression from GEO” library was downloaded from the Enrichr web tool [88] and was converted to Perseus-compatible annotation file for 1D annotation enrichment analysis. All biological terms were filtered for Benjamini–Hochberg FDR < 0.05. Heatmaps were plotted in Phantasus (https://artyomovlab.wustl.edu/phantasus/), and boxplots were plotted in R-Studio. S1 Data. Excel spreadsheet containing the numerical data used for the main and Support- ing information figures. S1 Data. Excel spreadsheet containing the numerical data used for the main and Support- ing information figures. PLOS Biology | https://doi.org/10.1371/journal.pbio.3000986 December 30, 2020 S1 Raw Images. Raw images of the western blots presented in Fig 2E, 2F and 2I–2K and S2A, S2G and S2I Fig. (PDF) S1 Fig. Kinetics of Tir-induced cell death throughout 8 h of infection. (A) Gbp2 expression level was measured by qRT-PCR in IFNγ-primed cells and normalised to the Gbp2 expression level in the untreated cells. Means ± SEM from n = 3 independent biological repeats. Statistical significance was determined using 2-tailed t test.  p  0.05. (B) PI uptake into unprimed SNU-C5 cells infected with EPEC WT, EPEC-0 and EPEC-1. Measurements were taken every 10 min. The time-course PI uptake was plotted. PI uptake results were normalised by UI. Means ± SEM from n = 5 independent biological repeats are shown. (C) PI uptake into IFNγ- PLOS Biology | https://doi.org/10.1371/journal.pbio.3000986 December 30, 2020 24 / 31 PLOS BIOLOGY EPEC Tir triggers calcium influx-dependent pyroptosis primed uninfected cells. PI uptake results were normalised to unprimed UI cells. Means ± SEM from n = 7 independent biological repeats are shown. The underlying data for this figure can be found in S1 Data. EPEC, enteropathogenic Escherichia coli; IFNγ, interferon gamma; PI, propidium iodide; qRT-PCR, real-time quantitative PCR; SEM, standard error of the mean; Tir, translocated intimin receptor; UI, uninfected; WT, wild-type. (TIF) S2 Fig. Apoptosis, NLRP3-dependent pyroptosis and necroptosis do not occur during EPEC-1 infection. (A) Cell lysates of primed SNU-C5 cells infected with EPEC WT, EPEC-0, and EPEC-1, or treated by STS for 20 h, were used for PARP1 western blot. Representative blots were shown. (B) PI uptake into THP1 cells treated by LPS for 3 h followed by nigericin for 8 h, with or without MCC950 treatment 30 min before nigericin addition. Means ± SEM from n = 3 independent biological repeats. (C) PI uptake into primed SNU-C5 cells infected with EPEC-1 treated with MCC950 30 min before infection. Means ± SEM from n = 3 inde- pendent biological repeats. (D, E) PI uptake into SNU-C5 cells treated by YVAD, Nec1 and NSA 30 min before LPS transfection (D) or STS and zVAD treatment (E). Means ± SEM from n = 3 independent biological repeats. (F) PI uptake into SNU-C5 cells infected with EPEC-1 treated with Nec1, NSA, zVAD, and a combination of zVAD, Nec1, and NSA 30 min before infection. Means ± SEM from n = 3 independent biological repeats. (G) Caspase-8 western blot of SNU-C5 cells transfected with caspase-8 siRNA. Representative blot from n = 3 independent biological repeats are shown. S1 Raw Images. Raw images of the western blots presented in Fig 2E, 2F and 2I–2K and S2A, S2G and S2I Fig. (PDF) (H) PI uptake into SNU-C5 cells transfected by caspase-4, GSDMD or caspase-8 siRNA or combinations of them and infected with EPEC-1. Means ± SEM from n = 3 independent biological repeats. (I) RIPK3 western blot of SNU-C5 cells transfected with RIPK3 siRNA. Representative blot from n = 3 independent biological repeats are shown. (J) PI uptake into SNU-C5 cells transfected by RIPK3 siRNA treated by STS and zVAD. Means ± SEM from n = 3 independent biological repeats. (K) PI uptake into SNU-C5 cells transfected by caspase-4, GSDMD or RIPK3 siRNA or combinations of them and infected with EPEC-1. Means ± SEM from n = 3 independent biological repeats. Statistical significance was determined using 2-tailed t test (B, C, J) and 1-way ANOVA with Tukey post- test (D, E, F, H, K).  p  0.05;  p  0.01;  p  0.001. The underlying data for this figure can be found in S1 Data. ANOVA, analysis of variance; EPEC, enteropathogenic Escherichia coli; GSDMD, gasdermin D; LPS, lipopolysaccharide; NLRP3, NLR family pyrin domain con- taining 3; NSA, necrosulfonamide; PARP1, poly [ADP-ribose] polymerase 1; PI, propidium iodide; SEM, standard error of the mean; siRNA, small interfering RNA; STS, staurosporine; WT, wild-type; YVAD, z-YVAD-fmk; zVAD, z-VAD-fmk. ( ) S3 Fig. EPEC adheres to and Tir induces actin polymerisation in SNU-C5, HT-29, SNU-C2B and COLO-320-HSR. Immunofluorescence staining of SNU-C5, HT-29, SNU-C2B and COLO-320-HSR cells were infected with EPEC-0 and EPEC-1 for 4 h. DAPI: blue; EPEC: green; Phalloidin: red. Representative images from n = 3 independent biological repeats are shown. Error bar: 5 μm. The underlying data for this figure can be found in S1 Data. EPEC, enteropathogenic Escherichia coli; Tir, translocated intimin receptor. (TIF) S3 Fig. EPEC adheres to and Tir induces actin polymerisation in SNU-C5, HT-29, SNU-C2B and COLO-320-HSR. Immunofluorescence staining of SNU-C5, HT-29, SNU-C2B and COLO-320-HSR cells were infected with EPEC-0 and EPEC-1 for 4 h. DAPI: blue; EPEC: green; Phalloidin: red. Representative images from n = 3 independent biological repeats are shown. Error bar: 5 μm. The underlying data for this figure can be found in S1 Data. EPEC, enteropathogenic Escherichia coli; Tir, translocated intimin receptor. (TIF) S4 Fig. Tir-dependent cell death in unprimed SNU-C5 is inhibited by YVAD and EGTA. (A, B) PI uptake into unprimed SNU-C5 cells infected with EPEC-1 with 30 min pretreatment with YVAD (A) or EGTA (B). PLOS Biology | https://doi.org/10.1371/journal.pbio.3000986 December 30, 2020 S1 Raw Images. Raw images of the western blots presented in Fig 2E, 2F and 2I–2K and S2A, S2G and S2I Fig. (PDF) Means ± SEM from n = 3 independent biological repeats. (C) PI uptake into unprimed SNU-C5 cells infected with EPEC-0, EPEC-2, EPEC-1-TirAA-EspZ, EPEC-1 and EPEC-1-TirAA. Means ± SEM from n = 5 independent biological. Statistical significance was S4 Fig. Tir-dependent cell death in unprimed SNU-C5 is inhibited by YVAD and EGTA. (A, B) PI uptake into unprimed SNU-C5 cells infected with EPEC-1 with 30 min pretreatment with YVAD (A) or EGTA (B). Means ± SEM from n = 3 independent biological repeats. (C) PI uptake into unprimed SNU-C5 cells infected with EPEC-0, EPEC-2, EPEC-1-TirAA-EspZ, EPEC-1 and EPEC-1-TirAA. Means ± SEM from n = 5 independent biological. Statistical significance was 25 / 31 PLOS Biology | https://doi.org/10.1371/journal.pbio.3000986 December 30, 2020 PLOS BIOLOGY EPEC Tir triggers calcium influx-dependent pyroptosis determined using 2-tailed t test (A, B) and 1-way ANOVA with Tukey posttest (C).  p  0.05;  p  0.01;  p  0.001. The underlying data for this figure can be found in S1 Data. ANOVA, analysis of variance; EPEC, enteropathogenic Escherichia coli; PI, propidium iodide; SEM, stan- dard error of the mean;Tir, translocated intimin receptor; YVAD, z-YVAD-fmk. (TIF) S5 Fig. Illustration of the proteomics workflow used in this study. (A) IFNγ-primed and unprimed SNU-C5 cells were infected with the indicated EPEC strains for 2 h. Extracted pro- teins were digested with trypsin and peptides were labelled with the TMT 11plex reagents in 2 separate replicate batches. TMT-labelled peptides were subjected to offline high-pH reversed- phase fractionation followed by LC–MS analysis. (B) PCA of proteomics data. EPEC, entero- pathogenic Escherichia coli; IFNγ, interferon gamma; LC–MS, liquid chromatography–mass spectrometry; PCA, principal component analysis; TMT, tandem mass tag. (TIF) S5 Fig. Illustration of the proteomics workflow used in this study. (A) IFNγ-primed and unprimed SNU-C5 cells were infected with the indicated EPEC strains for 2 h. Extracted pro- teins were digested with trypsin and peptides were labelled with the TMT 11plex reagents in 2 separate replicate batches. TMT-labelled peptides were subjected to offline high-pH reversed- phase fractionation followed by LC–MS analysis. (B) PCA of proteomics data. EPEC, entero- pathogenic Escherichia coli; IFNγ, interferon gamma; LC–MS, liquid chromatography–mass spectrometry; PCA, principal component analysis; TMT, tandem mass tag. (TIF) S6 Fig. Proteomic differences of IFNγ-primed and unprimed cells. Heatmap of the differen- tially regulated proteins between IFNγ-primed and unprimed SNU-C5 cells (t test, FDR < 0.05, absolute log2 ratio versus uninfected > 0.5). S1 Raw Images. Raw images of the western blots presented in Fig 2E, 2F and 2I–2K and S2A, S2G and S2I Fig. (PDF) The underlying data for this figure can be found in S1 Data. FDR, false discovery rate; IFNγ, interferon gamma. (TIF) S7 Fig. Kinetics of Tir-induced Ca2+ influx over 1 h of infection. Fluo-4 assay performed on primed (A, C) and unprimed (B) SNU-C5 cells infected with EPEC-0 (A, B), EPEC-1 (A-C) and EPEC-1-TirAA (A, B) with or without 30 min pretreatment with EGTA (A, B) and YVAD (C). Means ± SEM from n = 3 independent biological repeats. The underlying data for this fig- ure can be found in S1 Data. EPEC, enteropathogenic Escherichia coli; SEM, standard error of the mean; Tir, translocated intimin receptor; YVAD, z-YVAD-fmk. (TIF) S8 Fig. EPEC-1 induces cell death independent of ROS- or mitochondrial respiration- dependent cell death. (A–C) PI uptake into primed SNU-C5 cells infected with EPEC-1, with 30 min pretreatment with DMTU (A), Tiron (B) or oligomycin (C). Means ± SEM from n = 3 independent biological. (D) OCR measurement was performed on SNU-C5 cells before or after oligomycin treatment. Means ± SEM from n = 3 independent biological repeats are shown. Statistical significance was determined using 2-tailed t test. ns, nonsignificant;  p  0.05;  p  0.01;  p  0.001. The underlying data for this figure can be found in S1 Data. DMTU, N,N0-dimethylthiourea; EPEC, enteropathogenic Escherichia coli; OCR, oxygen consumption rate; PI, propidium iodide; SEM, standard error of the mean. (TIF) S1 Table. Lists of strains, plasmids, oligonucleotide sequences, and antibodies used in this study. (A) Bacterial strains. (B) Plasmids. (C) Primer sequences. (D) siRNA and miRNA30E sequences. (E) Immunofluorescence antibodies and reagents. (F) Western blot antibodies. siRNA, small interfering RNA. (DOCX) Author Contributions Author Contributions Conceptualization: Gad Frankel, Jyoti S. Choudhary. Data curation: Qiyun Zhong, Theodoros I. Roumeliotis, Zuza Kozik. Formal analysis: Qiyun Zhong, Theodoros I. Roumeliotis, Zuza Kozik, Chris Bakal. Funding acquisition: Gad Frankel, Jyoti S. Choudhary. Investigation: Qiyun Zhong, Theodoros I. Roumeliotis, Zuza Kozik. Methodology: Qiyun Zhong, Theodoros I. Roumeliotis, Zuza Kozik, Massiel Cepeda-Molero, Avinash R. Shenoy, Chris Bakal, Gad Frankel, Jyoti S. Choudhary. Project administration: Gad Frankel, Jyoti S. Choudhary. Supervision: Theodoros I. Roumeliotis, Luis A´ngel Ferna´ndez, Avinash R. Shenoy, Chris Bakal Gad Frankel Jyoti S Choudhary Conceptualization: Gad Frankel, Jyoti S. Choudhary. Data curation: Qiyun Zhong, Theodoros I. Roumeliotis, Zuza Kozik. Formal analysis: Qiyun Zhong, Theodoros I. Roumeliotis, Zuza Kozik, Chris Bakal. Funding acquisition: Gad Frankel, Jyoti S. Choudhary. Investigation: Qiyun Zhong, Theodoros I. Roumeliotis, Zuza Kozik. Methodology: Qiyun Zhong, Theodoros I. Roumeliotis, Zuza Kozik, Massiel Cepeda-Molero, Avinash R. Shenoy, Chris Bakal, Gad Frankel, Jyoti S. Choudhary. Project administration: Gad Frankel, Jyoti S. Choudhary. Supervision: Theodoros I. Roumeliotis, Luis A´ngel Ferna´ndez, Avinash R. Shenoy, Chris Bakal, Gad Frankel, Jyoti S. Choudhary. Validation: Qiyun Zhong. Validation: Qiyun Zhong. Visualization: Qiyun Zhong, Theodoros I. Roumeliotis. Visualization: Qiyun Zhong, Theodoros I. Roumeliotis. Writing – original draft: Qiyun Zhong, Theodoros I. Roumeliotis, Zuza Kozik, Gad Frankel, Jyoti S. Choudhary. Writing – review & editing: Qiyun Zhong, Theodoros I. Roumeliotis, Zuza Kozik, Avinash R. Shenoy, Chris Bakal, Gad Frankel, Jyoti S. Choudhary. Acknowledgments We thank Enkai Jin (MRC CMBI) for EPEC binding assays, Gerald Larrouy-Maumus for pro- viding access of the Agilent Seahorse XFp Analyzer, the CMBI High throughput single-cell analysis facility (HTSCAF), and Pascal Meier for critical reading of the manuscript. PLOS Biology | https://doi.org/10.1371/journal.pbio.3000986 December 30, 2020 26 / 31 PLOS BIOLOGY EPEC Tir triggers calcium influx-dependent pyroptosis Author Contributions Conceptualization: Gad Frankel, Jyoti S. Choudhary. Data curation: Qiyun Zhong, Theodoros I. Roumeliotis, Zuza Kozik. Formal analysis: Qiyun Zhong, Theodoros I. Roumeliotis, Zuza Kozik, Chris Bakal. Funding acquisition: Gad Frankel, Jyoti S. Choudhary. Investigation: Qiyun Zhong, Theodoros I. Roumeliotis, Zuza Kozik. Methodology: Qiyun Zhong, Theodoros I. Roumeliotis, Zuza Kozik, Massiel Cepeda-Molero, Avinash R. Shenoy, Chris Bakal, Gad Frankel, Jyoti S. Choudhary. Project administration: Gad Frankel, Jyoti S. Choudhary. Supervision: Theodoros I. Roumeliotis, Luis A´ngel Ferna´ndez, Avinash R. Shenoy, Chris Bakal, Gad Frankel, Jyoti S. Choudhary. Validation: Qiyun Zhong. Visualization: Qiyun Zhong, Theodoros I. Roumeliotis. Writing – original draft: Qiyun Zhong, Theodoros I. Roumeliotis, Zuza Kozik, Gad Frankel, Jyoti S. Choudhary. Writing – review & editing: Qiyun Zhong, Theodoros I. Roumeliotis, Zuza Kozik, Avinash R. Shenoy, Chris Bakal, Gad Frankel, Jyoti S. Choudhary. PLOS Biology | https://doi.org/10.1371/journal.pbio.3000986 December 30, 2020 References Adhesion of enteropathogenic Escherichia coli to human intestinal enterocytes and cultured human intestinal mucosa. Infect Immun. 1987; 55(1):69–77. https://doi.org/ 10.1128/IAI.55.1.69-77.1987 PMID: 3539808 14. Kenny B, DeVinney R, Stein M, Reinscheid DJ, Frey EA, Finlay BB. Enteropathogenic E. coli (EPEC) transfers its receptor for intimate adherence into mammalian cells. 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Notes ON THE SUPERSTITIONS OF MENSTRUATION.
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ANCIENT VIEWS ON MENSTRUATION. The wise men of old stood baffled before the mystery of the menses. Pythagoras called menstrual blood the froth of the blood, and held that it con- tained the superfluity of the aliment. Aristotle explained menstruation as a process necessary to get rid of the excess (7r&eacute;p&iacute;TTWJLU) of blood that had accumulated in the interior of the body of the woman, and his theory is ardently supported 1500 years later by his great commentator Averrhoes. Can it be honestly said that, apart from some suggestive evidence as to the interdependence of ovarian activity and catamenial function, medicine of to-day has any more satisfying explanation of the purpose of menstruation than theirs? purpose Where science has failed to establish a proof religion has seldom failed to establish a theory. The Levitical ritual was in no doubt that impurities collected in the woman’s blood were discharged in the menstrual flux, so that she underwent a recur- ring series of purifications. To the men of the monasteries menstruation was a symbol of the essential sinfulness and inferiority of woman, polluted alike and polluting. Men of such widely different stamp as Paracelsus and de Graaf both upheld this view of a menstrual purification, and I seem to hear some echo of it in the modern sug- gestion that the uterine mucosa, by expulsion of its surface layer, is preparing a sterilised bed for the fertilised ovum. Laboriously acquired histo- logical knowledge competes on unequal terms with a plausible bacteriological preconception. MAGICAL BELIEFS. Back of all these scientific and religious theories, and not infrequently casting a shadow upon them, stands a congeries of magical beliefs, which constitute the main subject of our present inquiry. The menstruous woman is possessed by an evil spirit; the spirit resides in her blood, and by the medium of her menstrual blood may exert its infiuence, for good or for harm, on her environ- ment. The evil spirit may effect its entry into the woman in the form of a bird, or a, lizard, or a serpent; hence the folk association of these animals with menstruation. It was the serpent that marred the primordial bliss of Eden. In Portugal women during menstruation wear drawers to prevent the access of lizards. On the Pennefather river the menses are said to be produced by a curlew operating on woman. , PHYSICIAN TO KING’S COLLEGE HOSPITAL AND KING GEORGE’S MILITARY HOSPITAL; FELLOW OF KING’S COLLEGE, LONDON, ETC. SUPERSTITIONS concerning the menses loom so large in the customs of primitive peoples and are so far from extinct among the lower orders of civilised nations as to be worthy of serious consideration. Many a farmer’s wife in this country will still assure you that milk handled by a menstruous woman cannot be churned to butter or that hams will not take salt at her hands. That she has demonstrated the contrary in a series of experiments, month by month, year in and year out, has left unshaken the pillars of her faith: the foundations of faith stand firm in shifting soil. operating It is of the nature of magic, having postulated an all-sufficing cause, to take but little account of the intervening steps in the production of its effect. But in the matter of menstruation it has not been wholly silent. The natives on the Tully river in North Queensland attribute it to the breaking and discharge of the liver; what causes the breakage they do not know, but believe that menstruation has nothing to do with pregnancy. They are so far right in this that it is quite certain repeated pregnancy may occur in those who have never menstruated. Segregation of women from the men during menstruation would lend colour to the belief that menstruation is not necessarily con- nected with conception. Among the Sandwich Islanders menstruation is believed to be the result of coition. Notes ON THE SUPERSTITIONS OF MENSTRUATION. We see how it came about that Aristotle applied to the menses the self-same explanation of a super- fluity, that Hippocrates had adopted in the case of the male semen. DECEMBER 18, 1915. DECEMBER 18, 1915. never takes place. Women of this nature, however, are incapable of bearing children, because it is of this substance that the infant is formed. The seed of the male, acting as a sort of leaven, causes it to unite and assume a form, and in due time it acquires life and assumes a bodily shape. Notes ON THE SUPERSTITIONS OF MENSTRUATION. Read before the Historical Section of the Royal Society of Medicine, on Dec. 15th, 1915, BY RAYMOND CRAWFURD, M.A., M.D.OXON., F.R.C.P. LOND., PHYSICIAN TO KING’S COLLEGE HOSPITAL AND KING GEORGE’S MILITARY HOSPITAL; FELLOW OF KING’S COLLEGE, LONDON, ETC. SUPERSTITIONS CONNECTED WITH THE MILK- SUPPLY. Many superstitions centre around the milk- supply ; much light has been thrown upon them by the masterly handling of Frazer.3 Among the kaffir tribes of South Africa the menstruous woman must not drink milk, for if she do the cows would die. Even the maidens who attend a girl at her first menstruation are forbidden to drink milk for the same reason. Seclusion of the woman from the cattle and the taboo on milk last a fortnight, double the period demanded in the case of many other prohibitions. prohibitions. Among the Bahimas a woman may not drink milk at her monthly.times, but must eat vegetables and drink beer; the vegetarian diet implies a taboo on the flesh of the animals that yield the milk. Exception is made in favour of a girl at her first menstruation; her father then sets apart for her use an old cow, which is of small value, as it will soon pass out of milk. The conception that underlies these prohibitions is that of a sympathetic bond between the cow and its milk, which abides after the milk has left its body. Harm done to the milk inevitably involves the cow. plausible bacteriological preconception. It must be remembered that till the middle of the seventeenth century, when the work of Harvey, de Graaf, and other pioneer embryologists led up to the discovery of the ovum in the human female, it was generally believed that the male semen and the female menses were the constituents out of which the child was formed, and by most the leading part was assigned to the menses. Pliny defines the general belief in quite unequivocal language :- No 4816 g q q g g The .discharge, which is productive of such great and singular effects, occurs in women every thirty days, and in a greater degree every three months. In some individuals it occurs oftener than once a month, and in others again it inevitably Again a menstruous woman is forbidden to cross those parts of the kraal frequented by the cattle, for if a drop of blood were to fall on such a path 1332 and in Sumatra they are forbidden the rice fields for fear of damaging the crop. BELIEFS ABOUT THE CROPS. Superstitions such as we have already cited are in the nature of things most prevalent among the unsophisticated children of the soil, and wear a rural aspect. Others centre about the crops. Thus Pliny 4 says :- y y If a woman strips herself naked while she is menstruating and walks round a field of wheat, the caterpillars, worms, beetles, and other vermin will fall off from the ears of corn. Metrodorus, of Scepsos, tells us that the discovery was first made in Cappadocia, and that, in consequence of such multitudes of cantharides being found to breed there, it is the practice for women to walk through the middle of the fields with their garments tucked up above the thighs. In other places again it is the usage for women to go barefoot, with the hair dishevelled and the girdle loose ; due precau- tion must be taken, however, that this is not done at sunrise, for, if so, the crop would wither and dry up. y A mare big with foal, if touched by a woman in this state, will be sure to miscarry; nay, even more than this, at the very sight of a woman, though seen even at a distance, should she happen to be menstruating for the first time after the loss of her virginity, or for the first time while in a state of virginity. So pernicious are the properties of the menstrual discharge that women themselves, the source from which it is derived, are far from being proof against its effects : a pregnant woman, for instance, if touched with it, or if indeed she so much as steps over it, will be liable to miscarry.15 . Columella and Aelian both testify to the efficacy of this procedure, which finds a modern echo in Hiawatha’s injunction to Minnehaha :- Pliny further records its value as an ingredient of abortifacient remedies and as a means of pre- venting impregnation. We have the recent testi- mony of Dr. T. G. Morton, of Philadelphia, that ignorant female abortionists still vend a nostrum for defeating pregnancy, one of the component parts of which is catamenial fluid. SUPERSTITIONS CONNECTED WITH THE MILK- SUPPLY. The huts of a kafnr kraal are usually arranged in a circle with the cattle-fold in the centre, and the circuitous paths taken by the women, in passing from hut to hut, may be seen outside the circle of huts, between them and the palisade which sometimes surrounds the kraal; but there is no such restriction on the walks of women who are past child-bearing, as they have ceased to be a source of danger. Nor is it only menstrual blood that is feared, for a Zulu custom forbids a wounded man to drink milk until he has submitted to a prescribed ritual. p esc bed Among many pastoral tribes of South Africa and India, women are either entirely prohibited from milking the cows, or are permitted to do so only in accordance with certain strict rules of ceremonial purification. Among the Todas of South India the men alone undertake the operations of milking and churning. The women may go to the dairy to fetch butter-milk, but they must approach it by an appointed path and stand at an appointed place to receive the milk. Such scrupulous care to shield the cattle and their milk from harm is commensurate with their importance in the economy of a pastoral people. It is interesting to encounter these primi- tive attempts at the provision of a pure milk- supply, and to consider the different character of the impurities to be feared, then and now. SUPERSTITIONS CONNECTED WITH THE MILK- SUPPLY. At Saigon no woman is employed in the opium factory lest she should turn the opium bitter, while Annamese women say that it is very difficult for them to prepare opium pipes during the catamenial period." Even the lowlier forms of life are activated by this uncanny influence, so that beer and wine turn sour and vinegar will not keep. Clearly in this state she must be secluded from the food-supply. We have seen that she is forbidden to take milk or meat at these times for fear of damaging the flocks. Some Central Australian 10 tribes will not allow a menstruous woman to gather a certain bulb, which is a staple food, for fear the crop of bulbs should fail. Again, no woman was formerly admitted to the great sugar refineries in the North of France during the boiling or cooling processes on the ground that the sugar would blacken if a woman were to enter during her period." In Syria to this day women are not allowed to salt or pickle when their courses are on them, as it is believed that whatever they salt or pickle will not keep ; and in Brunswick people think that if a menstruous woman assists at the killing of a pig the flesh will putrefy.l any cow passing over it would run great risk of dying from disease. The huts of a kafnr kraal are usually arranged in a circle with the cattle-fold in the centre, and the circuitous paths taken by the women, in passing from hut to hut, may be seen outside the circle of huts, between them and the palisade which sometimes surrounds the kraal; but there is no such restriction on the walks of women who are past child-bearing, as they have ceased to be a source of danger. Nor is it only menstrual blood that is feared, for a Zulu custom forbids a wounded man to drink milk until he has submitted to a prescribed ritual. any cow passing over it would run great risk of dying from disease. MALIGN INFLUENCE ON ANIMAL LIFE. With an ever-widening circumference the malign influence affects all living things about the home- stead. Bees will forsake their hives at her touch, for they have a special aversion to a thief and a menstruous woman, and a glance of her eyes suffices to kill a swarm of bees (Pliny- Among the Indians an Omaha 14 woman may not approach a horse during her courses or she will grievously harm the animal. Pliny says :- BELIEFS ABOUT THE CROPS. That it should possess this powerful ecbolic faculty is a mere matter of sympathetic influence, and for the same reason- j You shall bless to-night the cornfields, Draw a magic circle round them To protect them from destruction, Blast of mildew, blight of insect. In the night, when all is darkness, Rise up from your bed in silence, Lay aside your garments wholly, Walk around the fields you planted, Round the borders of the cornfields, Covered with your tresses only, Robed with darkness as a garment. co e ds, Covered with your tresses only, Reduced to ashes and mixed with oil of roses it is very useful; applied to the forehead, for allaying headache, particularly in women (Pliny).17 your only, Robed with darkness as a garment. For the most part, however, the influence of the menstruous woman on vegetation is harmful. According to Pliny- particularly (Pliny).17 Dogs which may have tasted of the matter so discharged are seized with madness, and their bite is venomous and incurable. H Seeds which are touched by her become sterile, grafts wither away, garden plants are parched up, and the fruit will fall from the tree beneath which she sits. 6 And again in a later passage Pliny says :- CUSTOMS AMONG PRIMITIVE YEOPLES. ( y ) The powers of Nature even own the menstrual sway- CUSTOMS AMONG PRIMITIVE YEOPLES. Among the Australian 26 blacks the boys are taught from early childhood that if they set eyes on menstruous blood their hair will turn grey and their vigour abate prematurely. Consequently, the women must live apart at these times, and if a boy 3r young man is seen to approach she must call 3ut at once, so that he may avoid her; he must not 3ven cross the tracks made by women at such times. rhe woman is forbidden under pain of death to jouch anything that the men use or to walk on ihe paths they frequent. An Australian black milled his wife when he found that she had lain on lis blanket during her menstrual period and died )f terror within a fortnight. Among the Bhinyan )f South Mirzapur every house has two doors, one )f which is used exclusively by the women during nenstruation. These superstitious terrors help is to appreciate the mentality of those eminent cientists who find the contiguity of a women an mpediment to serious work, and who knows but hat their unconscious influence may explain the rade unions hostility to the collaboration of vomen in the workshop. For in the first place hailstorms, they say, whirlwinds and lightning even will be scared away by a woman uncovering her body, while her monthly courses are upon her. The same too with all other kinds of tempestuous weather ; and out at sea a storm may be lulled by a woman merely uncovering her body, even though not menstruating at the time (Pliny 2). Seasonal and other influences are held to heighten the virulence of the discharge. Aelian lays it down that during menstruation woman is in communica- tion with the starry bodies. Aristotle 24 says that wiseacres assert that the moon is female, because the menstrual flux and the waning of the moon both take place towards the end of the month, and after the wane and the discharge both become whole again. Yet one more instance may be taken from Pliny of its influence on animal life :- p pp g The menstruating woman has the divine gift of kings, for by her touch she can cure not only scrofulous ulcers, but " imposthumes of the parotid glands, inflamed tumours, erysipelas, boils, and defluxions of the eyes." 26 Avicenna and Pliny both recommend menstrual blood as an application to all sores. In Bavaria it is held even to have prophy- lactic power against wounds and to be able to extinguish fires. It was given internally for erysipelas and calculi, and was esteemed a good cosmetic to drive away pimples. In the Middle Ages it was a sovereign cure for leprosy.2.> That the ant, an insect so extremely minute, is sensible of its presence and rejects the grains, which it has been carrying, and will not return to them again.2" CUSTOMS AMONG PRIMITIVE YEOPLES. According to Pliny- g g y If the menstrual discharge coincides with an eclipse of the moon or sun, the evils resulting from it are irremediable ; and no less so, when it happens while the moon is in con- junction with the sun ; congress with a woman at such a period being noxious and attended with fatal ejects to the man. At this period also, the lustre of purple is tarnished by the touch of a woman, so much more baneful is her influence at this time than at any other. 23 s t 1 y ] a ] j l E p Among the people of the Lower Congo 27 a woman, uring menstruation, may not cook her husband’s ood nor any other man’s, neither anything belong- ng to men, and must not return the salutations of ny man. If a woman in this condition has to pass ear some men who are likely to give her the quivalents of "Good morning" or "Good even- 19" " she will deliberately put her pipe in her iouth as a sign that she cannot answer because lie is unclean. Virginity too exerts a special power. Pliny say that ’’ the nature of the discharge is most virulen in females, whose virginity has been lost solely by lapse of time." 28 The first menstruation also, as well as the first after loss of virginity, possess a special potency. And again in a later passage Pliny says :- g y It is universally agreed that, when a person has been bitten by a dog and manifests a dread of water and of all kinds of drink, it will be quite sufficient to put under his cup a strip of cloth that has been dipped in this fluid, the result heing that hydrophobia will immediately disappear. This arises, no doubt, from that powerful sympathy which has been so much spoken of by the Greeks, and the existence of which is proved by the fact already mentioned, that dogs become mad on tasting this fluid." Young vines, too, it is said, are injured irremediably by the touch of a woman in this state ; and both rue and ivy, plants possessed of highly medicinal virtues, will die instantly upon being touched by her. "( upon g by All plants will turn of a yellow complexion on the approach of a woman who has the menstrual discharge upon her. 8 Frazer says that in Galela women at their monthly periods may not enter a tobacco field, OTHER PHENOMENA ATTRIBUTED TO THE CATAMENIAL STATE. Household articles enjoy no immunity. Aristotle ,says that the glance of a menstruous woman takes the polish out of a mirror and that the next person looking into it will be bewitched. Pliny says that the tarnished mirror will recover its bright- ness if the same woman looks steadily upon the back of it. Her glance also blunts the edge of steel and takes away the polish from ivory. Lausent 21 enumerates a whole catalogue of un- canny phenomena attributed to the catamenial state. Two girls believed that their shoes, stockings, and clothes stuck to them so closely that they needed help to remove them. There are many tales of the snapping of strings of harps and violins during the menses; also of the readiness with which women break things at these times, needles snapping, glasses breaking, and clocks stopping if touched. Iron wiil become rusty on contact with her, copper coated with verdigris, razors blunted, and linen boiling in the cauldron will turn black (Pliny 22). ges g leprosy.2. The wise women of Pliny’s day recommended it to be placed on a thread or the wool of a black sheep and worn in a bracelet as an amulet in tertian or quartan fevers. The midwife Sotira considered that better results were obtained by rubbing the soles of the patient’s feet with it, and if possible this should be done by the woman her- self without the patient’s knowledge. She had also proved the efficacy of this method in reviving a person from an epileptic seizure. For quartan fever Ictidas, the physician, recommended sexual intercourse with a woman just beginning to men- struate. This is but one instance of the profound influence on males attributed to the menstrual state. water and laid on the part; the discharge was also used as a topical application in acute gout. Yet one more instance may be taken from Pliny of its influence on animal life :- CURATIVE PROPERTIES, when about to compound their medicines, were in the habit of making a saving clause that the remedy would be effective, provided no menstruating woman approached their chamber during the com- pounding process. Surely nowadays the altogether inordinate apprehensions of both nurse and surgeon as to the evils of operation during the menstrual state are tinctured witli the savour of this same superstitious fear. superstitious Among the native Australians 2H the woman may not eat fish, or cross the water, or even go near it, or it will spoil the fishermen’s luck; she may not even fetch water for the camp. Mrs. James Smith, in her account of the Roandik tribes, says that men have a special objection to setting eyes on menstrual blood, because it would enfeeble them in their fight against enemies and they would be killed; and the Aruntas 2D of Central Australia believe that a draught of woman’s blood would kill the strongest man. In Polynesia the vessels, gar- ments, and other property of a menstruating woman may not be used by others on pain of disease and death. proven The ordinances of religion, with regard to the segregation of the menstruous woman, fall into line with the customs of magic. As Frazer has said, the divinely inspired law-givers mistook their own ideas for revelations from heaven. The Hindoo law-giver, Manu, who professed to have received his institutes from the Creator, Brahman, informs us that the wisdom, the energy, the strength, the sight, and the vitality of a man who approaches a woman in her courses will utterly perish; whereas, if he avoids her, his wisdom, energy, strength, sight, and vitality, will all increase. i LOVE CHARMS AND OTHER MAGICAL POWERS. Menstrual fluid has always been reckoned to possess a powerful influence over the affections of men, and has held pride of place as an ingredient of the love-charms administered by girls to would-be lovers. CURATIVE PROPERTIES, In modern Germany girls have administered drops of menstrual blood in coffee to their sweet- hearts to make sure of retaining their affections- a characteristic display of " frightfulness." Accord- ing to Pliny its action is effective on either sex, for " if a man takes a frog and transfixes it with a reed entering its body at the sexual parts and coming out of the mouth, and then dips the reed in the menstrual discharge of his wife, she will be sure to conceive an aversion for all paramours." So, too, the Persian law-giver, Zarathustra, who claims to have derived his code from the mouth of the Supreme Being, Ahura Mazda, declares menstruation to be the work of Ahriman, the Devil, and lays down injunctions similar to those which the Hebrew law-giver Moses claimed to have derived from Jehovah. THE PURPOSE OF THE TABOOS. Just as the horror of menstrual blood is merely a special aspect of the horror of blood in general, so the taboos by which it is sought to segregate the menstrual woman are but a special application of the taboos by which any other supernatural agent undergoes segregation. The purpose that underlies them all is the isolation of the tabooed person from society. The primitive mind discriminates little between good and evil spirits, between holiness and pollution, for in each condition there exists a powerful supernatural force, be it active for good or for evil. So comes it, as Frazer shows, that the same taboos as apply to the menstruous woman are applied to divine kings or priests. Thus in many primitive races the divine personage may not touch the ground with his foot, and the sun may not shine upon him. We find him carried on men’s shoulders, or stepping only on carpets or mats ; and in many nations confined in palace, temple, or underground dwelling: the Mikado of Japan formerly observed both these taboos. Nowhere is he so free from danger and so harmless, as suspended between heaven and earth, in contact with neither. In electrical language he is insulated. He can neither discharge his supernatural energy with harmful violence, nor can it be drained from him to the detriment of that magical power on which the well-being of his people depends. paramours. Menstrual fluid has other intimate relations to magic. Thus Pliny says that if it is sprinkled on the door-posts it will render ineffectual all the spells of the magicians. Josephus speaks of plants acquiring magical properties by watering them with menstruous blood and the urine of a woman-a radio-active influence, no doubt! and by sym- pathetic affinity stains made on a garment by menstrual blood can only be removed by the urine of the same woman. CURATIVE PROPERTIES, Among the beneficent properties of the menstrua discharge must be ranked its power to cure various ailments. The doctrine that the various secretions and excretions are imbued with the same properties as the body from which they are liberated led to their wholesale adoption into the primitive phar- Diacopoeia. The scatophagy, at which Aristophanes jibed, was the parent of modern organotherapy. The first napkin used by a healthy virgin was preserved for use in cases of plague, malignant carbuncles. and other diseases ; it was damped with ir J 1 v f: c p t k If in this state she will pollute mere man, still tore will she pollute a sacred man. A menstruating swish woman was formerly forbidden to shake ands with a Rabbi, and women at these times ere excluded from the Jewish synagogues and om the communion table of the early Christian Lurch. The Parsees, who reverence fire, will not ermit a woman then even to look on a lighted per ; in Rome, too, it was the vestal virgins who :pt alive the sacred flame. Medicine men, too, 1334 a fringe of shells or bones hanging from her fore- head so as to cover her eyes, as she is susceptible to any malicious sorcerer at such times. Among the Carrier Indians 132 menstruous women may not cut up the grebes, which are caught in great numbers, as by sympathy their blood would cause an unnaturally prolonged flux in the transgressor. Similarly, Shushap women are forbidden, during their seclusion at puberty, to eat anything that bleeds. There are numerous instances of women refraining, during menstruation, from fish and flesh, and confining themselves to a vegetable diet for the same reason. It is probably in obedience to the dictates of sympathetic magic that the physician of to-day administers preparations of blood for the cure of amenorrhcea in place of the old and proven haematinics. when about to compound their medicines, were in the habit of making a saving clause that the remedy would be effective, provided no menstruating woman approached their chamber during the com- pounding process. Surely nowadays the altogether inordinate apprehensions of both nurse and surgeon as to the evils of operation during the menstrual state are tinctured witli the savour of this same superstitious fear. THE UNDERLYING IDEA. Zulu girls believe that they would shrivel to skeletons if the sun were to shine on them at puberty, and in some Brazilian tribes the girls think that a trans- gression of the rules would entail sores on the neck and throat. We find the same taboos as apply to menstruous girls enjoined on warriors who have shed blood, and on young warriors at the time of their first campaign. They may not feed themselves; they may not scratch their heads except with a stick, for their hands are defiled with blood, and they need protection from themselves.34 tc intervening stages by which the one has been transformed into the other. Each condition is due to ovarian activity, and comes to an end with com- plete removal of the ovaries and any accessory ovarian tissue; the medium of stimulation is almost certainly an internal secretion of the ovary. In each condition the changes that occur in the external and internal generative organs may be broadly described as " active congestion," and are similar in character. The congestive phenomena in the external genitals are far more marked in animals than in the human female, though occa- sionally marked swelling and congestion may also be seen in the latter. But in this and other points, when animals have been submitted to ages of domestic influence, there is a tendency to approxi- mate to the human type. Domesticity has done for animals what civilisation has done for man. Speaking generally the human female, in the absence of pregnancy, menstruates once a month throughout her sexually effective life, and is capable of conception at any time. The nearest approach to this is the case of the monkey, who has a regular series of monthly periods, as does the human female, but has a limited breeding season-i.e., a limited period during which concep- tion is possible. Both man and monkey have developed sexual activity to a maximum, but in the matter of reproductive capacity man seems to tJ.LLv .LLLQJUUt;JL V.1. LICJ..I:-’LVUu..B.IU.L Y V B.IaJ’paJB.I.LIJJ OJ.LL OvvO UV have outstripped the monkey; thus the latter occupies a position midway between man and other animals. Mares, cows, sheep, and pigs exhibit a series of sexual periods during such limited portion of the year as constitutes their appropriate sexual season. The wild cat has one sexual season each year, but the domestic cat has three or four. THE UNDERLYING IDEA. The idea underlying these customs is that there resides in the menstrual blood some agent powerful for good or for harm ; most of all is its power over the virility of the male, enfeebling him in the fierce struggle for the survival of the fittest. The spirit of the woman resides in her blood and is transferable therewith to the male. It is a special aspect and a special applica- tion of the universal primitive blood-horror, founded on this same belief that the spirit of the person resides in his blood, and emerges with it for good or for harm. In addition, there is some- thing unintelligible about menstruation, something uncanny, and the primitive mind explains it as due to some spiritual agency, which may harm the woman if she does not take certain precautions, and may pass from her and harm others if they neglect the prescribed precautions. g p p depe ds. In like manner in some tribes the menstruous woman is compelled to dwell apart in a dark hut specially prepared for her use, or even in a hole in the ground or a dark cage.3s When confined in holes in the ground they are sometimes covered with dirt, or sand, or a shelter of branches, or with mats. Special attendants are told off to wait p p ecaut o s As examples of the need of self-protection, among the Tinneh Indians..31 a menstruating girl may not touch her own head with her hand, and, if it itches, she must use a stick to scratch it. In some races she is not allowed to feed herself. She must wear 1335 upon them and supply their needs, and they may not eat any food which by sympathy would damage similar food-supplies. In some races the girls at these times screen themselves from the sun by wearing huge hats. Elsewhere they are sewn up in hammocks, as though dead, leaving only a small hole for breathing. The rules that the girl may not touch the ground nor see the sun keep her suspended between heaven and earth; she can poison neither of these great sources of life. But not only the safety of others, but her own safety is assured, if she be thus perfectly insulated. THE UNDERLYING IDEA. The wild wolf has but one sexual period a year, but his kinsman the domestic dog has two or more sexual seasons each year. In all these animals conception is strictly limited to the sexual season. It must be remembered that the monkey, though not strictly speaking a domestic animal, has developed a high degree of domesticity of its own, and it is at least possible that the semi-erect posture may be an accessory factor in approximating its sexual features to the human. y p We have spoken of the universal horror of blood, which finds its highest expression in the lowest orders of civilisation, and which extends also to, at any rate, the herbivorous group of higher animals. This instinctive horror primitive man explains and excuses by asserting the presence of a spirit in the blood; the blood is the life. Blood is therefore taboo, and taboos all that comes in contact with it. As one reflects on the horror of menstrual blood, which in all primitive communities has led to prohibition, sometimes under penalty of death, of sexual intercourse during the menstrual period, the question inevitably suggests itself to one’s mind whether the horror may not be the parent of the prohibition. The extreme divergence that sub- sists between the habit of man and the habit of animals in the period of sexual intercourse can hardly have failed to puzzle each of us at one time or another. Esthetic and hygienic considerations have come to dominate the mind of highly civilised man to such an extent that we are apt to look no further for an explanation. Yet it is just in those lowly stages of civilisation, in which aesthetic and hygienic considerations are of least account, that obedience to the prohibition is most strict; it is, indeed, a fundamental feature of savage ritual. With the development of civilisation medical writers, from Hippocrates onward, actually recom- mended sexual intercourse during menstruation for sterility and other ailments; and in the Middle Ages it had become so common that preachers found it necessary to warn their congregations against it.3" . SEXUAL SEASON IN THE HUMAN FEMALE. ] j ] { he Now there is evidence that in former times the sexual season of the human female was restricted, as is the case with most mammals; indeed, it is not improbable that the original type was universal in all primitive species and consisted of a single sexual season each year. Cook noted that not more than 10 per cent. of Esquimaux women menstruated during the long winter months; and as though heightened desire were correlated to less frequent menstruation, a sexual season of great intensity set in at the first reappearance of the sun, and little else was thought of for some time afterwards. Caldwell states that the Queensland natives have a distinct sexual season in September-i.e., in spring -and that they cannot be prevailed upon to do any work for some weeks at that time of the year. Wiltshire iJ6 quotes authorities to show that in a lowly state of civilisation, such as exists among the women of Lapland, Greenland, the Faroe Islands, Tierra del Fuego, and the Guaranis of Paraguay menstruation takes place only at intervals of several months. He suggests that the infrequent menstrua- tion of girls at puberty-at intervals of 3, 4, and 6 months-and the recurrence of a similar periodicity tt the end of sexual life may be a reversion to a estricted ancestral type. against The belief, of very ancient lineage, that coitus during menstruation led to the birth of monsters was formerly very widespread. Ambroise Pare affirmed his belief, and rested it on a dictum of the non-canonical Book of Esdras, that " menstruous women shall bring forth monsters." Ancient mythology attributed the deformity of Vulcan to the union of Jupiter with Juno during her men- strual period. The Talmud went further and assigned disorders of the mind, such as epilepsy, cretinism, and insanity to the same cause. The belief is not wholly dead nowadays. SEXUAL ACTIVITY, HUMAN AND ANIMAL. SEXUAL ACTIVITY, HUMAN AND ANIMAL. The identity of oestrus or " heat " in animals and of menstruation in the human female admits of no doubt ; it is, indeed, possible to trace many of the There is evidence also that in former ages the period during which conception was possible to the human female in each year was limited. SEXUAL SEASON IN THE HUMAN FEMALE. In different countries there is a marked excess of births at some or other period of the year, and in general terms it may be stated that the tendency is for women both to commence men- struation and to bring forth children in the warmer months. Westermarck considers that the evidence points to two special seasons of sexual activity- spring and autumn-and that the activity is greatest towards the end of spring in the south of Europe and rather later in the north. for you will find yourself confronted by the puzzling fact that with some domesticated animals, notably the bitch, there is a strong tendency, not more, to reject intercourse during the period of substantial flux, some preferring the cominence- ment, but most the actual termination of the period of heat. - MM!OSMp/;</.&mdash;1. Nat. Hist., vii., 13. 2. Hartland : Primitive Paternity, ii., 273. 3. Folk-lore in the Old Test,ament ; and Golden Bough. 4. Nat. Hist., xxviii., 23. 5. loiigfc,.Ilow: Hiawatha, Canto xiii. 6. Nat. Hist., vii., 13. 7. Ibid.. xxviii.. 23. 8. Ibid., xix., 57. 9. Havelock Ellis: Psychology of Sex, vol. ii. 10. Frazer: Gulden Bough. 11. Havelock Ellis. Psvchology ot Sex, vol. ii. 12. Golden Bough, vol. iii., 302. seq. 13. Pliny: Nat. Hist., vii., 13, and xi., 15. 14. Golden Bough, Ibid. 15. Pliny: Nat. Hist., xxviii., 23. 16. Bourke: Scatologic ]{!tes of All Nations. 17. Nat. Hist., Ibid. 18. Nat. Hist., vii., 13. 19. Ibid., xxviii., 23. 20. Ibid., vii.. 13. 21. Annales des Sciences Psychiques, September-October, 1897. 22. Pliny: Nat. Hist., vii., 13. 23. Ibid., xxviii, 23. 24. Aristotle: Hist. Anim., vii., 2. 25. H. L. Strack: Der Blutaberglaube in der Menschheit, eri. 1892, pp. 14-18. 26. Frazer: Golden Bough, iii., 202. 27. Folklore, vol. xix., p. 418; J. H. Weeks: Customs of the Lower Congo Pfople. 28. Golden Bough, i., 323. 29. Ibid., iii., 331. 30. Nat. Hist., xxiii., 13. 31. Golden Bough, i., ?25. 32. Ibid , iii., 202. 33. Ibid., i., 325. 34. Ibid., iii., 208. 35. Havelock Ellis: Psychology of Sex, vol. ii. 36. Brit. Med. Jour., 1883, The Comparative Physiology of Menstruation. 37. Martin : Hirst’s System of Obstetrics. Though closely assodiated, conception and men- struation are separable functions and not neces- sarily interdependent, for conception may occur in the prolonged absence of menstruation. THE EARLY OPERATIVE TREATMENT OF GUNSHOT WOUNDS OF THE ALIMENTARY CANAL. BY CUTHBERT WALLACE, B.S. LOND., F.R.C.S. ENG., CONSULTING SURGEON TO THE EXPEDITIONARY FORCE IN FRANCE. The advantage to primitive man and to wild animals of a limited sexual season is that the young may be born at the most propitious season of the year, when food is most abundant and climate most favourable. Once an artificial supply of food and of warmth is assured, independent of season, the necessity for a special season of sexual desire and a special breeding season disappears, and gives place to a type of greater frequency, regardless of season. It is just these things that domestication and civilisation afford. THE EARLY OPERATIVE TREATMENT OF GUNSHOT WOUNDS OF THE ALIMENTARY CANAL. , CONSULTING SURGEON TO THE EXPEDITIONARY FORCE IN FR ABDOMINAL wounds have always aroused great, interest not only in the professional but also in the lay mind. There has always been a tendency to demand more care for these cases than for any other class of injury. It is well, therefore, to inquire into the frequency of abdominal compared with other wounds, for in war the medical service must try to do the greatest good to the greatest number; q it must not waste effort on a particular class of case to the detriment of a larger but possibly less interesting series of wounded. Under domestication the frequency of cestrus is increased, and all the evidence goes to show that food and temperature are the chief efficient agents in producing the change. As to temperature, it is a familiar fact that in the bitch winter oestrus does not last so long as summer oestrus, and in the human female the menstrual flux is more readily established in summer than in winter. We may well look in the same direction for an explanation of the fact that, other things being equal, town-bred girls commence menstruation several months in advance of those reared in the country. Hard fare and laborious life tend to diminish the menstrual flow, while luxury and ease tend to increase it. In some parts of this country the hard-working farm girls still wear no protection during menstruation. Blood in the discharge is far more abundant in domesticated animals than in wild. g Relative Freq2cency of Abdominal Wounds. q y The actual number of men wounded in the abdomen will never be known. The number of abdominal wounds admitted to field ambulances and casualty clearing stations are, subject to certain error, known. The error is caused by the impossi- bility of accurately diagnosing a perforating wound. The numbers vary with each observer, and the surgeon who believes in the expectant treatment will be inclined to reckon as perforating, wounds which a believer in operative treatment would class as non-perforating. The following are figures taken in certain casualty clearing stations by careful observers :- We see, then, that the circumstances of domesti- cation and civilisation tend to an increase and a greater frequency both of oestrus and of menstrua- tion ; perhaps one other parallel condition may help us a stage further in the attempt to unravel the process of transformation. We know that in most mammals non-satisfaction of sexual desire by intercourse tends to a quicker return of heat than normal; and it may be that the greater fre- quency of menstruation in the human female may in part be referable to the fact that civilisation has brought it about that in the human female sexual desire is often compelled to go unsatisfied.&bgr;7 tage of Abdonzinal Perforatin!1 TJrowuls to Total Wounds. A = 1-88 per cent. C 0 75 per cent. B = 1-5 ., D = 062 " If we take the statistics from 9 field ambulances and from 7 casualty clearing stations for a period of six months we get the following figures :- Percentage of Abdominal Wounds to Total Wounds. Field ambulances ............ 192 per cent. Casualty clearing stations ...... 0-72 " Percentage of Abdominal Wounds to Total Wounds. Field ambulances ............ 192 per cent. Casualty clearing stations ...... 0-72 " SEXUAL SEASON IN THE HUMAN FEMALE. But statistics show an enormous preponderance of con- ceptions as occurring immediately after cessation of the menses, while a minimum is reached just before their commencement. INFLUENCE OF DOMESTICATION AND CIVILISATION. INFLUENCE OF DOMESTICATION AND CIVILISATION. CONCLUSION. These general considerations, and much more besides, are a necessary prelude to any reasoned attempt to answer the question whether the universal human horror of menstrual blood may have initiated the practice of abstinence from intercourse during the menses. Give an affirma. tive reply and you are still little nearer a solution, The difference is probably accounted for in two ways : 1. The mortality among abdominal cases at field ambulances is very heavy. 2. There is more time for accurate diagnosis at the casualty stations. The important fact brought out is the relatively
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EGFR/MET promotes hepatocellular carcinoma metastasis by stabilizing tumor cells and resisting to RTKs inhibitors in circulating tumor microemboli
Cell death and disease
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www.nature.com/cddis ARTICLE OPEN EGFR/MET promotes hepatocellular carcinoma metastasis by stabilizing tumor cells and resisting to RTKs inhibitors in circulating tumor microemboli Shouyang Song1, Zhen Yu1, Yajing You1, Chenxi Liu1, Xiaoyu Xie1, Huanran Lv2, Feng Xiao1, Qiang Zhu ✉ Chengyong Qin 1,2,4 1,2,3,4 ✉ and 1234567890();,: © The Author(s) 2022 The receptor tyrosine kinases (RTKs) family is well-recognized as vital targets for the treatment of hepatocarcinoma cancer (HCC) clinically, whereas the survival benefit of target therapy sorafenib is not satisfactory for liver cancer patients due to metastasis. EGFR and MET are two molecules of the RTK family that were related to the survival time of liver cancer patients and resistance to targeted therapy in clinical reports. However, the mechanism and clinical therapeutic value of EGFR/MET in HCC metastasis are still not completely clarified. The study confirmed that EGFR/MET was highly expressed in HCC cells and tissues and the phosphorylation was stable after metastasis. The expression of EGFR/MET was up-regulated in circulating tumor microemboli (CTM) to accelerate IL-8 production and resistance to the lethal effect of leukocytes. Meanwhile, highly expressed EGFR/MET effectively regulated the Ras/MAPK pathway and stabilized suspended HCC cells by facilitating proliferation and inhibiting apoptosis. Moreover, EGFR/MET promoted phosphorylation of hetero-RTKs, which was dependent on high-energy phosphoric acid compounds rather than their direct interactions. In conclusion, highly expressed EGFR/MET could be used in CTM identification and suitable for preventing metastasis of HCC in clinical practice. Cell Death and Disease (2022)13:351 ; https://doi.org/10.1038/s41419-022-04796-8 INTRODUCTION Liver cancer is one of the most lethal cancers, causing the 6th carcinoma incidence and 4th cancer mortality in the world [1]. Hepatocellular carcinoma (HCC) is the most common type of liver cancer in clinical with high relapse and mortality rates [2]. Respectively, as the first-line drug for the treatment of liver cancer targeted to PDGFR/VEGFR family, sorafenib extends the survival time of patients by 2.8 or 2.3 months in two clinical trials among Caucasians and Asians, providing modest survival benefit [3, 4]. Recurrence and metastasis are responsible for the death of HCC patients [5]. Research has confirmed that the lung was the most common site of extrahepatic metastasis and significantly reduces the survival time of patients [6]. However, the mechanism in extrahepatic metastases of HCC remains unclear. According to several studies, tumor cells in the circulating system are precursors of metastasis and may contribute to tumor progression or metastasis [7, 8]. Also, anoikis resistance is essential for tumor metastasis [9, 10]. Multiply molecules and pathways including the Ras/MAPK pathway, contribute to stabilizing tumor cells in suspension [11]. However, mechanisms of survival and hematogenous metastasis of suspended HCC cells need to be completely explored. Receptor tyrosine kinases (RTKs) are currently considered as critical roles in malignant transformation and cancer metastasis [12]. The human genome encodes for 58 RTKs, which can be divided into 20 subfamilies, and certain RTKs like VEGFR/PDGFR families, are used for clinical target therapy of primary HCC [13]. They belong to the same membrane protein family which can catalyze transferring of the γ phosphate of ATP to hydroxyl groups of tyrosinases on target proteins and play important roles in the control of most fundamental cellular processes including cell cycle, migration, metabolism, proliferation, differentiation and survival [14]. We have confirmed activation of VEGFR-1, one of the RTKs which leads to tumor angiogenesis and induces MMP-9dependent invasion in HCC [15]. Some RTKs namely EGFR/MET can participate in metastasis of tumors by forming a molecular complex with integrin, mutation, amplification, and drug resistance [16–18]. RTKs may be vital for tumor cell survival in the circulating system and distal metastasis. For example, overphosphorylation of EGFR in tumor cells cultured in suspension was observed [7, 10]. EGFR and Her2 are also identified as a biomarker on the surface of circulating tumor cells [19]. Additionally, MET-amplification has been discovered in the CTC of NSCLC patients [20]. However, the function of RTKs expressed in CTM is not completely clear. Expression and phosphorylation of 1 Shandong Provincial Hospital, Cheeloo College of Medicine, Shandong University, Jinan 250021 Shandong, P.R. China. 2Shandong Provincial Hospital Affiliated to Shandong First Medical University, Jinan 250021 Shandong, P.R. China. 3The First Affiliated Hospital of Xinjiang Medical University, Urumqi 830054 Xinjiang, P.R. China. 4Shandong Provincial Engineering and Technological Research Center for Liver Diseases Prevention and Control, Jinan 250021 Shandong, P.R. China. ✉email: zhuqiang@sdu.edu.cn; chengyong_qin@126.com Received: 2 November 2021 Revised: 25 March 2022 Accepted: 29 March 2022 Official journal of CDDpress S. Song et al. 2 Fig. 1 EGFR/MET were highly expressed in HCC tissue and HCC cell lines compared to other RTKs. A, B Luminex and ELISA assay were used for detection of expression of RTKs in HCC tissues and HCC cell lines. Data of tissues are shown as the mean ± SD. C Expression and phosphorylation of EGFR/MET in several HCC cell lines were determined by Western blot. D Expression of EGF and HGF in carcinoma tissue and HCC cells. Tissues and cells were lysis and Luminex was used for detection of concentration. Data of tissues are shown as the mean ± SD. Patient information for detecting concentration of RTKs and EGF/HGF in tissues is suppled in Table 4. RTKs are quite different in various tumors [21]. It is therefore that targeting the most expressional and functional RTKs is the key to the treatment of HCC. In the present study, we determined the expression of RTKs proteins in CTM and cells and verified the important roles of EGFR/ MET and the possibility of being used as a clinical treatment target. Furthermore, we identified the mechanism that EGFR/MET promotes metastasis and stabilizes HCC cell lines in hematogenous metastasis. These findings provide mechanistic evidence for us to design HCC medications targeting EGFR/MET, thereby benefiting patients with HCC in reducing the incidence of metastasis and prolonging survival time. RESULTS EGFR/MET is highly expressed in human HCC tissues and cell lines compared to other RTKs families We firstly explored the expressions of RTKs in HCC tissues and HCC cell lines. Interestingly, the expressions of RTKs in HCC tissues and HCC cell lines varied greatly. Certain RTKs namely M-CSFR were highly expressed in HCC tissues whereas poorly expressed in cell lines. However, other RTKs namely the HER family were highly expressed in both HCC tissues and cell lines. Compared to the other RTKs we detected, the concentration of EGFR/MET was high expressional RTKs both in HCC tissues and HCC cell lines. Especially in HCC cells, expressions of EGFR and MET in MHCC97H were 54,548.83 and 607,470.8 pg/mg, respectively, and those of SK-HEP1 were 103,099.9 and 38,652.84 pg/mg, respectively, higher in expression than other RTKs (Fig. 1A, B). Additionally, both EGFR/MET in MHCC97H/LM3 cell lines with MET amplification were highly phosphorylated. In contrast, low phosphorylation of EGFR/MET was detected in PLC/PRF/5 and SK-HEP1 (Fig. 1C). Interestingly, the high expression level of HGF and low expression of EGF were observed in HCC tissue, though low concentration of EGF and HGF were expressed in HCC cells of tissues (Fig. 1D, Supplementary Fig. 1A). Low concentrations of EGF and HGF were observed in HCC cell lines, while HGF expression in MHCC97H was higher than SK-Hep 1 (Fig. 1D, Supplementary Fig. 1B). In brief, the EGFR/MET of the RTKs family was significantly expressed in HCC tissues and cells, and phosphorylation of EGFR/MET was related to the expression of MET. Expression of EGFR/MET is up-regulated in CTM but downregulated in lung metastasis Next, we verified whether the expressions of EGFR/MET using a xenograft model for lung metastasis of mice were stable during metastasis. In this model, Hepa1-6 cells were successfully metastasized from the left lobe of the liver to the lungs (Fig. 2A). Compared with the primary tumor, there was no significant difference in expression and phosphorylation levels of EGFR/MET in metastatic tumors (Fig. 2B). The expression of EGFR in HCC was markedly lower than that in peritumor tissues, while the expression of MET was substantially higher. However, the expression in lung metastases was just reversed compared with normal lung tissues (Fig. 2C). Interestingly, we found the presence of CTM in mice with widespread lung metastasis, and the expression level of EGFR/MET was higher than that in primary tumor and lung metastasis (Fig. 2D). In scRNA-seq, cells were divided into clusters for analysis (Fig. 2E). We found EGFR/MET were mainly expressed in HCC cells of carcinoma tissue, which differed from some RTKs for clinical targets expressed mainly in peritumor tissue such as the PDGFR family (Fig. 2F). However, expression of EGFR/MET in lung metastasis was lower than that in primary tumor, which was in agreement with most of the highly expressed RTKs (Fig. 2G). The ratios of RTKs expression in carcinoma tissue compared to whole tissues in primary tumor and lung metastasis were provided in Table 1. Collectively, expression of EGFR/MET increased during metastasis and decreased after metastasis, but phosphorylation of EGFR/MET kept stable after metastasis. It suggested that EGFR/MET was an important marker for the detection of CTM. As stable phosphorylation of EGFR/MET is suitable for the treatment of lung metastasis, targeting EGFR/MET in CTM may be more effective in the treatment of HCC. Anoikis resistance is related to up-regulation in expression and phosphorylation of EGFR/MET Since metastasis of CTM was closely related to anoikis resistance, we next observed the role of EGFR/MET in HCC cell lines of Cell Death and Disease (2022)13:351 S. Song et al. 3 Fig. 2 Expression of EGFR/MET is higher in CTM but lower in lung metastasis. A Gross image of HCC and lung metastasis in mice 60 days after injection of Hepa1-6 cell line. B Mice Phosphorylation Array was used for detection of RTK phosphorylation of HCC tissue separated from liver and lung. C Immunofluorescence analyzed the differences of EGFR/MET expression in cancer tissues and peritumor tissues in liver and lung. D Immunohistochemistry analyzed the differences of EGFR/MET expression in cancer tissues and CTM. E UMAP showed different clusters in carcinoma tissues of liver and lung. F Distribution of RTKs were showed in each cell of primary tumor and lung metastasis with Single cell RNA sequence by heatmap. G Difference of RTKs with high expression in carcinoma tissue between primary tumor and lung metastasis were showed. Data are shown as the mean ± SD, *P < 0.05. suspension. Phosphorylation RTKs array reported that phosphorylation of EGFR/MET in suspended HCC cell lines was enhanced and Western blot assays indicated that peak point appeared at 24–48 h (Fig. 3A, B). IL-8 synthesis/secretion could be interrupted by EGFR/MET inhibitor, which was observed in MHCC97H cell lines with high phosphorylation of EGFR/MET. However, in PLC/PRF/5 HCC cell lines with lower phosphorylation of EGFR/MET, the Cell Death and Disease (2022)13:351 inhibitory effect of EGFR/MET inhibitors was greatly reduced, and secretion of IL-8 could interrupt by EGFR inhibitor was relatively obvious (Fig. 3C). Also, inhibition of EGFR/MET made HCCLM3 cell lines more easily killed by leukocytes and more leukocytes could survive (Fig. 3D), which were not beneficial to hematogenous metastasis of HCC cell lines. The findings suggested that EGFR/ MET played an important role in the metastasis of HCC cells. S. Song et al. 4 Table 1. Expression of RTKs in carcinoma tissue. Liver Lung GENE Expressed in tumor Expressed totally Ratio Expressed in tumor Expressed totally Ratio P Value ****EGFR 4536 4722 0.96061 1780 2703 0.658528 <0.0001 ****MET 5143 5396 0.953113 1766 2174 0.812328 <0.0001 AXL 3536 5604 0.630978 1991 8497 0.234318 *DDR1 2276 2641 0.861795 1259 1997 0.630446 0.107143 EPHA1 16 29 0.551724 3 28 ****EPHA2 9029 9507 0.949721 3394 4141 0.819609 EPHA3 0 14 0 3 114 0.026316 EPHA4 10 41 0.243902 30 285 0.105263 EPHA5 2 6 0.333333 5 8 0.625 EPHB1 0 6 0 0 37 0 EPHB2 148 159 0.930818 53 318 0.166667 EPHB3 66 75 0.88 113 262 0.431298 ****EPHB4 5616 6007 0.934909 2236 2613 0.855721 EPHB6 847 914 0.926696 378 485 0.779381 **ERBB2 1031 1085 0.95023 489 632 0.773734 ERBB3 174 271 0.642066 192 306 0.627451 ERBB4 0 6 0 0 3 0 ****FGFR1 3603 4064 0.886565 1512 2961 0.510638 FGFR2 165 199 0.829146 79 265 0.298113 FGFR3 135 230 0.586957 96 280 0.342857 ****FGFR4 1257 1326 0.947964 555 606 0.915842 FLT1 119 1597 0.074515 108 2701 0.039985 FLT3 31 882 0.035147 26 890 0.029213 FLT4 1415 2320 0.609914 916 1255 0.72988 IGF1R 20 761 0.026281 45 2199 0.020464 IGF2R 660 1477 0.446852 464 2272 0.204225 INSR 2362 3133 0.75391 1022 2073 0.493005 KDR 32 2086 0.01534 17 707 0.024045 MCSFR 186 3752 0.049574 228 7354 0.031004 MST1R 22 23 0.956522 65 161 0.403727 MUSK 11 12 0.916667 28 39 0.717949 NGFR 45 77 0.584416 431 499 0.863727 PDGFRA 191 235 0.812766 119 2844 0.041842 PDGFRB 5 200 0.025 23 1477 0.015572 RET 0 28 0 2 73 0.027397 ROR1 463 496 0.933468 360 755 0.476821 ROR2 41 47 0.87234 110 263 0.418251 ***RYK 3315 3880 0.854381 1766 2935 0.601704 TIE1 11 345 0.031884 15 435 0.034483 0.0317 <0.0001 <0.0001 0.0011 <0.0001 <0.0001 0.0007 RTKs Ratio > 85% expression in carcinoma cells of liver and copy > 1000 was used for comparing difference in carcinoma tissue before and after metastasis. Mann-Whitney test was used for analysis. * < 0.05, ** < 0.01, *** < 0.001, **** < 0.0001. EGFR/MET stabilizes suspended HCC cells and avoids the killing of leukocytes by regulating the Ras/MAPK pathway As the Ras/MAPK pathway regulated by RTKs played an important role in tumor metastasis, the function of EGFR/MET in regulating the Ras/MAPK pathway will be further investigated in our subsequent research. Firstly, like phosphorylation of EGFR/MET, we found Ras-GTP was up-regulated in HCC cell lines in suspension (Fig. 4A). Both EGF and HGF could activate the corresponded receptor and lead to the activation of the Ras/MAPK pathway in HCC cell lines with low phosphorylation of EGFR/MET and activation of EGFR were was substantially promoted in the regulation of the Ras/MAPK pathway. However, HCC cell lines with high phosphorylation of EGFR/MET were not sensitive to EGF and HGF. In contrast, the inhibitor of EGFR/MET could down-regulate the Ras/MAPK pathway in HCC cell lines with high phosphorylation of EGFR/MET instead of a condition of low phosphorylation of EGFR/MET. In brief, the Ras/MAPK pathway could be regulated by the basal phosphorylation levels of EGFR/MET (Fig. 4B, C). While fully activating the corresponding RTKs by a sufficient amount of ligand, we found that MET worked more effectively inactivating Cell Death and Disease (2022)13:351 S. Song et al. 5 Fig. 3 Expression and phosphorylation of EGFR/MET was up-regulated in suspended HCC cells. A RTK array was used to measure phosphorylation of RTKs in suspended HCC cell lines. B Western blot was used to analyze phosphorylation of EGFR/MET in HCC cell lines with 0, 24, 48, 72 h of suspension. Low concentration of suspended HCC cells was used in prevention of cluster formation. C ELISA was used to detect IL-8 secretion of MHCC97H and PLC/PRF/5 cell lines with EGFR/MET inhibitor. D Immunofluorescence analyzed the interactions between MHCC97H cells and leukocyte with or without EGFR/MET inhibitor. Data are shown as the mean ± SD, n = 3. *P < 0.05. the Ras/MAPK pathway than other RTKs in treating clinical HCC (Fig. 4D). However, simple knockdown of EGFR/MET, EGFR in particular, could directly inhibit the Ras/MAPK pathway, which could barely be restored by activating other RTKs (FGFR, VEGFR, and PDGFR) based on their low expression. Moreover, the Ras/ MAPK pathway could not respond to activation of RTKs (PDGFR and VEGFR) poorly expressed (Fig. 4E–G). However, based on high expression and activation of EGFR or MET could offset the effect of knockdown in each other on the Ras/MAPK pathway to some degree (Fig. 4H, I). EGFR and MET synergistically regulated the Ras/ MAPK pathway. EGFR inhibitors could decrease the phosphorylation level of MET that was activated endogenously or exogenously in HCC cell lines which were sensitive to EGFR inhibitors, but it did not work effectively in HCC cell lines with low phosphorylation of EGFR (Fig. 4J, K). Moreover, MET knockdown could weaken the effect of EGFR inhibitors on the interruption of the Ras/MAPK pathway (Fig. 4L). Different expressions of EGF/HGF indicated that activation of EGFR/MET relied on different ways in vivo and higher levels of HGF in HCC tissue allowed MET to play an important role in the regulation of the Ras/MAPK pathway. Next, we analyzed the activation of ERK in suspended HCC cell lines. The findings were identical to Ras-GTP indicating that the phosphorylation level of ERK was up-regulated in suspended HCC cell lines (Fig. 5A). Following the inhibition of the Ras/MAPK pathway, DNA synthesis and G2/S-phase of the cell cycle were Cell Death and Disease (2022)13:351 decreased and the G1-phase of the cell cycle increased in HCC with or without suspension. Moreover, S-phase increased in suspended HCC cells, indicating that the Ras/MAPK pathway maintained the growth of HCC cell lines (Fig. 5B, C). Flow cytometry confirmed that inhibition of the Ras/MAPK pathway in HCC cells with or without suspension could promote the increase of apoptosis in HCC cell lines, but the effect in HCC cells with suspension was significant (Fig. 5D). We also found all cyclin proteins were down-regulated followed by Ras/MAPK inhibition. In detail, Cyclin A/B were less interfered with by Ras/ MAPK inhibitors and poorly expressed in suspended HCC cell lines. Conversely, Cyclin E was highly expressed in suspended HCC cell lines and could be easily down-regulated by Ras/MAPK inhibitors. Both cultured in suspension and Ras/MAPK inhibitors could activate PARP, a protein major in DNA repairing in HCC cell lines [22]. Moreover, a combination of Ras/MAPK inhibitor and suspension caused higher activation of PARP in HCC cell lines, which was related to higher apoptosis of HCC cell lines (Fig. 5E). Also, inhibition of the Ras/MAPK pathway made suspended HCC cells more easily to be killed by leukocytes (Fig. 5F). Collectively, the data indicated that the Ras/MAPK pathway induced by EGFR/MET stabilized suspended HCC cell lines and prevented the lethal effect of leukocytes. Moreover, it was more difficult for suspended HCC cells to survive under a condition of poorly phosphorylated ERK. S. Song et al. 6 Fig. 4 EGFR/MET were powerful in regulation of Ras/MAPK pathway based on high expression. A Ras Activation Assay Kit was used for detection of Ras-GTP in suspended HCC cell lines. Low concentration of suspended HCC cells was prevented from cluster formation and used for detection of Ras activation in 0, 24, 48 and 72 h. B Western Blot was used to confirm the regulation of EGFR/MET in Ras/MAPK pathway, including sensitivity of Ras/MAPK pathway to EGFR/MET inhibitors and C EGF/HGF in activation of Ras/MAPK pathway in different HCC cell lines. D 50 ng/ml of recombined ligands was used for RTKs phosphorylation and cells were harvested after 1 h for comparing activation of Ras/ MAPK pathways. (E) VEGF, (F) PDGF-BB and (G) FGF2 were verified in restoration of Ras/MAPK pathway activation in PLC/PRF/5 cell line with EGFR/MET knock-down. H EGF/HGF was detected in activation of Ras/MAPK pathway of PLC/PRF/5 with MET knock-down and I EGFR knockdown. J Western Blot showed the effect of EGFR on MET down-regulation in EGFR inhibitor in PLC/PRF/5 cell line with exogenous/ endogenous HGF stimulation. K EGFR inhibitor was used in detection of MET inhibition in SK-Hep1 cell line, which is not sensitive to EGFR inhibitor. L Effect of EGFR inhibitor was verified on PLC/PRF/5 cell line with MET knock-down. EGFR/MET participates in the phosphorylation of multiple RTKs family members Based on the high expression of EGFR/MET in HCC tissue/cell lines and stable expression in metastasis, the regulatory effects of EGFR/MET on the phosphorylation level of other RTKs were investigated. In this study, Lenvatinib, as an inhibitor of PDGFRβ/ VEGFR/FGFR/RET, was effective in suppressing EGFR/MET and targeting RTKs in MHCC97H and HCCLM3, but less effective on PLC/PRF/5. In contrast, Sorafenib, as an inhibitor of PDGFRβ/ VEGFR/Raf, had less effect on EGFR/MET, and barely suppressed the function of PDGFRβ in MHCC97H and LM3 (Fig. 6A). Also, we found that high phosphorylation of EGFR/MET could maintain the hyperphosphorylation status of some RTKs which were clinical targets for HCC treatment, such as PDGFRβ and FGFR1, and it was obvious in HCC cell lines with MET amplification. Briefly, both EGFR/MET inhibitors could reduce phosphorylation of ErbB family, MET family, and RET, and MET inhibitors could additionally interfere with the InsR family (Fig. 6B). MET knockdown exhibited the same effect as MET inhibitors in the suppression of phosphorylation of multiple RTKs (Fig. 6C). Additionally, EGFR/ MET inhibitors could effectively interrupt phosphorylation of FGFR, VEGFR, and PDGFRβ which were for clinical therapy, even stimulated by RTK ligand (Fig. 6D). Similarly, MET knock-down also inhibited phosphorylation of those RTKs (Fig. 6E). EGFR/MET had an impaired ability to influence the phosphorylation of hetero-RTKs in HCC cell lines with low phosphorylation (Fig. 6F). In brief, EGFR/MET also affected the inhibitory effect of clinical antiHCC drugs on RTKs, which might lead to treatment failure and extrahepatic metastasis. Phosphorylation of EGFR/MET is related to high-energy phosphate compounds, not EGFR/MET hetero-dimer Next, we focused on possible mechanisms related to the phosphorylation of EGFR/MET. Interestingly, we found that phosphorylation of MET was affected by MET amplification (Fig. 7A). Phosphorylated EGFR/MET, either endogenous or induced by ligands, tended to form homo-dimers and it was almost impossible for EGFR/MET to form hetero-dimer, whether EGFR/MET was phosphorylated or not (Fig. 7B, C). It suggested that phosphorylation of EGFR/MET was not affected by hetero-dimer. In further research, we found sufficient ATP in the solution could effectively promote phosphorylation of most RTKs, even in HCC cell lines with METamplification, but MET was not affected (Fig. 7D). MET phosphorylation was affected by GTP rather than ATP (Fig. 7E). Phosphorylation of EGFR induced by ATP was not affected by MET, but the factor creatine kinase that degraded ATP could reduce the level of EGFR phosphorylation (Fig. 7F). It suggested that EGFR could be directly phosphorylated by high-energy phosphate compounds in the absence of ligands, but MET relied on GTP, MET amplification, or ligand induction (Fig. 8). Cell Death and Disease (2022)13:351 S. Song et al. 7 Fig. 5 Ras/MAPK pathway stabilized suspended HCC cell lines and avoid killing of leukocytes. Cells were divided in 4 groups: (a) cultured without suspension (b) cultured with suspension (c) cultured without suspension, 10 µM of AZ628 were used for Ras/MAPK pathway inhibition (d) cultured with suspension, 10 µM of AZ628 were used for Ras/MAPK pathway inhibition. Cells in different group were detected in (A) phosphorylation of ERK by Western blot, (B) cell cycle by flow cytometry, (C) DNA synthesis by EDU, (D) apoptosis by flow cytometry and (E) Cyclin, CDK4 and PARP by Western blot. F Immunofluorescence was used in determining interactions between MHCC97H and leukocyte with or without Ras/MAPK inhibitor. Data of flow cytometry are shown as the mean ± SD, n = 3. *P < 0.05. DISCUSSION RTKs nowadays are considered to be critical in tumorigenesis, intrahepatic metastasis, and distal metastasis of liver cancer [23]. The correlation exists between multiply RTKs and recurrence times of liver cancer [13]. Currently, the clinical targeted drugs for liver cancer are mainly aimed at RTKs. For example, sorafenib, as a multi-RTKs inhibitor that exerts an antiangiogenic effect by VEGFR 2/3 and PDGFR-β, is typically used as the first-line therapy in patients with primary advanced HCC [24, 25]. However, sorafenib resistance can cause therapeutic failure and metastasis of HCC [26]. Research has shown that RTKs EGFR/MET are responsible for sorafenib resistance [27, 28] and they were highly phosphorylated in multiply carcinoma cells including the lungs, breasts, and pancreas [21]. Interestingly, highly expressed RTKs VEGFR1 and PDGFRβ in HCC tissues were barely expressed in HCC cell lines in the present study. Instead, RTKs EGFR/MET, which was targeted for HCC treatment, were highly expressed in both HCC tissues and HCC cell lines. Especially in HCC cell lines, the EGFR/MET levels were higher than other common RTKs. It suggested that some RTKs inhibitors sorafenib, tended to be effective to primary HCC mass by interruption of neoplastic angiogenesis, but low Cell Death and Disease (2022)13:351 expression of VEGFR/PDGFRβ or compensation effect from EGFR/MET in HCC cell lines were invalid in metastasis. Thus, discovering the mechanism of EGFR/MET promoting distal metastasis may be helpful for the treatment and recurrence of HCC. Extrahepatic metastasis is easily observed in patients with HCC [29]. Because of the high level of expression, EGFR/MET has been reported as a hopeful target for HCC and combination therapy may benefit patients with prolonged survival time by interaction with multiply molecules like PARP [22]. Lung metastasis is most frequently observed in extrahepatic metastasis [6]. In the C57 xenograft model, CTM with high EGFR/MET expression was detected in the blood vessels of lung metastasis mice, which was higher than that in tumor tissues, indicating EGFR/MET as a suitable marker for CTC/CTM detection. Meanwhile, stable phosphorylation of EGFR/MET makes targeted therapy possible for lung metastases. However, expression of EGFR/MET in CTM is higher than that in lung metastasis, suggesting that it is necessary to target CTM. EGFR/MET may participate in the metastasis of carcinoma through circulating tumor cells. For example, EGFR has been identified as a biomarker on the surface of circulating tumor S. Song et al. 8 Fig. 6 EGFR/MET participated in regulation of phosphorylation of hetero-RTKs. A Western Blot was used to observe phosphorylation of RTKs in HCCLM3, MHCC97H and PLC/PRF/5 cell lines with Lenvatinib or Sorafenib added. B Western blot was used to determine the effect of down-regulation of EGFR/MET on hetero-RTKs, including: Phosphorylation of EGFR, MET, Her3, RON, IR/IGF-1R and RET in HCC cell lines with EGFR/MET inhibitor added. C Phosphorylation of EGFR, Her3, RON, IR/IGF-1R and RET in HCC cell lines with MET knock-down. D Phosphorylation of FGFR1, VEGFR2 and PDGFRβ with ligand stimulated in HCC cell lines with EGFR/MET inhibitor added. E Phosphorylation of FGFR1, VEGFR2 and PDGFRβ with ligand stimulated in HCC cell lines with MET knock-down. F Phosphorylation of RTKs in PLC/PRF/5 cell line with EGFR or MET knock-down. cells [19]. Over phosphorylation of EGFR has been observed in suspended carcinoma cells such as lung and gastric cancer cells [7, 10]. Also, a clinical trial has shown that MET inhibition is related to reducing CTC [30]. Cancer cells with anoikis resistance are suitable for entering the circulating or lymphatic system, which is essential for successful cancer metastasis [31], and this may be one mechanism of tumor metastasis promotion. Interference function of EGFR/MET attenuated anoikis resistance of tumor cells [32, 33]. Similarly, our data showed the phosphorylation of EGFR/ MET phosphorylation was keeping stable or up-regulated in HCC cell lines cultured at beginning of suspension in this study. As the suspension time passed by, phosphorylated EGFR/MET decreased. Most tumor cells in the circulating system survive for only a few minutes and living cells are prone to combine with other tumor cells and non-tumor cell types in peripheral blood, which are more aggressive than single cells in cancer metastasis [34, 35]. EGFR/ MET was also related to cytokines synthesis/secretion and resistance to leukocytes of HCC cell lines and inhibition of EGFR/ MET could interrupt metastasis of HCC cell lines, especially in CTM with higher expression of EGFR/MET. The Ras/MAPK pathway is considered a survival factor for carcinoma cells, and RTKs produce a similar mechanism in regulating the Ras/MAPK pathway [14, 36]. Increased expression and activity of the Ras/MAPK pathway have been observed in mouse HCC models and human HCC tissues and they have been reported as important factors for anoikis resistance and metastasis [36, 37]. Over-activated Ras/MAPK pathway is considered to be associated with tumorigenesis and metastasis [38]. We found that the ability of RTKs to regulate the Ras/MAPK pathway was correlated with phosphorylation and expression levels in cells. Moreover, the RTKs family has cross-regulation on the Ras/MAPK pathway and RTKs inhibitors can sometimes cause Ras/MAPK re-activation, namely IR/IGF-1R knockdown leading to up-regulation of EGFR [39]. On the other hand, for RTKs with extremely low expression levels, even sufficient ligands cannot trigger the regulation of the Ras/MAPK pathway. Thus, EGFR and MET play important roles in the regulation of the Ras/MAPK pathway based on their high expression in the RTKs family. Same as the result of phosphorylation of EGFR/MET, we found that ERK was highly phosphorylated in suspended HCC cell lines. Inhibition of Ras/MAPK pathway promoted apoptosis, declination of DNA synthesis, and interfered cell cycle, which was observed in HCC cell lines both cultured without and with suspension. Also, overactivation of ERKs in suspended HCC cell lines could offset but not completely reverse the damage from the suspension environment, thereby maintaining the stability of suspended cells and preventing lethal effect of leukocytes in degree with mild activation of PARP. Based on the previously described results, EGFR/MET stabilized HCC cell lines and promoted distal metastasis by activating the Ras/MAPK pathway. RTKs have a similar effect on the activation of downstream signaling pathway Ras/MAPK pathway [14]. Our previous experiments have confirmed the advantage in the concentration of EGFR/MET in highly aggressive HCC cell lines and MET amplification is one of the important reasons for the hyper-phosphorylation of EGFR. It has been reported that EGFR/MET may be involved in resistance of RTKs target therapy [40, 41], and MET amplification is important in tumor metastasis [42]. In our result, EGFR/MET could indirectly regulate the phosphorylation of multiple RTKs and the effect was superior to their ligands in cells with MET amplification. Thus, highly phosphorylated EGFR or MET could easily induce resistance to RTKs inhibitors. Interestingly, EGFR/MET didn’t directly induce phosphorylation of hetero-RTKs via the action of kinases. High-energy phosphate compounds represented by ATP played important roles in RTKs phosphorylation [13]. Also, EGFR/ MET over-phosphorylation might cause regulation disorder of high-energy phosphate compounds, thereby promoting heteroRTKs phosphorylation. EGFR could be easily phosphorylated by ATP/GTP in the absence of EGF, that is, EGFR might be more dependent on high-energy phosphate compounds in the cell than Cell Death and Disease (2022)13:351 S. Song et al. 9 Fig. 7 Phosphorylation of EGFR/MET was related to high-energy phosphate compounds but not EGFR/MET hetero-dimer. A MET phosphorylation ratio was compared in different HCC cell. Co-immunoprecipitation was used for detection of crosstalk in (B) EGFR/p-EGFR or MET/p-MET after stimulation of ligand and (C) EGFR-EGFR, MET-MET and EGFR-MET in cells with MET amplification. D RTKs Phosphorylation Array was used for detection of phosphorylation of RTKs after incubation with 0.2 mM ATP in PLC/PRF/5 and MHCC97H cell lines. E Western Blot was used for detection of phosphorylation of EGFR/MET stimulated by ATP/GTP. 0.2 mM of ATP/GTP was incubated with cell lysate of PLC/ PRF/5 with kinase buffer for 1 h at 37 °C. F Phosphorylation of EGFR in PLC/PRF/5 cell lysis with 0.2 mM of ATP with creatine kinase or MET knock-down was detected by Western Blot. Table 2. Sequences of EGFR/MET SiRNAs. Gene Target sequence EGFR GCAGUCUUAUCUAACUAUGAUGCAA MET SiRNA1 CUGGUUUUGUCGACGUAAA MET SiRNA2 CGAGGGAAUCAUCAUGAAA MATERIALS AND METHODS Cell preparation Human HCC cell lines MHCC-97H (Zhong Qiao Xin Zhou Biotechnology Co., Ltd, China), PLC/PRF/5 (Hongbo Biotechnology, China), HCCLM3, SK-Hep1, and mouse HCC cell line (Procell, China) were cultured in DMEM containing high glucose (Biological Industries, Israel) with 10% fetal bovine serum (Biological Industries, Israel) at 37 °C in a humidified atmosphere containing 5% CO2. 100 mg/mL penicillin G and 50 μg/mL streptomycin (Biological Industries, Israel) were added. All cell lines were identified by STR and mycoplasma tests and all were used within 3 months after thawing early passage cells. Fig. 8 A proposed model of EGFR/MET-mediated in HCC and metastasis. After metastasis to blood, both expression and phosphorylation of EGFR/MET was up-regulated in CTM. Based on high expression, EGFR/MET could induce phosphorylation of other RTKs via ATP production, and over-phosphorylation of RTKs were independent to RTKs ligand and resistant to RTKs inhibitors. Compared to other RTKs, EGFR/MET could more effectively activate Ras/MAPK pathway. Activation of Ras/MAPK pathway participate in cell proliferation, resistance to apoptosis and stabilization of suspended HCC cells. Also, EGFR/MET mediated in helping CTM to avoid killing effect of leukocytes. ligands in phosphorylation. Moreover, MET was not sensitive to ATP, and HGF not EGF was over-expressed in cell lines with MET amplification and HCC tissues, suggesting that MET was easier to perform biological functions than EGFR in terms of liganddependent phosphorylation. Thus, dysfunctional RTKs could definitely affect the functions of the Ras/MAPK pathway. In conclusion, we have demonstrated that EGFR/MET are as the two of the most expressed in the RTKs family in both HCC tissue and cells. The study also clarifies the relationship between EGFR/ MET and CTM. Activation of EGFR/MET caused by complicated factors helps suspended HCC cell lines to maintain cell stability and distal metastasis. Targeting EGFR/MET may provide opportunities for the prevention of metastasis of HCC. Cell Death and Disease (2022)13:351 Suspension culture Of 3% Poly-HEMA (Sigma, MO, USA) was dissolved in ethanol for preventing HCC cell lines adhesion. Tissue culture plates were coated with 1 ml of Poly-HEMA solution and dried overnight at room temperature in a laminar flow hood. All cells were sub-cultured, re-plated in suspension condition, and harvested at indicated time points for detection. EGFR/MET knockdown cell construction For EGFR/MET knockdown, siRNAs were transfected into MHCC97H/ HCCLM3/PLC-PRF-5 cells using Lipofectamine 3000 (Thermo Fisher, MD, USA). Cells were cultured for further experiments after 48 h. The sequences of siRNAs were listed in Table 2. RTKs and Ras/MAPK inhibitors and RTKs ligands Sorafenib (VEGFR and PDGFRβ), Lenvatinib (RET, VEGFR, PDGFRβ, and FGFR), Erlotinib (EGFR), Afatinib (EGFR), Crizotinib (MET), and AZ628 (Ras/ MAPK) were purchased from Selleck (TX, USA) and dissolved in DMSO. Recombined EGF, HGF, FGF2, PDGF-BB, and VEGF were purchased from PeproTech (NJ, USA), and recombined insulin was bought from Selleck (TX, USA). Western blot and co-immunoprecipitation (Co-IP) The composition of Co-IP buffer was 50 mM Tris, 150 mM NaCl, 1% NP40, 0.5% sodium deoxycholate, 1 mM EDTA, 1 mM PMSF and 1× protease inhibitor cocktail (CWBIO, China). Co-IP and Western blot were performed S. Song et al. 10 Table 3. Purchase source and catalog ID of antibodies used. Antibody for Western blot Assay Table 4. Clinicopathologic characteristics of HCC patients in cohorts for IHC and RTKs concentrations. Cohort 1 (n = 23, for IHC) No. of patients (%) Cohort 2 (n = 16, for Luminex detecting RTKs and EGF/HGF) No. of patients (%) 22 (95.7%) 14 (87.5%) 1 (4.3%) 2 (12.5%) <60 13 (56.5%) 10 (62.5%) ≥60 10 (43.5%) 6 (37.5%) Characteristics Enterprise Detail EGFR Abcam ab52894 pEGFR CST 3777T MET CST/Abcam 8198T/ab51067 Gender pMET CST 3077T Male pHer3 Abcam ab255607 Female pMST1R Abcam Ab124671 Age pIGF1R/pInsR CST 3021T pRET Abcam ab51103 pFGFR Abcam ab173305 HBV infection pVEGFR2 CST 2478T Yes 12 (52.2%) 12 (75%) pPDGFRβ Abcam ab218534 No 11 (47.8%) 4 (25%) MEK Abcam ab178876 Tumor size p-MEK CST 9154T <5 cm 5 (21.7%) 10 (62.5%) ERK Abcam ab184699 ≥5 cm 18 (78.3%) 6 (37.5%) p-ERK CST 4370T Receiving anti-HBV treatment before resection Ras CST 3339T Entecavir 0 (0%) 2 (12.5%) CDK4 CST 12790T Adefovir Dipivoxil 0 (0%) 1 (6.3%) CyclinA CST 4656T Lamivudine 1 (4.3%) 0 (0%) CyclinB CST 12231T Recall lacking 2 (8.6%) 0 (0%) CyclinD CST 2978T No CyclinE CST 4129T Receiving non-resection treatment before resection C-PARP Abcam ab32064 TACE 1 (4.3%) 2 (12.5%) B-Actin ProteinTech Cat No. 66009-1-Ig 0 (0%) ProteinTech Cat No. 60004-1-Ig Microwave Ablation 1 (4.3%) GAPDH 20 (87%) 21 (91.4%) 13 (81.3%) Antibody For Co-immunoprecipitation No Assay Enterprise Detail Receiving any RTKs inhibitors treatment before resection 14 (87.5%) EGFR santa sc-373746 Yes 0 (0%) 0 (0%) 23 (100%) 16 (100%) MET CST sc-8057 No IgG santa(M)/CST(R) sc-2025/3900S HCC metastasis existence before resection p-EGFR CST 3777T Intrahepatic thrombus 0 (0%) 1 (6.3%) pMET CST 3077T Retroperitoneal 0 (0%) 1 (6.3%) 1 (4.3%) 0 (0%) Antibody For Immunofluorescence CD45 Abcam 52894 Common bile duct EpCAM Abcam 187372 No 22 (95.7%) 14 (87.5%) Antibody For Immunohistochemistry EGFR santa sc-373746 MET santa sc-514148 Flow cytometry as described [15] and all antibodies used were listed in Table 3. Markers were used for indicating protein lower than 180 KD (PageRuler 26616, Thermo Scientific, MA, USA) and more than 180 KD (PL00003, ProteinTech, China). All HCC cell lines were harvested and resuspended at a concentration of 105/ml. Trypsin digestion was performed in cells with or without suspension at the same time to dissociate clusters into single cells and reduce interference from trypsin. PI/RNAse was used for detecting cell cycle and Annexin V/PI was used for detecting apoptosis of HCC cell lines. Flow cytometry was performed according to the manufacturer’s instructions. Luminex, ELISA, Array kit, and Ras Activation Assay Kit Luminex Kit (R&D system, CA, USA) was coated with RTKs (VEGFR1-3, ErbB2-3, Tie, EphA2, and MCSFR) and RTKs ligands (EGF and HGF). ELISAs (EGFR, MET, PDGFRβ, IGF-1R, and InsR) were used to detect the concentration of RTKs which Luminex Kit didn’t contain. Phosphorylation of RTKs was performed by Human/Mice Phospho-RTKs Array Kit (R&D system, CA, USA) according to the manufacturer’s instructions. Image pro plus 6.0 was used for intensity quantification. Ras Activation Assay Kit (Sigma, MO, USA) was used for the detection of Ras activation. Immunohistochemistry/Immunofluorescence Paraffin specimens from patients with HCC and carcinoma tissues of mice were performed according to the manufacturer’s instructions. Immunofluorescence assays were conducted in a manner blinded to the sample identity. Cells cultured with or without suspension were harvested and dried on coverslips with Poly-L-lysine (Beyotime, China) coated. EDU assay (KeyGEN, China) was used for detecting DNA synthesis of HCC cell lines. Cell Death and Disease (2022)13:351 S. Song et al. 11 Co-culture of HCC and leukocytes Leukocytes were collected from healthy donor blood by gradient centrifugation with leukocytes separation (Solarbio Technologies, China). After being cultured with DMSO or inhibitors for 24 h, HCC cells lines were re-suspended and the supernatant was discarded. Then, equal leukocytes were added to the culture medium with MHCC97H cell line and were cocultured for 24 h in suspension. Cell mixtures were collected, dried on coverslips with Poly-L-lysine coated, and observed by immunofluorescence with EpCAM + EGFR and CD45 staining. Animal xenograft model 5-week-old C57 male mice (Charles River Ltd, Beijing, China) were employed to construct xenograft tumor models. The mice were anesthetized with 50 mg/kg Zoletil®50 (VIRBAC, France) combined with 5 mg/kg Xylazine hydrochloride (Selleck, TX, USA) by intraperitoneal injection, and 105 Hepa1-6 cells were injected into the left lobe of the liver in each mouse. The animals were sacrificed after 60 days and the presence of any extrahepatic metastasis except lungs was considered to be a failure, even lung metastasis model was successfully established. Tumor tissues from the liver and lung in the same mouse were collected for phosphorylation detection, immunohistochemistry, immunofluorescence, and scRNA-seq. Single-cell RNA sequence (scRNA-seq) and analysis ScRNA-seq was performed by Annoroad Gene Tech (Beijing, China). Carcinoma tissues were separated from the xenograft model in 48 h and a library was constructed as manufacturer’s instructions. CellRanger was applied to identify cell barcodes in reads1 and UMI marks in different transcripts, and the corresponding reads2 were compared referring to genome through STAR to determine source gene of the reads and complete quantification of gene expression. RBC in cells containing <200 expressed genes or mitochondria UMI rate >80% were excluded. Then, mitochondrial genes were removed in the expression table. After normalizing UMI data of gene expression in each cell by Seurat analysis and UMI data dimension reduction by PCA, the top 20 principal components were selected for cell classification and UMAP construction based on graph-based clustering method. FindAllMarkers function by the Wilcox rank-sum test algorithm (lnFC > 0.25; p-value < 0.05; min. pct > 0.1) was used for marker gene identification. Clinical HCC specimens Paraffin specimens and HCC resections of HCC tissues were collected from the Department of Hepatobiliary Surgery, Shandong Provincial Hospital, Cheeloo College of Medicine, Shandong University. Paraffin specimens of HCC resection for IHC were obtained from patients with further HCC metastasis within ten years. RTKs concentration of HCC resections was determined by Luminex and Elisa. Details of the clinicopathologic characteristics of the recruited HCC patients were shown in Table 4. Statistical analysis GraphPad Prism 8 software (GraphPad, CA, USA) was used for data analysis. The statistical significance of differences between different experimental cell groups was determined by t-tests. Except results of scRNA-seq failed in conforming to Gaussian distribution, all data were estimated of variation within each group of data, and P-value used in all analyses was two-tailed, and the value <0.05 was considered statistically significant. As for the results of scRNA-seq, a Mann-Whitney test was used for analyzing gene expression differences among samples. DATA AVAILABILITY The datasets used and/or analyzed during the current study are available from the corresponding author on reasonable request. REFERENCES 1. Bray F, Ferlay J, Soerjomataram I, Siegel RL, Torre LA, Jemal A. Global cancer statistics 2018: GLOBOCAN estimates of incidence and mortality worldwide for 36 cancers in 185 countries. CA Cancer J Clin. 2018;68:394–424. Cell Death and Disease (2022)13:351 2. 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FUNDING This work was supported in part by grants from the National Natural Science Foundation of China (81772626, 82160124 and 81770607), the Major Special Plan of Science and Technology of Shandong Province (2015ZDXX0802A01), the Clinical Medical Science and Technology Innovation Program (202019094), and WBE Liver Fibrosis Foundation (CFHPC2021011). COMPETING INTERESTS The authors declare no competing interests. CONSENT TO PARTICIPATE Protocols dealing were in accordance with guidelines of the Institutional Animal Care and Use Committee and were approved by the Medical Ethics Committee of Shandong Provincial Hospital, Cheeloo College of Medicine, Shandong University. ETHICS APPROVAL Written informed consents were obtained from all patients before the research was initiated. Protocols were in accordance with the Helsinki Declaration and approved by the Medical Ethics Committee of Shandong Provincial Hospital, Cheeloo College of Medicine, Shandong University. 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